A molecular approach to describe the role of glutamate NMDA receptor in memory formation and behaviour of an insect model: the honeybee Apis mellifera
Final Report Abstract
The main goal was to better understand the glutamatergic neurotransmission of the honeybee by focusing on the NR1 subunit of NMDA receptors. We identified the NR1 subunit and we localized its expression sites in the brain. It is widespread in all neuropiles except in the mushroom body. RNAi was induced against the NR1 subunit. The RNAi response was characterized by an inhibition restricted to the MB region that persists up to 2 hours after conditioning. The inhibition of the NR1 subunit selectively affects specific memory phases. Mid-term and early long-term memory tests were affected whereas late long-term memory was not. The fact that late long-term memory was intact suggests that the acquisition of the association was unaffected. Thus the impairment of memory performances during the acquisition suggests that a short-term memory phase was affected. These results are surprising because it is believed that the receptor is required during the acquisition phase for the formation of all memory phases. The limitation of the RNAi effect in time and space might explain the selective effect on memory. Alternatively, late long-term memory might be independent of the NMDA receptor. In addition, the effect on memory is seasonal. It was observed at the beginning (April) and at the end (September, October) of the season. In the meantime (from May to August), only a small reduction of memory performances was observed during the acquisition phase. At the beginning and the end of the season, colonies are composed of older individuals and the resources in nutrients are scarce. We showed that NR1 subunit levels are elevated in young foragers and low in older ones. We also analyzed the effect of the RNAi treatment on in-hive behaviour. We showed that foragers treated with dsRNA against the NR1 subunit disappeared more from the colony than negative controls. This effect was more pronounced in older individuals. Thus, the age of the animal seems to be the principal factor influencing the behavioural effect induced by RNAi against the NR1 subunit. We extended the analysis of the glutamatergic neurotransmission by focusing on glutamate chloride channel subunit α (GluClα). The receptor is localised in most brain regions, in the antennae and in muscles. We applied our RNAi strategy to evaluate the role of the receptor in olfactory memory. The RNAi effect induced a reduction of the GluClα that was limited to the day following the injection. The inhibition of the receptor was associated with an impairment of memory retrieval. Memory performances were not affected when the expression of the subunit returned to normal levels. These results show that different components of the glutamatergic neurotransmission are playing a distinct role in memory processes. In a study on the visual system, we reported a method that allows bees to associate a colour with a reward just after a few training trials. By using this paradigm, with showed that that faster colour discrimination learning was correlated with reduced colour similarity between stimuli. In another study, we applied the RNAi technique to the long-wave length (LWL) opsin. The RNAi effect was demonstrated at the mRNA and the protein levels. We confirmed that high concentrations of dsRNA are required and that the effect is limited in space and time. In addition, the RNAi effect could be induced only at a certain time of the day. Unfortunately, this manipulation was not associated with a behavioural effect. We also worked on the development of in vivo electroporation. We were able to induce recombinant protein expression lasting several days. Only sparse clusters of cells were electroporated. Glia cells but also neurons expressed the recombinant protein. This limitation of the technique is incompatible with functional analyses but is probably of interest for neuroanatomical and optophysiological studies. We also tested the efficiency of lentiviruses to induced recombinant protein expression. The first experiments showed that cells were efficiently infected, probably glial cells and not neurons. Finally, I participated to the annotation of the honeybee genome which is an important source of information for molecular studies. We focused our attention on the PKA family and on the CREB/CREM family.
Publications
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“Insights into social insects from the genome of the honeybee Apis mellifera”. (2006) – Nature – 443: 931-949
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“The PKA-CREB system encoded by the honeybee genome”. (2006) – Insect Mol. Biol. – 15(5): 551-561
Eisenhardt D., Kühn C. and Leboulle G.
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“Fast learning but coarse discrimination of colours in restrained honeybees”. (2009) – J. Exp. Biol. – 212(9): 1344-1350
Niggebrügge, C., Leboulle, G., Menzel, R., Komischke B. and de Ibarra NH.
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“Acute Disruption of the NMDA Receptor Subunit NR1 in the Honeybee Brain Selectively Impairs Memory Formation”. (2010) – J. Neuroscience – 30(23): 7817-7825
Müßig L., Richlitzki A., Rößler R., Eisenhardt D., Menzel R., Leboulle G.
