Subcellular Peptide Localization in Single Identified Neurons by Capillary Microsampling Mass Spectrometry.

Single cell mass spectrometry (MS) is uniquely positioned for the sequencing and identification of peptides in rare cells. Small peptides can take on different roles in subcellular compartments. Whereas some peptides serve as neurotransmitters in the cytoplasm, they can also function as transcription factors in the nucleus. Thus, there is a need to analyze the subcellular peptide compositions in identified single cells. Here, we apply capillary microsampling MS with ion mobility separation for the sequencing of peptides in single neurons of the mollusk Lymnaea stagnalis, and the analysis of peptide distributions between the cytoplasm and nucleus of identified single neurons that are known to express cardioactive Phe-Met-Arg-Phe amide-like (FMRFamide-like) neuropeptides. Nuclei and cytoplasm of Type 1 and Type 2 F group (Fgp) neurons were analyzed for neuropeptides cleaved from the protein precursors encoded by alternative splicing products of the FMRFamide gene. Relative abundances of nine neuropeptides were determined in the cytoplasm. The nuclei contained six of these peptides at different abundances. Enabled by its relative enrichment in Fgp neurons, a new 28-residue neuropeptide was sequenced by tandem MS.

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.

Susceptibility of memory consolidation during lapses in recall.

Memories that can be recalled several hours after learning may paradoxically become inaccessible for brief periods after their formation. This raises major questions about the function of these early memory lapses in the structure of memory consolidation. These questions are difficult to investigate because of the lack of information on the precise timing of lapses. However, the use of a single-trial conditioning paradigm in Lymnaea solves this problem. Here we use electrophysiological and behavioural experiments to reveal lapses in memory recall at 30 min and 2 h post conditioning. We show that only during these lapses is consolidation of long-term memory susceptible to interruption by external disturbance. These shared time points of memory lapse and susceptibility correspond to transitions between different phases of memory that have different molecular requirements. We propose that during periods of molecular transition memory recall is weakened, allowing novel sensory cues to block the consolidation of long-term memory.

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.

Balanced plasticity and stability of the electrical properties of a molluscan modulatory interneuron after classical conditioning: a computational study.

The Cerebral Giant Cells (CGCs) are a pair of identified modulatory interneurons in the Central Nervous System of the pond snail Lymnaea stagnalis with an important role in the expression of both unconditioned and conditioned feeding behavior. Following single-trial food-reward classical conditioning, the membrane potential of the CGCs becomes persistently depolarized. This depolarization contributes to the conditioned response by facilitating sensory cell to command neuron synapses, which results in the activation of the feeding network by the conditioned stimulus. Despite the depolarization of the membrane potential, which enables the CGGs to play a key role in learning-induced network plasticity, there is no persistent change in the tonic firing rate or shape of the action potentials, allowing these neurons to retain their normal network function in feeding. In order to understand the ionic mechanisms of this novel combination of plasticity and stability of intrinsic electrical properties, we first constructed and validated a Hodgkin-Huxley-type model of the CGCs. We then used this model to elucidate how learning-induced changes in a somal persistent sodium and a delayed rectifier potassium current lead to a persistent depolarization of the CGCs whilst maintaining their firing rate. Including in the model an additional increase in the conductance of a high-voltage-activated calcium current allowed the spike amplitude and spike duration also to be maintained after conditioning. We conclude therefore that a balanced increase in three identified conductances is sufficient to explain the electrophysiological changes found in the CGCs after classical conditioning.

Sensory driven multi-neuronal activity and associative learning monitored in an intact CNS on a multielectrode array.

The neuronal network controlling feeding behavior in the CNS of the mollusc Lymnaea stagnalis has been extensively investigated using intracellular microelectrodes. Using microelectrodes however it has not been possible to record from large numbers of neurons simultaneously and therefore little is known about the population coding properties of the feeding network. Neither can the relationships between feeding and neuronal networks controlling other behaviors be easily analyzed with microelectrodes. Here we describe a multielectrode array (MEA) technique for recording action potentials simultaneously from up to 60 electrodes on the intact CNS. The preparation consists of the whole CNS connected by sensory nerves to the chemosensory epithelia of the lip and esophagus. From the buccal ganglia, the region of the CNS containing the feeding central pattern generator (CPG), a rhythmic pattern of activity characteristic of feeding was readily induced either by depolarizing an identified feeding-command neuron (the CV1a) or by perfusing the chemosensory epithelia with sucrose, a gustatory stimulus known to activate feeding. Activity induced by sucrose is not restricted to the buccal ganglia but is distributed widely throughout the CNS, notably in ganglia controlling locomotion, a behavior that must be coordinated with feeding. The MEA also enabled us to record electrophysiological consequences of the associative conditioning of feeding behavior. The results suggest that MEA recording from an intact CNS enables distributed, multiple-source neural activity to be analyzed in the context of biologically relevant behavior, behavioral coordination and behavioral plasticity.

Different phases of long-term memory require distinct temporal patterns of PKA activity after single-trial classical conditioning.

The cAMP-dependent protein kinase (PKA) is known to play a critical role in both transcription-independent short-term or intermediate-term memory and transcription-dependent long-term memory (LTM). Although distinct phases of LTM already have been demonstrated in some systems, it is not known whether these phases require distinct temporal patterns of learning-induced PKA activation. This question was addressed in a robust form of associative LTM that emerges within a matter of hours after single-trial food-reward classical conditioning in the pond snail Lymnaea stagnalis. After establishing the molecular and functional identity of the PKA catalytic subunit in the Lymnaea nervous system, we used a combination of PKA activity measurement and inhibition techniques to investigate its role in LTM in intact animals. PKA activity in ganglia involved in single-trial learning showed a short latency but prolonged increase after classical conditioning. However, while increased PKA activity immediately after training (0-10 min) was essential for an early phase of LTM (6 h), the late phase of LTM (24 h) required a prolonged increase in PKA activity. These observations indicate mechanistically different roles for PKA in recent and more remote phases of LTM, which may underpin different cellular and molecular mechanisms required for these phases.

Dynamic control of a central pattern generator circuit: a computational model of the snail feeding network.

Central pattern generators (CPGs) are networks underlying rhythmic motor behaviours and they are dynamically regulated by neuronal elements that are extrinsic or intrinsic to the rhythmogenic circuit. In the feeding system of the pond snail, Lymnaea stagnalis, the extrinsic slow oscillator (SO) interneuron controls the frequency of the feeding rhythm and the N3t (tonic) has a dual role; it is an intrinsic CPG interneuron, but it also suppresses CPG activity in the absence of food, acting as a decision-making element in the feeding circuit. The firing patterns of the SO and N3t neurons and their synaptic connections with the rest of the CPG are known, but how these regulate network function is not well understood. This was investigated by building a computer model of the feeding network based on a minimum number of cells (N1M, N2v and N3t) required to generate the three-phase motor rhythm together with the SO that was used to activate the system. The intrinsic properties of individual neurons were represented using two-compartment models containing currents of the Hodgkin-Huxley type. Manipulations of neuronal activity in the N3t and SO neurons in the model produced similar quantitative effects to food and electrical stimulation in the biological network indicating that the model is a useful tool for studying the dynamic properties of the feeding circuit. The model also predicted novel effects of electrical stimulation of two CPG interneurons (N1M and N2v). When tested experimentally, similar effects were found in the biological system providing further validation of our model.

Phase-dependent molecular requirements for memory reconsolidation: differential roles for protein synthesis and protein kinase A activity.

After consolidation, a process that requires gene expression and protein synthesis, memories are stable and highly resistant to disruption by amnestic influences. Recently, consolidated memory has been shown to become labile again after retrieval and to require a phase of reconsolidation to be preserved. New findings, showing that the dependence of reconsolidation on protein synthesis decreases with the age of memory, point to changing molecular requirements for reconsolidation during memory maturation. We examined this possibility by comparing the roles of protein synthesis (a general molecular requirement for memory consolidation) and the activation of protein kinase A (PKA) (a specific molecular requirement for memory consolidation), in memory reconsolidation at two time points after training. Using associative learning in Lymnaea, we show that reconsolidation after the retrieval of consolidated memory at both 6 and 24 h requires protein synthesis. In contrast, only reconsolidation at 6 h after training, but not at 24 h, requires PKA activity, which is in agreement with the measured retrieval-induced PKA activation at 6 h. This phase-dependent differential molecular requirement for reconsolidation supports the notion that even seemingly consolidated memories undergo further selective molecular maturation processes, which may only be detected by analyzing the role of specific pathways in memory reconsolidation after retrieval.

Cyclic AMP response element-binding (CREB)-like proteins in a molluscan brain: cellular localization and learning-induced phosphorylation.

The phosphorylation and the binding to DNA of the nuclear transcription factor, cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) are conserved key steps in the molecular cascade leading to the formation of long-term memory (LTM). Here, we characterize, for the first time, a CREB1-like protein in the central nervous system (CNS) of Lymnaea, a model system used widely for the study of the fundamental mechanisms of learning and memory. We demonstrate cAMP response element (CRE)-binding activity in CNS protein extracts and show that one of the CRE-binding proteins is recognized by a polyclonal antibody raised to mammalian (human) CREB1. The same antibody detects specific CREB1 immunoreactivity in CNS extracts and in the nuclei of most neurons in the brain. Moreover, phospho-CREB1-specific immunoreactivity is increased significantly in protein extracts of the CNS by forskolin, an activator of adenylate cyclase. The forskolin-induced increase in phospho-CREB1 immunoreactivity is localized to the nuclei of CNS neurons, some of which have an important role in the formation of LTM. Significantly, classical food-reward conditioning increases phospho-CREB1 immunoreactivity in Lymnaea CNS protein extracts. This increase in immunoreactivity is specific to the ganglia that contain the feeding circuitry, which undergoes cellular changes after classical conditioning. This work establishes the expression of a highly conserved functional CREB1-like protein in the CNS of Lymnaea and opens the way for a detailed analysis of the role of CREB proteins in LTM formation in this model system.

Loss of self-inhibition is a cellular mechanism for episodic rhythmic behavior.

BACKGROUND: Rhythmic motor behaviors can be generated continuously (e.g., breathing) or episodically (e.g., locomotion, swallowing), when short or long bouts of rhythmic activity are interspersed with periods of quiescence. Although the mechanisms of rhythm generation are known in detail in many systems, there is very little understanding of how the episodic nature of rhythmic behavior is produced at the neuronal level. RESULTS: Using a well-established episodic rhythm-generating neural circuit controlling molluscan feeding, we demonstrate that quiescence between bouts of activity arises from active, maintained inhibition of an otherwise rhythmically active network. We show that the source of the suppressive drive is within the circuit itself; a single central pattern generator (CPG) interneuron type that fires tonically to inhibit feeding during quiescence. Suppression of the tonic activity of this neuron by food is sufficient to change the network from an inactive to a rhythmically active state, with the cell switching function to fire phasically as part of the food-evoked rhythmogenesis. Furthermore, the absolute level of intrinsic suppressive control is modulated extrinsically by the animal's behavioral state (e.g., hunger/satiety), increasing the probability of episodes of feeding when the animal is hungry. CONCLUSIONS: By utilizing the same intrinsic member of a CPG network in both rhythm-generation and suppression, this system has developed a simple and efficient mechanism for generating a variable level of response to suit the animal's changing behavioral demands.