Vertebrate-like effects of drug treatment in honeybees

7.1.1 Glutamate in the honeybee CNS and memory

The use of targeted drugs such as memantine, L-rrajis-2,4-PDC and MK-

801 in Chapter 2 revealed that a disruption of the glutamergic signalling pathways

in the honeybee CNS could lead to a reduction in the recall of long-term olfactory

associative memory. This result lends further support to the idea that L-glutamate

might play as important a role in the insect CNS, as it has long been known to in

vertebrates. The importance of glutamate in the vertebrate CNS cannot be

understated - it is the major excitatory neurotransmitter in the CNS, and is vital to

the normal development of the CNS, being involved in processes such as synaptic

plasticity and long-term potentiation (Danbolt, 2001). In addition, the NMDA

receptor is considered to be a ‘classic’ learning and memory receptor, due to its

requiring a simultaneous depolarisation as well as a ligand (e.g. L-glutamate).

Until very recently, the status and role of L-glutamate in the insect CNS was

practically unknown. The use of the glutamate transporter blocker L-trans-2,4-

PDC gave one of the first clues to the function of this neurotransmitter in the

learning and memory pathways of the honeybee brain (Maleszka et al., 2000).

Two metabotropic glutamate receptor subtypes were also recently cloned from the

brain of the honeybee, and were found to be expressed, like the glutamate

transporter AmEAAT (Kucharski et al., 2000), in the Kenyon cells of the

mushroom bodies (Funada et al., 2004). This further suggests a role for glutamate

in higher cognitive functions like learning and memory. The results described in

Chapter 2 support the conclusions drawn by Maleszka et al (2000), and show that

other drugs, which specifically target the NMDA receptor in vertebrates, have

behavioural effects in honeybees similar to those observed in rats, mice and even

humans.

The choice of glutamergic drugs used in this study is significant. MK-801,

the high-affinity NMDA antagonist, was first developed as an anti-Alzheimer

drug. In spite of promising results in vitro, however, the drug yielded poor clinical

results due to the blocking of normal physiological activity, and the resulting

severe side effects, such as hallucinations, ataxia and memory loss (Farlow, 2004). Consistent with this, the loss of long-term olfactory memory was certainly

observed in the honeybees treated with this drug prior to training and testing.

Memantine, on the other hand, is a promising new anti-Alzheimer drug, which, in

recent clinical trials, has been shown to be useful in treating patients with

moderate to severe AD, while increasing their autonomy and being clinically well

tolerated (Parsons et al., 1999; Palmer and Widzowski, 2000; Rive et al., 2004).

At the receptor level, memantine is a medium-affinity antagonist of the NMDAR.

By preferentially blocking open channels (Parsons et al., 1999), memantine plays

memantine, the restoration of the recall of long-term memory in bees treated with

L-/rrms-2,4-PDC is strong evidence in favour of the involvement of glutamate in

learning and memory in this insect. Based on these findings, a likely scenario

would be an abnormal elevation in L-glutamate in the synaptic cleft, brought

about by the blocking of glutamate transporter proteins by L-rra«s-2,4-PDC. The

resulting overstimulation of NMDAR on the postsynaptic neuron would induce

excitotoxicity, and impair the recall of long-term memory. The administration of

memantine under such conditions would have the effect of temporarily blocking

the open NMDA channels, thereby reversing the effect of L-mms-2,4-PDC, and

facilitating recall.

Three different glutamergic drugs (two of which specifically target

NMDAR) with three different modes of action were used in this study, and were

found to produce behavioural changes in the honeybee similar to those observed in vertebrates. These results speak strongly in favour of a learning and memory-

related glutamergic pathway in the honeybee brain, and perhaps in the CNS of other insect species as well.

7.1.2 Caffeine and arousal

Strikingly human-like motivation-enhancing effects were observed in

caffeine-treated bees used in the experiments reported in Chapter 5. The range of

behavioural and cognitive changes brought about by the drug was as large and

diverse as that reported from studies on humans - nevertheless, there was a clear

enhancement of the arousal state of treated bees, as evidenced by the increased

frequency of visits to the feeder, independent of the experimental paradigm.

Caffeine improved performance in a DMTS task, and has been shown in a

previous experiment to dramatically accelerate the development of young worker

bees, allowing them to learn an olfactory associative task as early as 3 days post­

emergence (control bees could only learn the task at 6 days; Maleszka et al.,

unpublished data). In contrast, the results of the dance experiment showed that

caffeine also suppresses the probability of foraging and dancing. This is probably

a result of the high concentration of drug administered to the experimental bees,

and represents a toxic effect that inhibits various behaviours. While the dance

experiment needs to be repeated with a lower concentration of caffeine, it is

noteworthy that similar negative effects have also been reported from studies on

vertebrates (Lorist and Tops, 2003; Howell and Landrum, 1994).

Although the drugs administered in this thesis were developed specifically

for vertebrate nervous systems, they have been shown to have largely the same effects in honeybees as in vertebrates. While there are clearly differences in the

gross anatomical organisation of the insect and vertebrate nervous systems

(Menzel and Giurfa, 2001), it is really the frequent similarities in the biochemistry

and cognitive abilities of the two groups that are most striking (Kandel and Abel,

1995; Bicker, 1999; Müller, 2000; Giurfa, 2003). Indeed, it has been shown that

the developmental genetic mechanisms in vertebrate and invertebrate brains are

highly conserved, with homeotic {Hox) genes being expressed in a virtually

col inear anteroposterior pattern in the developing posterior brain of insects and

recent surge of information on the (vertebrate-like) cognitive abilities of the

honeybee, as well as the pharmacological and behavioural data presented in this

thesis, strongly suggest the existence of common learning and memory-related

signalling pathways between the two lineages.