Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrated that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we showed that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accordance with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger-induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which has an impact on the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia.

To elucidate mechanisms that underlie the profound physiological effects of the monoamine precursors 5-hydroxy-L-tryptophan (5-HTP) and L-3,4-dihydroxyphenylalanine (L-DOPA), we examined their action on single monoaminergic neurons isolated from the ganglia of the gastropod snail Lymnaea stagnalis. In isolated serotonergic PeA motoneurons, 5-HTP produced excitation. The effect was mimicked by serotonin at 0.5-1 microM, masked by pretreatment with serotonin at higher concentrations, and abolished by the inhibitor of aromatic amino acid decarboxylase (AAAD) m-hydroxybenzylhydrazine (NSD-1015), the inhibitor of the vesicular monoamine transporter reserpine or the serotonin receptor antagonist mianserin. Exposure of the dopaminergic interneurons RPeD1 to L-DOPA caused a biphasic effect composed of a depolarization followed by a hyperpolarization. AAAD inactivation with NSD-1015, as well as the blockade of dopamine receptors with sulpiride, resulted in the enhancement of the excitatory effect, and the abolition of the inhibitory effect. Dopamine caused hyperpolarization and masked the inhibitory phase of L-DOPA action. The results show that precursors affect the rate of firing of isolated monoaminergic neurons and that their effect is completely or partially mediated by the enhanced synthesis of the respective neurotransmitter, followed by extrasynaptic release of the latter and activation of extrasynaptic autoreceptors.

Although predictions from the past about the future have been of major interest to current neuroscience, how past and present behavioral experience interacts at the level of a single neuron remains largely unknown. Using the pond snail Lymnaea stagnalis we found that recent experience of terrestrial locomotion (exercise) results in a long-term increase in the firing rate of serotonergic pedal (PeA) neurons. Isolation from the CNS preserved the “memory” about previous motor activity in the neurons even after the animals rested for two hours in deep water after the exercise. In contrast, in the CNS, no difference in the firing rate between the control and “exercise-rested” (ER) neurons was seen. ER snails, when placed again on a surface to exercise, nevertheless showed faster locomotor arousal. The difference in the firing rate between the control and ER isolated neurons disappeared when the neurons were placed in the microenvironment of their home ganglia. It is likely that an increased content of dopamine in the CNS masks an increased excitation of PeA neurons after rest: the dopamine receptor antagonist sulpiride produced sustained excitation in PeA neurons from ER snails but not in the control. Therefore, our data suggest the involvement of two mechanisms in the interplay of past and present experiences at the cellular level: intrinsic neuronal changes in the biophysical properties of the cell membrane and extrinsic modulatory environment of the ganglia.

The involvement of serotonin in mediating hunger-related changes in behavioral state has been described in many invertebrates. However, the mechanisms by which hunger signals to serotonergic cells remain unknown. We tested the hypothesis that serotonergic neurons can directly sense the concentration of glucose, a metabolic indicator of nutritional state. In the snail Lymnaea stagnalis, we demonstrate that completely isolated pedal serotonergic neurons that control locomotion changed their biophysical characteristics in response to glucose application by lowering membrane potential and decreasing the firing rate. Additionally, the excitatory response of the isolated serotonergic neurons to the neuroactive microenvironment of the pedal ganglia was significantly lowered by glucose application. Because hunger has been reported to increase the activity of select neurons and their responses to the pedal ganglia microenvironment, these responses to glucose are in accordance with the hypothesis that direct glucose signaling is involved in the mediation of the hunger-related behavioral state.

The buccal ganglia of the mollusc, Lymnaea stagnalis, contain two distinct but interacting rhythm-generating units: the central pattern generator for the buccal rhythm and nitrergic B2 neurons controlling gut motility. Nitric oxide (NO) has previously been demonstrated to be involved in the activation of the buccal rhythm. Here, we found that NO-generating substances (SNP and SNAP) activated the buccal rhythm while slowing the endogenous rhythm of B2 bursters. The inhibitor of NO-synthase, L-NNA, the NO scavenger PTIO, or the inhibitor of soluble guanylyl cyclase, ODQ, each produced opposite, depolarising effects on the B2 neuron. In isolated B2 cells, only depolarising effects of substances interfering with NO production or function (PTIO, L-NNA and ODQ) were detected, whereas the NO donors had no hyperpolarising effects. However, when an isolated B2 cell was placed close to its initial position in the ganglion, hyperpolarising effects could be obtained with NO donors. This indicates that extrasynaptic release of some unidentified factor(s) mediates the hyperpolarising effects of NO donors on the B2 bursters. The results suggest that NO is involved in coordination between the radula and foregut movements and that the effects of NO are partially mediated by the volume chemical neurotransmission of as yet unknown origin.