Uli Müller

Prolonged Activation of cAMP-Dependent Protein Kinase during Conditioning Induces Long-Term Memory in Honeybees

To investigate the function cAMP-dependent protein kinase (PKA) exerts in the induction of long-term memory, changes in PKA activity induced by associative learning in vivo were measured in the antennal lobes (ALs) of honeybees. The temporal dynamics of PKA activation depend on both the sequence of conditioned and unconditioned stimuli and the number of conditioning trials. Only multiple-trial conditioning, which induces long-term memory (LTM), leads to a profound prolongation of PKA activation mediated by the NO/cGMP system. Imitation of this prolonged PKA activation in the ALs in combination with single-trial conditioning is sufficient to induce LTM. These findings not only demonstrate the close connection between conditioning procedure and temporal dynamics in PKA activation but also reveal that already during conditioning a distinct temporal pattern of PKA activation is critical for LTM induction in intact animals.

Inhibition of nitric oxide synthase impairs a distinct form of long-term memory in the honeybee, Apis mellifera.

Nitric oxide has been shown to be implicated in neural plasticity that underlies processes of learning and memory. In the honeybee, studies on the role of nitric oxide in associative olfactory learning reveal its specific function in memory formation. Inhibition of nitric oxide synthase during olfactory conditioning impairs a distinct long-term memory that is formed as a consequence of multiple learning trials. Acquisition or retrieval of memory or memory formation induced by a single learning trial is not affected by blocking of nitric oxide synthase. This finding provides a first step toward dissection of molecular mechanisms involved in memory formation, in general, and the special function of nitric oxide synthase in particular.

Serotonin Induces Temporally and Mechanistically Distinct Phases of Persistent PKA Activity in Aplysia Sensory Neurons

The cAMP signaling cascade has been implicated in several stages of memory formation. We have examined activation of this cascade by serotonin (5-HT) in the sensory neurons of Aplysia. We find that different patterns of 5-HT exposure induce three distinct modes of PKA activation. First, a single 5 min pulse induces transient (5 min) PKA activation that requires neither transcription nor translation. Second, 4-5 pulses induce intermediate-term persistent activation (3 hr duration) that requires translation but not transcription. Third, 5 pulses of 5-HT, as well as continuous (90 min) exposure, induce long-term persistent activation 20 hr later, which requires both transcription and translation. Thus, in the sensory neurons, different patterns of 5-HT give rise to three independent phases of PKA activation that differ in their induction requirements, their temporal profiles, and their molecular mechanisms.

Ca2+/Calmodulin-dependent Nitric Oxide Synthase in Apis mellifera and Drosophila melanogaster

NADPH diaphorase (NADPHd) is a marker enzyme for nitric oxide (NO)‐producing cells in vertebrates. This paper investigates the relationship between NADPHd and the NO‐producing enzyme NO synthase (NOS) in neuronal tissue of Apis and Drosophila, two insects used for studying learning. First, the NOS and the NADPHd in both species were characterized biochemically. The fixation‐insensitive NADPHd activity, which accounts for the staining in NADPHd histochemistry, co‐purifies with the insect Ca2+/calmodulin‐dependent NOS. Formation of NO from l‐arginine depends on NADPH, and half‐maximal stimulation is observed with 0.3 μM Ca2+. NOS is competitively inhibited by methyll‐arginine and nitro‐l‐arginine, with Kiof 1.7 and 1.9 μM, respectively. The co‐purification and the competitive inhibition of NOS by the NADPHd substrate, nitro blue tetrazolium (NBT), are proof that in insects the enzyme responsible for fixation‐insensitive NADPHd activity is nitric oxide synthase. Second, the NOS activity was quantified in distinct neuropiles and the NO‐producing neuropiles were visualized using NADPHd histochemistry. In both species the highest NOS activity is found in the chemosensory neuropiles of the antenna1 lobes, intermediate activity in the neuropiles of the central brain and by far the lowest NOS activity in the visual neuropiles. Although in both species the Kenyon cell somata of the mushroom bodies show no detectable staining, the neuropiles of the mushroom bodies of Drosophila and Apis show a distinct staining. The staining pattern of NOS in both species is different to that of all known neurotransmitters.

The Nitric Oxide/cGMP System in the Antennal Lobe of Apis mellifera is Implicated in Integrative Processing of Chemosensory Stimuli

The high concentration and the localization of nitric oxide synthase in the olfactory system of both vertebrates and invertebrates suggest that the diffusible messenger nitric oxide plays a central role in the processing of chemosensory information. This paper describes the nitric oxide releasing system in the antenna and the antennal lobes of Apis mellifera using the NADPH diaphorase technique, and analyses the contribution of the nitric oxide system in the neuronal processing of chemosensory signals using a behavioural assay in vivo. In the antenna the strongest NADPH diaphorase staining is found in non‐neuronal auxiliary and/or epithelial cells, while the sensory cells and the antennal nerve are stained at a low level. At the major site of chemosensory signal integration, the antennal lobes, the highest nitric oxide synthase activity is located in the glomeruli, which are ideally suited to act as diffusion compartments. We demonstrate that inhibition of nitric oxide synthase in the antennal lobes specifically interferes with neuronal processing of repetitive chemosensory stimuli but does not affect the response to single stimuli, and is independent of parameters such as satiation level, stimulus strength, interstimulus interval and duration of sensory stimuli. Since inhibition of the soluble guanylate cyclase, a major target of nitric oxide, also particularly affects the adaptive component, the physiological effects of nitric oxide appear to be mediated by the action of cGMP. These findings suggest that the nitric oxide/cGMP system in the antennal lobes is a component of the molecular machinery involved in adaptive and/or integrative mechanisms during chemosensory information processing in vivo.

Learning at Different Satiation Levels Reveals Parallel Functions for the cAMP–Protein Kinase A Cascade in Formation of Long-Term Memory

Learning and memory formation in intact animals is generally studied under defined parameters, including the control of feeding. We used associative olfactory conditioning of the proboscis extension response in honeybees to address effects of feeding status on processes of learning and memory formation. Comparing groups of animals with different but defined feeding status at the time of conditioning reveals new and characteristic features in memory formation. In animals fed 18 hr earlier, three-trial conditioning induces a stable memory that consists of different phases: a mid-term memory (MTM), translation-dependent early long-term memory (eLTM; 1–2 d), and a transcription-dependent late LTM (lLTM; ≥3 d). Additional feeding of a small amount of sucrose 4 hr before conditioning leads to a loss of all of these memory phases. Interestingly, the basal activity of the cAMP-dependent protein kinase A (PKA), a key player in LTM formation, differs in animals with different satiation levels. Pharmacological rescue of the low basal PKA activity in animals fed 4 hr before conditioning points to a specific function of cAMP–PKA cascade in mediating satiation-dependent memory formation. An increase in PKA activity during conditioning rescues only transcription-dependent lLTM; acquisition, MTM, and eLTM are still impaired. Thus, during conditioning, the cAMP–PKA cascade mediates the induction of the transcription-dependent lLTM, depending on the satiation level. This result provides the first evidence for a central and distinct function of the cAMP–PKA cascade connecting satiation level with learning.

Neuronal cAMP-dependent protein kinase type II is concentrated in mushroom bodies of Drosophila melanogaster and the honeybee Apis mellifera

In both Drosophila melanogaster and the honeybee Apis mellifera, cyclic adenosine monophosphate (cAMP)-dependent processes have been implicated in mechanisms of learning. This study characterizes the type II cAMP-dependent protein kinase (PKAII), the major target of cAMP in adult animals. In both species, PKAII is restricted to neuronal tissue, in which it accounts for more than 90% of total PKA activity. Although the intensity of PKAII immunoreactivity differs between distinct brain regions, labeling is detectable in all neuropiles and most somata. While the visual neuropiles, the antennal lobes, and structures of the central brain exhibit intermediate immunostaining, the mushroom bodies show high labeling and contain a three- to fourfold higher PKA activity compared to other neuropiles. Since the mushroom bodies are central sites of olfactory learning mediated via cAMP-dependent signaling, the modulatory functions of transmitters on PKA activity in Kenyon cells from the honeybee were tested. Agents which elevate cytoplasmic Ca2+ levels have no effects on PKA activity in cultured Kenyon cells. Dopamine, serotonin, and octopamine, however, cause an increase in PKA activity in Kenyon cells. The modulation of PKA activity by octopamine, the putative transmitter of the unconditioned stimulus in associative olfactory learning in the honeybee, together with the findings on the central role of the cAMP cascade in Drosophila mushroom bodies, suggests a major implication of PKAII-mediated phosphorylation in learning and memory in both Drosophila and Apis.

Focal and Temporal Release of Glutamate in the Mushroom Bodies Improves Olfactory Memory in Apis mellifera

In contrast to vertebrates, the role of the neurotransmitter glutamate in learning and memory in insects has hardly been investigated. The reason is that a pharmacological characterization of insect glutamate receptors is still missing; furthermore, it is difficult to locally restrict pharmacological interventions. In this study, we overcome these problems by using locally and temporally defined photo-uncaging of glutamate to study its role in olfactory learning and memory formation in the honeybee, Apis mellifera. Uncaging glutamate in the mushroom bodies immediately after a weak training protocol induced a higher memory rate 2 d after training, mimicking the effect of a strong training protocol. Glutamate release before training does not facilitate memory formation, suggesting that glutamate mediates processes triggered by training and required for memory formation. Uncaging glutamate in the antennal lobes shows no effect on memory formation. These results provide the first direct evidence for a temporally and locally restricted function of glutamate in memory formation in honeybees and insects.

Neuroactive Substances in the Antennal Lobe

Understanding the neural network of a particular brain area like the insect antennal lobe (AL) requires not only in-depth analysis of the anatomical and electrophysiological properties of its constituent neurons, but also information about the nature, distribution, and modes of action of the chemical substances involved in neuronal communication. Immunocytochemistry has become a powerful tool, not only for identifying candidate neurotransmitters in the AL, but also for elucidating their distribution down to the single-cell level, and, in many instances, to the level of intracellular vesicles. These studies have revealed an unforeseen variety and complexity of transmitter-related signalling mechanisms in the AL, the physiological significance of which is just beginning to be explored. Selective staining of neurons through immunocytochemistry has led to the discovery of anatomically and functionally novel types of interneurons in the AL (Chap. 4), and, finally, immunocytochemical markers have proved to be valuable tools for studies of AL development (Chap. 8).

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.