A novel and unique refraction-based optical recording system for pharmacological investigations on isolated muscle preparations.

In the field of neuroscience and ecotoxicology, there is a great need for investigating the effect(s) of a variety of different chemicals (e.g., pharmacologically active compounds, pesticides, neurotransmitters, modulators) at different biological levels. Different contractile tissue preparations have provided excellent model systems for in vitro pharmacological experiments for a long time. However, such investigations usually apply mechanical force transducer-based approaches. Thus, a rapid, easy, cheap, digital, and reproducible in vitro pharmacological method based on an effective, 'non-invasive' (compared to the force-transducer approaches), refraction-based optical recording approach and isolated heart preparations was developed.•A versatile and unique refraction-based optical recording system with a Java application was developed.•The recording system was tested and validated on isolated heart preparations obtained from the widely used invertebrate model organism, the great pond snail (Lymnaea stagnalis).•The recording system illustrates the progression of technology from the mechanical force transducer system and can represent a suitable tool in ecotoxicology or neuroscience.

Interneuronal mechanisms for learning-induced switch in a sensory response that anticipates changes in behavioral outcomes.

Sensory cues in the natural environment predict reward or punishment, important for survival. For example, the ability to detect attractive tastes indicating palatable food is essential for foraging while the recognition of inedible substrates prevents harm. While some of these sensory responses are innate, they can undergo fundamental changes due to prior experience associated with the stimulus. However, the mechanisms underlying such behavioral switching of an innate sensory response at the neuron and network levels require further investigation. We used the model learning system of Lymnaea stagnalis1-3 to address the question of how an anticipated aversive outcome reverses the behavioral response to a previously effective feeding stimulus, sucrose. Key to the switching mechanism is an extrinsic inhibitory interneuron of the feeding network, PlB (pleural buccal4,5), which is inhibited by sucrose to allow a feeding response. After multi-trial aversive associative conditioning, pairing sucrose with strong tactile stimuli to the head, PlB's firing rate increases in response to sucrose application to the lips and the feeding response is suppressed; this learned response is reversed by the photoinactivation of a single PlB. A learning-induced persistent change in the cellular properties of PlB that results in an increase rather than a decrease in its firing rate in response to sucrose provides a neurophysiological mechanism for this behavioral switch. A key interneuron, PeD12 (Pedal-Dorsal 12), of the defensive withdrawal network5,6 does not mediate the conditioned suppression of feeding, but its facilitated output contributes to the sensitization of the withdrawal response.

Interneuronal mechanism for Tinbergen's hierarchical model of behavioral choice.

Recent studies of behavioral choice support the notion that the decision to carry out one behavior rather than another depends on the reconfiguration of shared interneuronal networks [1]. We investigated another decision-making strategy, derived from the classical ethological literature [2, 3], which proposes that behavioral choice depends on competition between autonomous networks. According to this model, behavioral choice depends on inhibitory interactions between incompatible hierarchically organized behaviors. We provide evidence for this by investigating the interneuronal mechanisms mediating behavioral choice between two autonomous circuits that underlie whole-body withdrawal [4, 5] and feeding [6] in the pond snail Lymnaea. Whole-body withdrawal is a defensive reflex that is initiated by tactile contact with predators. As predicted by the hierarchical model, tactile stimuli that evoke whole-body withdrawal responses also inhibit ongoing feeding in the presence of feeding stimuli. By recording neurons from the feeding and withdrawal networks, we found no direct synaptic connections between the interneuronal and motoneuronal elements that generate the two behaviors. Instead, we discovered that behavioral choice depends on the interaction between two unique types of interneurons with asymmetrical synaptic connectivity that allows withdrawal to override feeding. One type of interneuron, the Pleuro-Buccal (PlB), is an extrinsic modulatory neuron of the feeding network that completely inhibits feeding when excited by touch-induced monosynaptic input from the second type of interneuron, Pedal-Dorsal12 (PeD12). PeD12 plays a critical role in behavioral choice by providing a synaptic pathway joining the two behavioral networks that underlies the competitive dominance of whole-body withdrawal over feeding.

Reversal of age-related learning deficiency by the vertebrate PACAP and IGF-1 in a novel invertebrate model of aging: the pond snail (Lymnaea stagnalis).

With the increase of life span, nonpathological age-related memory decline is affecting an increasing number of people. However, there is evidence that age-associated memory impairment only suspends, rather than irreversibly extinguishes, the intrinsic capacity of the aging nervous system for plasticity (1). Here, using a molluscan model system, we show that the age-related decline in memory performance can be reversed by administration of the pituitary adenylate cyclase activating polypeptide (PACAP). Our earlier findings showed that a homolog of the vertebrate PACAP38 and its receptors exist in the pond snail (Lymnaea stagnalis) brain (2), and it is both necessary and instructive for memory formation after reward conditioning in young animals (3). Here we show that exogenous PACAP38 boosts memory formation in aged Lymnaea, where endogenous PACAP38 levels are low in the brain. Treatment with insulin-like growth factor-1, which in vertebrates was shown to transactivate PACAP type I (PAC1) receptors (4) also boosts memory formation in aged pond snails. Due to the evolutionarily conserved nature of these polypeptides and their established role in memory and synaptic plasticity, there is a very high probability that they could also act as "memory rejuvenating" agents in humans.

A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate.

Similar to other invertebrate and vertebrate animals, cAMP-dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) and for the AC-activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this "strong" food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a "weak" multitrial food-reward conditioning paradigm, lip touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multitrial tactile conditioning accelerated the formation of transcription-dependent memory. Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning.

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present and biochemically active in the central nervous system of the pond snail Lymnaea stagnalis.

PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.

G-protein coupled receptor kinase-like immunoreactivity in the snail, Helix pomatia, neurons.

GPRCs are regulated via phosphorylation by different protein kinases including GRKs and PKA and PKC. The purpose of this study was to determine the presence and physiological role of GRKs in the tissues of the snail, Helix pomatia. Here we report that immunoblotting of brain homogenate with anti-GRK2/3 antibody prepared from mammalian tissue can be detected in snail GRK-like immunoreactivity. The GRK2/3 immunoreactivity was found at approximately 80 kDa in a variety of cells, including salivary duct, salivary gland and eye. Intracellular injection of the anti-GRK2/3 prevented the neuron from desensitization and agonist-induced activation augmented the phosphorylated GRKs in the membrane fraction suggesting that GRKs may have a functional role in the neuropeptide receptor desensitization in snail.

Mechanism of 4-aminopyridine block of the transient outward K-current in identified Helix neuron.

The block of the transient outward K-current, I(K(A)) by 4-aminopyridine (4-AP) and blood-depressing substances (BDS) was investigated in identified Helix pomatia neurons (LPa3) using the two microelectrode voltage-clamp technique. The present study shows that 4-AP inhibits I(K(A)) in snail neurons in a voltage- and concentration-dependent manner. The 4-AP block of I(K(A)) involves the block of both open and closed states of the channel, however binding to open channels is preferred. It is suggested that 4-AP have two binding sites on the identified Helix neuron. One site causes an open channel block, which affects the N-type inactivation, and binding to the second site induces closed channel block, which affects C-type inactivation. In control solution the inactivating phase of the current is biexponential, suggesting simultaneous presence of two types of inactivation. The counterplay of these mechanisms results in the crossover of the current traces recorded from control and 4-AP blocked channels. It is assumed that use-dependence does not occur through blocker 'trapping', but rather by a different mechanism. BDS had no effect on Helix I(K(A)), suggesting that transient potassium channels in LPa3 neuron are not Kv3.4 type channels.