The Yerkes-Dodson law and appropriate stimuli for conditioned taste aversion in Lymnaea.

The pond snail Lymnaea stagnalis can learn conditioned taste aversion and then consolidate it into long-term memory (LTM). A high-voltage electric shock was used as the unconditioned stimulus, where we have previously used KCl. We varied the strength of both the conditioned and unconditioned stimuli to determine whether the so-called Yerkes-Dodson law prevailed. This is an empirical relationship between the state of arousal and LTM formation, showing that there is an optimal level of arousal leading to memory formation. However, too little or too much arousal results in poorer LTM. We found here that the most appropriate stimuli to use in taste aversion training in Lymnaea were a 10 mmol l(-1) sucrose solution as the conditioned stimulus and a 3 s electric shock as the unconditioned stimulus.

Memory block: a consequence of conflict resolution.

Food deprivation for 1 day in the pond snail Lymnaea stagnalis before aversive classical conditioning results in optimal conditioned taste aversion (CTA) and long-term memory (LTM) formation, whereas 5-day food deprivation before training does not. We hypothesize that snails do in fact learn and form LTM when trained after prolonged food deprivation, but that severe food deprivation blocks their ability to express memory. We trained 5-day food-deprived snails under various conditions, and found that memory was indeed formed but is overpowered by severe food deprivation. Moreover, CTA-LTM was context dependent and was observed only when the snails were in a context similar to that in which the training occurred.

Involvement of insulin-like peptide in long-term synaptic plasticity and long-term memory of the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA-LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA-LTM in the Lymnaea CNS.

An increase in insulin is important for the acquisition conditioned taste aversion in Lymnaea.

Conditioned taste aversion (CTA) in Lymnaea is brought about by pairing a sucrose solution (the conditioned stimulus, CS) with an electric shock (the unconditioned stimulus, US). Following repeated CS-US pairings, CTA occurs and it is consolidated into long-term memory (LTM). The best CTA is achieved, if snails are food-deprived for 1 day before training commences. With a longer period of food deprivation (5 days), learning and memory formation does not occur. It has been hypothesized that the levels of insulin in the central nervous system (CNS) are very important for CTA to occur. To test his hypothesis, we injected insulin directly into 5-day food-deprived snails. The injection of insulin, as expected, resulted in a decrease in hemolymph glucose concentration. Consistent with our hypothesis with insulin injection, learning and memory formation of CTA occurred. That is, the 'insulin spike' is more important than an increase in hemolymph glucose concentration for CTA-LTM. If we injected an insulin receptor antibody into the snails before the insulin injection, learning was formed but memory formation was not, which is consistent with our previous study. Therefore, a rise in the insulin concentration (i.e., insulin spike) in the CNS is considered to be a key determining factor in the process of CTA-LTM.

What are the elements of motivation for acquisition of conditioned taste aversion?

The pond snail Lymnaea stagnalis is capable of being classically conditioned to avoid food and to consolidate this aversion into a long-term memory (LTM). Previous studies have shown that the length of food deprivation is important for both the acquisition of taste aversion and its consolidation into LTM, which is referred to as conditioned taste aversion (CTA). Here we tested the hypothesis that the hemolymph glucose concentration is an important factor in the learning and memory of CTA. One-day food deprivation resulted in the best learning and memory, whereas more prolonged food deprivation had diminishing effects. Five-day food deprivation resulted in snails incapable of learning or remembering. During this food deprivation period, the hemolymph glucose concentration decreased. If snails were fed for 2days following the 5-day food deprivation, their glucose levels increased significantly and they exhibited both learning and memory, but neither learning nor memory was as good as with the 1-day food-deprived snails. Injection of the snails with insulin to reduce glucose levels resulted in better learning and memory. Insulin is also known to cause a long-term enhancement of synaptic transmission between the feeding-related neurons. On the other hand, injection of glucose into 5-day food-deprived snails did not alter their inability to learn and remember. However, if these snails were fed on sucrose for 3min, they then exhibited learning and memory formation. Our data suggest that hemolymph glucose concentration is an important factor in motivating acquisition of CTA in Lymnaea and that the action of insulin in the brain and the feeding behavior are also important factors.

Paired pulse ratio analysis of insulin-induced synaptic plasticity in the snail brain.

Insulin's action in the brain can directly alter cognitive functioning. We have recently shown that molluscan insulin-related peptides are upregulated following a conditioned taste aversion (CTA) training procedure. In addition, when mammalian insulin is superfused over the isolated Lymnaea stagnalis central nervous system, it elicits long-term synaptic enhancement at the monosynaptic connection between the cerebral giant cell and the buccal 1 (B1) motor neuron. This synaptic enhancement is thought to be a neural correlate of CTA. Here, we examined whether the observed changes in synaptic plasticity were the result of presynaptic and/or postsynaptic alterations using the paired pulse procedure. The paired pulse ratio was unaltered following insulin application, suggesting that insulin's effects on synaptic plasticity are mediated postsynaptically in the B1 motor neuron. Thus, it was suggested that postsynaptic changes need to be considered when insulin's actions on synaptic plasticity are examined.

Multiple subtypes of serotonin receptors in the feeding circuit of a pond snail.

In the central nervous system of the pond snail Lymnaea stagnalis, serotonergic transmission plays an important role in controlling feeding behavior. Recent electrophysiological studies have claimed that only metabotropic serotonin (5-HT(2)) receptors, and not ionotropic (5-HT(3)) receptors, are used in synapses between serotonergic neurons (the cerebral giant cells, CGCs) and the follower buccal motoneurons (the B1 cells). However, these data are inconsistent with previous results. In the present study, we therefore reexamined the serotonin receptors to identify the receptor subtypes functioning in the synapses between the CGCs and the B1 cells by recording the compound excitatory postsynaptic potential (EPSP) of the B1 cells evoked by a train of stimulation to the CGC in the presence of antagonists: cinanserin for 5-HT(2) and/or MDL72222 for 5-HT(3). The compound EPSP amplitude was partially suppressed by the application of these antagonists. The rise time of the compound EPSP was longer in the presence of MDL72222 than in that of cinanserin. These results suggest that these two subtypes of serotonin receptors are involved in the CGC-B1 synapses, and that these receptors contribute to compound EPSP. That is, the fast component of compound EPSP is mediated by 5-HT(3)-like receptors, and the slow component is generated via 5-HT(2)-like receptors.

Glutamate transporters in the central nervous system of a pond snail.

Previous studies on glutamate (GLU) and its receptors in the pond snail Lymnaea stagnalis have suggested that GLU functions as a neurotransmitter in various behaviors, particularly for generation of feeding rhythm. The uptake mechanism of GLU is not yet known in Lymnaea. In the present study, we characterized the GLU transporters and examined their functions in the feeding circuits of the central nervous system (CNS) in Lymnaea. First, measurement of the accumulation of (3)H-labeled GLU revealed the presence of GLU transport systems in the Lymnaea CNS. The highest accumulation rate was observed in the buccal ganglia, supporting the involvement of GLU transport systems in feeding behavior. Second, we cloned two types of GLU transporters from the Lymnaea CNS, the excitatory amino acid transporter (LymEAAT) and the vesicular GLU transporter (LymVGLUT). When we compared their amino acid sequences with those of mammalian EAATs and VGLUTs, we found that the functional domains of both types are well conserved. Third, in situ hybridization revealed that the mRNAs of LymEAAT and LymVGLUT are localized in large populations of nerve cells, including the major feeding motoneurons in the buccal ganglia. Finally, we inhibited LymEAAT and found that changes in the firing patterns of the feeding motoneurons that have GLUergic input were similar to those obtained following stimulation with GLU. Our results confirmed the presence of GLU uptake systems in the Lymnaea CNS and showed that LymEAAT is required for proper rhythm generation, particularly for generation of the feeding rhythm.

Raphe modulation of the pre-Bötzinger complex respiratory bursts in in vitro medullary half-slice preparations of neonatal mice.

Spontaneous respiratory bursts which begin in the pre-Bötzinger complex were recorded from the hypoglossal (XIIth) nerve rootlets of in vitro slices prepared from newborn mice. First, we examined the respiratory bursts before and after a midline or para-midline transection which spared the caudal raphe nuclei: the raphe obscurus and raphe pallidus. After a midline transection, the respiratory bursts in both half-slices were desynchronized and had slightly decreased amplitudes and frequencies. After a para-midline transection, the bursts continued with similar frequencies in the half slice containing the raphe obscurus and raphe pallidus. Second, to analyze the effects of modulation by the raphe obscurus and raphe pallidus, a dorsal or ventral midline lesion was used to damage either the raphe obscurus or raphe pallidus. After a dorsal lesion, the synchronized respiratory bursts persisted with slightly decreased frequencies. In contrast, after a ventral lesion, the bursts were almost completely abolished, but recovered significantly after the addition of 5-HT. The present results demonstrated that the pre-Bötzinger complex on each side of the medulla can independently generate rhythmic respiratory activity. It is suggested that the 5-HT released from the ventral part of the raphe nuclei (predominantly the raphe pallidus) plays a critical role in sustaining rhythmic respiratory bursts.

Upside-down gliding of Lymnaea.

The pond snail Lymnaea stagnalis can often be observed moving upside down on its back just below the surface of the water. We have termed this form of movement "upside-down gliding." To elucidate the mechanism of this locomotion, we performed a series of experiments involving behavioral analyses and microscopic observations. These experiments were designed (1) to measure the speed of this locomotion; (2) to determine whether the mucus secreted from the foot of Lymnaea repels water, thereby allowing the snail to exploit the surface tension of the water for upside-down gliding; and (3) to observe the beating of foot cilia in this behavior. The beating of these cilia is thought to be the primary driving force for upside-down gliding. Our results demonstrate that upside-down gliding is an efficient active process involving the secretion of mucus that floats up to the water surface to serve as a substrate upon which cilia beat to cause locomotion at the underside of the water surface.

Requirement of new protein synthesis of a transcription factor for memory consolidation: paradoxical changes in mRNA and protein levels of C/EBP.

Some specific transcription factors are essential for memory consolidation across species. However, it is still unclear whether only the activation of constitutively expressed forms of these conserved transcription factors is involved in memory consolidation or their de novo synthesis also occurs after learning. This question has remained unanswered partly because of the lack of an efficient method for the determination of copy numbers of particular mRNAs in single neurons, which allows the detection of new transcription at the cellular level. Here we applied a newly developed protocol of single-cell quantitative real-time polymerase chain reaction (qRT-PCR) to single neurons playing an important role in associative learning. Specifically, we examined the changes in the mRNA and protein expression levels of a highly conserved transcription factor, CCAAT/enhancer binding protein (C/EBP), in the paired B2 motoneurons of the pond snail Lymnaea stagnalis. These buccal neurons are involved in the motor control of feeding behavior, with a potentially important role in conditioned taste aversion (CTA). Single-cell qRT-PCR revealed a significant decrease in LymC/EBP mRNA copy numbers in the B2 motoneurons during memory consolidation after CTA training. By contrast, isoelectric focusing and immunoblotting of extracts of the buccal ganglia showed that translation and phosphorylation levels of LymC/EBP significantly increased during memory consolidation. The C/EBP-like immunoreactivity in the B2 motoneurons, which are the major immunopositive component in the buccal ganglia, also significantly increased during memory consolidation, suggesting that the main source of increase in the level of protein in the buccal ganglia are the B2 motoneurons. Thus, early memory consolidation after CTA learning in L.stagnalis involves both the rapid synthesis and phosphorylation of LymC/EBP as well as the rapid breakdown of LymC/EBP mRNA in the neural network controlling feeding, suggesting that all of these processes play a role in the function of C/EBP in memory consolidation.

Involvement of medullary GABAergic and serotonergic raphe neurons in respiratory control: electrophysiological and immunohistochemical studies in rats.

In the present study we first examined the possible involvement of the putative neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5-HT) in raphe-induced facilitatory or inhibitory effects on the respiratory activity of rats. Secondly, we investigated the possibility of spinal projections of GABAergic and serotonergic neurons from the medullary raphe nuclei to the phrenic motor nucleus (PMN). We observed that an intravenous (i.v.) injection of (+)-bicuculline, a GABA(A) receptor antagonist, significantly reduced respiratory inhibition induced by electrical stimulation of the raphe magnus (RM) or the raphe obscurus (RO). On the other hand, an i.v. injection of methysergide, a broad-spectrum 5-HT receptor antagonist, significantly reduced the respiratory facilitation induced by electrical stimulation of the raphe pallidus (RP) or RO. By using a combined method of retrograde tracing with Texas Red injected into the PMN region at segments C4 and C5 and immunohistochemical labeling, we observed that glutamic acid decarboxylase (GAD; a GABA synthesizing enzyme) immunopositive and Texas Red double labeled neurons were predominantly localized in the RM, and additionally in the RO. However 5-HT immunopositive and Texas Red double-labeled neurons were predominantly localized in the RP, and additionally in the RO and RM. These findings suggest that RM-, or RO-induced inhibitory effects, are transmitted, at least in part, to the PMN via a direct GABAergic descending pathway. The RP-, or RO-induced facilitatory effects in rats however, are transmitted via a serotonergic descending pathway.

Comparison of midazolam effects on inhibitory postsynaptic currents in hippocampal CA3 with those in CA1.

We compared the effects of midazolam, one of the popular benzodiazepines, on inhibitory postsynaptic currents in CA3 pyramidal cells with those in CA1 pyramidal cells in rat hippocampal slices. With all of the midazolam concentrations tested, the conductance of the evoked inhibitory postsynaptic current of the CA3 and CA1 pyramidal cells was significantly larger than the control, and increased in a dose-dependent manner. The normalized conductance of the inhibitory postsynaptic currents of the CA3 and CA1 pyramidal cells was not significantly different. However, we found that midazolam significantly prolonged the decay time of inhibitory postsynaptic currents of CA3 pyramidal cells. The results suggest that the differential effect of midazolam on the inhibitory postsynaptic currents could be due to the different decay kinetics, which depend on the gamma-aminobutyric acid type A receptor subunit compositions of the CA3 and CA1 pyramidal cells.

Distal forelimb cross-innervation effectively induces formation of corticorubral synapses.

We investigated whether cross-innervation of the distal forelimb extensor and flexor muscles effectively induced formation of corticorubral synapses in cats. The average time-to-peak of the corticorubral EPSPs in the red nucleus (RN) neurons innervating cervicothoracic segments (C-cells) of animals with distal forelimb cross-innervation was significantly less than that of animals with elbow cross-innervation. These fast-rising corticorubral EPSPs were shown to be induced by newly formed synapses located on the proximal soma-dendritic membrane of RN neurons, indicating that distal forelimb cross-innervation is more effective for inducing synapse formation in C-cells than elbow cross-innervation. Thus corticorubral synapse formation after forelimb cross-innervation appears to be related to direct rubrospinal connections to distal forelimb motoneurons and of functional importance to RN neurons for performing voluntary distal forelimb movement.

Non-ocular dermal photoreception in the pond snail Lymnaea stagnalis.

Based on the results of previous behavioral experiments, researchers believe that sensitivity to light stimuli is not restricted to the eyes in the pond snail Lymnaea stagnalis. To determine the presence of a non-ocular dermal photoreception system and to examine the synaptic connections between this peripheral system and the central nervous system, we electrophysiologically examined the activities of the pedal nerves in L. stagnalis by light stimulation. The results demonstrated that light stimulation evokes non-ocular dermal photosensitive responses in the foot, that these responses exert inhibitory, afferent influences through the inferior pedal nerves to the pedal ganglia, and that these responses were independent of the ocular photoreception system in L. stagnalis.

Differential effects of midazolam on inhibitory postsynaptic currents in CA1 pyramidal cells and dentate gyrus granule cells of rat hippocampal slices.

To examine regional differences of synaptic transmission, the effects of midazolam were observed on inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells (CA1-PCs) and dentate gyrus granule cells (DG-GCs) in rat hippocampal slices. Midazolam is one of the most popular benzodiazepines. The monosynaptic IPSCs in the CA1-PCs and DG-GCs were evoked by electrical stimulation of GABAergic interneurons and recorded by whole cell patch-clamp techniques. The effects of specific concentrations of midazolam (0.3, 1, 10 and 75 microM) on the IPSCs in CA1-PCs and DG-GCs were examined at particular membrane potentials (20 mV steps, from -120 to +40 mV). In all midazolam concentrations tested, the conductance of the IPSCs was significantly larger than that in control and was increased by increasing the concentration of midazolam in CA1-PCs (normalized conductance, 0.3 microM, 121%; 1 microM, 125%; 10 microM, 147%; 75 microM, 147%). However, midazolam did not significantly change the conductance of the IPSCs in DG-GCs (normalized conductance, 0.3 microM, 92%; 1 microM, 92%; 10 microM, 91%; 75 microM, 115%). The normalized conductance was significantly different between the CA1-PCs and DG-GCs in 1 and 10 microM midazolam. The results strongly suggest that the differential effects of midazolam on IPSCs in CA1-PCs and DG-GCs could be, at least in part, due to the different sensitivity to midazolam of the GABA(A) receptor subtypes.

Memory trace in feeding neural circuitry underlying conditioned taste aversion in Lymnaea.

BACKGROUND: The pond snail Lymnaea stagnalis can maintain a conditioned taste aversion (CTA) as a long-term memory. Previous studies have shown that the inhibitory postsynaptic potential (IPSP) evoked in the neuron 1 medial (N1M) cell by activation of the cerebral giant cell (CGC) in taste aversion-trained snails was larger and lasted longer than that in control snails. The N1M cell is one of the interneurons in the feeding central pattern generator (CPG), and the CGC is a key regulatory neuron for the feeding CPG. METHODOLOGY/PRINCIPLE FINDINGS: Previous studies have suggested that the neural circuit between the CGC and the N1M cell consists of two synaptic connections: (1) the excitatory connection from the CGC to the neuron 3 tonic (N3t) cell and (2) the inhibitory connection from the N3t cell to the N1M cell. However, because the N3t cell is too small to access consistently by electrophysiological methods, in the present study the synaptic inputs from the CGC to the N3t cell and those from the N3t cell to the N1M cell were monitored as the monosynaptic excitatory postsynaptic potential (EPSP) recorded in the large B1 and B3 motor neurons, respectively. The evoked monosynaptic EPSPs of the B1 motor neurons in the brains isolated from the taste aversion-trained snails were identical to those in the control snails, whereas the spontaneous monosynaptic EPSPs of the B3 motor neurons were significantly enlarged. CONCLUSION/SIGNIFICANCE: These results suggest that, after taste aversion training, the monosynaptic inputs from the N3t cell to the following neurons including the N1M cell are specifically facilitated. That is, one of the memory traces for taste aversion remains as an increase in neurotransmitter released from the N3t cell. We thus conclude that the N3t cell suppresses the N1M cell in the feeding CPG, in response to the conditioned stimulus in Lymnaea CTA.

Does conditioned taste aversion learning in the pond snail Lymnaea stagnalis produce conditioned fear?

In conditioned taste aversion (CTA) training performed on the pond snail Lymnaea stagnalis, a stimulus (the conditional stimulus, CS; e.g., sucrose) that elicits a feeding response is paired with an aversive stimulus (the unconditional stimulus, US) that elicits the whole-body withdrawal response and inhibits feeding. After CTA training and memory formation, the CS no longer elicits feeding. We hypothesize that one reason for this result is that after CTA training the CS now elicits a fear response. Consistent with this hypothesis, we predict the CS will cause (1) the heart to skip a beat and (2) a significant change in the heart rate. Such changes are seen in mammalian preparations exposed to fearful stimuli. We found that in snails exhibiting long-term memory for one-trial CTA (i.e., good learners) the CS significantly increased the probability of a skipped heartbeat, but did not significantly change the heart rate. The probability of a skipped heartbeat was unaltered in control snails given backward conditioning (US followed by CS) or in snails that did not acquire associative learning (i.e., poor learners) after the one-trial CTA training. These results suggest that as a consequence of acquiring CTA, the CS evokes conditioned fear in the conditioned snails, as evidenced by a change in the nervous system control of cardiac activity.

Learning-Dependent Gene Expression of CREB1 Isoforms in the Molluscan Brain.

Cyclic AMP-responsive element binding protein1 (CREB1) has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1) mRNA isoforms of spliced variants in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA) learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

Spontaneous respiratory rhythm generation in in vitro upper cervical slice preparations of neonatal mice.

Isolated upper cervical slice preparations were prepared from neonatal mice to examine whether spontaneous respiratory activity could be generated in the preparations. By using brainstem-spinal cord preparations, we first recorded from the cervical C1-C2 and C4 ventral roots rhythmic bursts which were synchronized with respiratory burst activity of the hypoglossal (XIIth) nerve. Following transection just above the C1 segment, smaller and slower rhythmic bursts still persisted in the C1/C2 ventral roots and these were synchronized with those in the C4 ventral root. The present result, that a bursting rhythm remained in the C1/C2 slices, suggests that the spinal neuronal circuit for generating respiratory rhythm is localized in the upper cervical segments which contain upper cervical inspiratory neurons.