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Associative Learning: Hebbian Flies

2005; Elsevier BV; Volume: 15; Issue: 11 Linguagem: Inglês

10.1016/j.cub.2005.05.028

1879-0445

David L. Glanzman,

Insect Utilization and Effects

Fruit flies can learn to associate an odor with an aversive stimulus, such as a shock. New findings indicate that disrupting the expression of N-methyl-d-aspartate (NMDA) receptors in flies impairs olfactory conditioning. The findings provide support for a critical role for NMDA receptors in associative learning. Fruit flies can learn to associate an odor with an aversive stimulus, such as a shock. New findings indicate that disrupting the expression of N-methyl-d-aspartate (NMDA) receptors in flies impairs olfactory conditioning. The findings provide support for a critical role for NMDA receptors in associative learning. Invertebrates, like the late comedian Rodney Dangerfield, don't get no respect, at least when it comes to their capacity for learning. This point was brought home to me one evening several years ago at a Japanese restaurant in Los Angeles, where I had taken our seminar speaker, Professor X, for dinner. I was describing the latest developments in my laboratory when the professor made a blunt pronouncement: "invertebrates don't learn", he told me. After I had recovered from choking on the piece of abalone sushi in my mouth, I asked X how he could hold such an absurd belief in the face of significant scientific evidence to the contrary. Blandly, X replied that the lives of invertebrates place such simple demands upon them that they do not require the ability to learn to survive. Although few neuroscientists who work on learning in vertebrates would publicly admit to a prejudice as extreme as that of Professor X, all too many seem to treat studies of invertebrate learning with benign neglect, if not mild contempt. To those of us who work on learning in invertebrate systems this situation is frustrating; higher invertebrates exhibit sophisticated learning abilities that, in some instances, represent true cognition [1Zhang S. Bock F. Si A. Tautz J. Srinivasan M.V. Visual working memory in decision making by honey bees.Proc. Natl. Acad. Sci. USA. 2005; 102: 5250-5255Crossref PubMed Scopus (96) Google Scholar]. But, surely, we have ourselves partly to blame. For over two decades, neurobiologists of invertebrate learning and memory have overemphasized the importance of simple presynaptic, or nonsynaptic, learning mechanisms [2Alkon D.L. Persistent calcium-mediated changes of identified membrane currents as a cause of associative learning.in: Alkon D.L. Farley J. Primary neural substrates of learning and behavioral change. (New York: Cambridge University), 1984: 291-323Google Scholar, 3Kandel E.R. The molecular biology of memory storage: a dialogue between genes and synapses.Science. 2001; 294: 1030-1038Crossref PubMed Scopus (2547) Google Scholar]. It is therefore not surprising that students of vertebrate learning might question the relevance of invertebrate studies for their own systems. As they well realize, the cell biology of vertebrate learning is far from simple; furthermore, it appears to comprise elegant mechanisms of associative plasticity, such as N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) [4Morris R.G. Moser E.I. Riedel G. Martin S.J. Sandin J. Day M. O'Carroll C. Elements of a neurobiological theory of the hippocampus: the role of activity-dependent synaptic plasticity in memory.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 773-786Crossref PubMed Scopus (382) Google Scholar, 5Tonegawa S. Nakazawa K. Wilson M.A. Genetic neuroscience of mammalian learning and memory.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 787-795Crossref PubMed Scopus (73) Google Scholar] and complex postsynaptic changes, such as modulation of the trafficking of postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors [6Malinow R. AMPA receptor trafficking and long-term potentiation.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 707-714Crossref PubMed Scopus (187) Google Scholar]. Enter the important new study by Xia et al. [7Xia S. Miyashita T. Fu T.-F. Lin W.-Y. Pyzocha L. Lin I.-R. Saitoe M. Tully T. Chiang A.-S. NMDA receptors mediate olfactory learning and memory in Drosophila.Curr. Biol. 2005; 15: 603-615Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar], reported recently in Current Biology. First, some background: fifty-six years ago the Canadian psychologist Donald Hebb [8Hebb D.O. The organization of behavior. (New York: Wiley), 1949Google Scholar] proposed a cellular model for how synapses in the brain might become strengthened during associative learning. His proposal, commonly known as 'Hebb's hypothesis', is familiar to every modern student of memory. Hebb's hypothesis proposes that when the activity of one neuron repeatedly causes, or contributes to, the firing of another neuron, the synapse between the two will become strengthened. The key idea here is the requirement for synchrony of presynaptic and postsynaptic activity. Of equal importance to Hebb's theoretical speculation was the discovery by Bliss and Lømo [9Bliss T.V.P. Lømo T. Long-lasting potentiation of synaptic transmission in the dentate area of the anesthetized rabbit following stimulation of the perforant path.J. Physiol. 1973; 232: 331-356PubMed Scopus (0) Google Scholar] in 1973 of long-term potentiation (LTP), a form of long-term synaptic enhancement. Because LTP was discovered at synapses in the hippocampus, a structure known from human clinical literature to be critical for certain forms of memory, many neuroscientists found attractive the idea that LTP might play a role in learning and memory. Interest in LTP further intensified following the recognition that activation of postsynaptic NMDA receptors was the mechanism of LTP induction at many hippocampal synapses [10Collingridge G.L. Kehl S.J. McLennan H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus.J. Physiol. 1983; 334: 33-46PubMed Google Scholar]. Soon after came the stunning confirmation from electrophysiological experiments that LTP in the hippocampus could be induced in precisely the manner Hebb had envisaged [11Brown T.H. Kairiss E.W. Keenan C.L. Hebbian synapses: biophysical mechanisms and algorithms.Annu. Rev. Neurosci. 1990; 13: 475-511Crossref PubMed Scopus (344) Google Scholar]. Almost immediately, the race was on to prove that NMDA receptor-dependent LTP mediated learning. A variety of experimental techniques have been used to link LTP with memory [4Morris R.G. Moser E.I. Riedel G. Martin S.J. Sandin J. Day M. O'Carroll C. Elements of a neurobiological theory of the hippocampus: the role of activity-dependent synaptic plasticity in memory.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 773-786Crossref PubMed Scopus (382) Google Scholar]; to many, however, the most persuasive evidence that LTP is crucial to memory has come from studies of knockout mice missing the gene for the NR1 subunit of the NMDA receptor [5Tonegawa S. Nakazawa K. Wilson M.A. Genetic neuroscience of mammalian learning and memory.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 787-795Crossref PubMed Scopus (73) Google Scholar]. These mutant mice lack NMDA receptor-dependent LTP in their hippocampi, and exhibit deficient spatial learning. But skeptics have found the NR1 knockout mice data unconvincing. Because the NR1 protein is missing throughout development, the brains of these mice may develop abnormally. Although the gross neuroanatomy of the NR1 knockouts appears normal, it is difficult to prove beyond doubt that the neural circuits that mediate spatial learning — which are poorly understood — are functionally intact in the mutants. Also, the learning task used in the NR1 knockout mouse studies — place navigation in the Morris water maze [4Morris R.G. Moser E.I. Riedel G. Martin S.J. Sandin J. Day M. O'Carroll C. Elements of a neurobiological theory of the hippocampus: the role of activity-dependent synaptic plasticity in memory.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 773-786Crossref PubMed Scopus (382) Google Scholar] —is complex, and the precise role of LTP in this task is unclear [12Saucier D. Cain D.P. Spatial learning without NMDA receptor-dependent long-term potentiation.Nature. 1995; 378: 186-189Crossref PubMed Scopus (279) Google Scholar]. Recently, an inducible genetic technique has been used to eliminate NR1 from the forebrain of adult mice [13Cui Z. Wang H. Tan Y. Zaia K.A. Zhang S. Tsien J.Z. Inducible and reversible NR1 knockout reveals crucial role of the NMDA receptor in preserving remote memories in the brain.Neuron. 2004; 41: 781-793Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar]. (This inducible genetic lesion requires 5 days.) After induction of the knockout the mice are impaired in contextual Pavlovian fear conditioning, a hippocampal-dependent form of learning. Nonetheless, doubts persist concerning the data from transgenic mouse studies of memory. In their study, Xia et al. [7Xia S. Miyashita T. Fu T.-F. Lin W.-Y. Pyzocha L. Lin I.-R. Saitoe M. Tully T. Chiang A.-S. NMDA receptors mediate olfactory learning and memory in Drosophila.Curr. Biol. 2005; 15: 603-615Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar] asked whether NMDA receptors participate in a form of Pavlovian conditioning in Drosophila [14Dubnau J. Tully T. Gene discovery in Drosophila: new insights for learning and memory.Annu. Rev. Neurosci. 1998; 21: 407-444Crossref PubMed Scopus (293) Google Scholar]. As it happens, NMDA receptors are not unique to vertebrates: molecular and/or pharmacological evidence exists for NMDA receptors in several invertebrate phyla, including mollusks [15Dale N. Kandel E.R. L-glutamate may be the fast excitatory transmitter of Aplysia sensory neurons.Proc. Natl. Acad. Sci. USA. 1993; 90: 7163-7167Crossref PubMed Scopus (138) Google Scholar], arthropods [16Ultsch A. Schuster C.M. Laube B. Betz H. Schmitt B. Glutamate receptors of Drosophila melanogaster: primary structure of a putative NMDA receptor protein expressed in the head of the adult fly.FEBS Lett. 1993; 324: 171-177Abstract Full Text PDF PubMed Scopus (90) Google Scholar], annelids [17Burrell B.D. Sahley C.L. Multiple forms of long-term potentiation and long-term depression converge on a single interneuron in the leech CNS.J. Neurosci. 2004; 24: 4011-4019Crossref PubMed Scopus (34) Google Scholar] and nematodes [18Brockie P.J. Mellem J.E. Hills T. Madsen D.M. Maricq A.V. The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion.Neuron. 2001; 31: 617-630Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar]. Xia et al. cloned two Drosophila NMDA receptor genes, dNR1 [16Ultsch A. Schuster C.M. Laube B. Betz H. Schmitt B. Glutamate receptors of Drosophila melanogaster: primary structure of a putative NMDA receptor protein expressed in the head of the adult fly.FEBS Lett. 1993; 324: 171-177Abstract Full Text PDF PubMed Scopus (90) Google Scholar] and dNR2, the protein products of which show significant amino acid sequence similarity to vertebrate NR1 and NR2 subunits, and both of which are expressed in neurons in the fly's brain. Xia et al. [7Xia S. Miyashita T. Fu T.-F. Lin W.-Y. Pyzocha L. Lin I.-R. Saitoe M. Tully T. Chiang A.-S. NMDA receptors mediate olfactory learning and memory in Drosophila.Curr. Biol. 2005; 15: 603-615Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar] next produced mutants with disruptions of dNR1, and tested the effect of the mutations on the flies' ability to associate an odor with an electrical shock (Figure 1A ). The mutant flies exhibited reduced learning, as indicated by their lack of avoidance of the odor that had been previously paired with shock. This result parallels the earlier studies using NR1 knockout mice [5Tonegawa S. Nakazawa K. Wilson M.A. Genetic neuroscience of mammalian learning and memory.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 787-795Crossref PubMed Scopus (73) Google Scholar]. They then used a clever technique to induce rapid, conditional disruptions of NMDA receptor function. Mutant flies were generated with a transposable P element carrying an enhancer and basal promoter (EP) inserted downstream of, and in the opposite orientation to, the transcription start site of dNR1. The activity of the EP element could be regulated because it contained an enhancer sequence responsive to the yeast transcription factor GAL4. These flies were crossed with transgenic flies containing a GAL4 gene under the control of a heat-shock promoter. Offspring heterozygous for the EP element insertion and the GAL4-heat shock system — EP/+, hs-GAL4/+ flies — were raised to adulthood at their normal temperature (18°), and then warmed to 30° for several hours. Heat shock induced an antisense transcript of dNR1 in these transgenic flies, resulting in a significant reduction in dNR1 protein in the fly brains when measured 15 hours after heat shock. The acute disruption of NMDA receptors produced deficient Pavlovian learning, compared to both wild-type and transgenic flies not subjected to heat shock. Furthermore, learning was normal in heat-shocked flies without the transgene. Impressively, when the EP/+, hs-GAL4/+ flies were trained 36 hours following heat shock, their learning was normal, presumably because the dNR1 protein had returned to pre-heat-shock levels. In further experiments, Xia et al. [7Xia S. Miyashita T. Fu T.-F. Lin W.-Y. Pyzocha L. Lin I.-R. Saitoe M. Tully T. Chiang A.-S. NMDA receptors mediate olfactory learning and memory in Drosophila.Curr. Biol. 2005; 15: 603-615Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar] found that long-term memory (LTM) — memory lasting at least a day, which can be induced in normal flies by training with multiple trials spaced in time of odor plus shock — is impaired in the transgenic flies when they are trained starting 15 hours after heat shock. Previous results have implicated the cyclic (c)AMP response element binding protein (CREB) in LTM in flies [14Dubnau J. Tully T. Gene discovery in Drosophila: new insights for learning and memory.Annu. Rev. Neurosci. 1998; 21: 407-444Crossref PubMed Scopus (293) Google Scholar]; thus, spaced training may cause NMDA receptor-dependent activation of CREB in the Drosophila brain. The study by Xia et al. [7Xia S. Miyashita T. Fu T.-F. Lin W.-Y. Pyzocha L. Lin I.-R. Saitoe M. Tully T. Chiang A.-S. NMDA receptors mediate olfactory learning and memory in Drosophila.Curr. Biol. 2005; 15: 603-615Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar] is a tour de force. Their results eliminate developmental abnormalities as an explanation for the results of their transgenic fly experiments, and provide convincing evidence that NMDA receptors are critical for both learning and LTM. But a great deal of work remains to be done. First, the critical Hebbian synapses for olfactory conditioning have not been identified in the fly's brain [19Yu D. Ponomarev A. Davis R.L. Altered representation of the spatial code for odors after olfactory classical conditioning; memory trace formation by synaptic recruitment.Neuron. 2004; 42: 437-449Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar] (Figure 1B). Interestingly, Xia et al. [7Xia S. Miyashita T. Fu T.-F. Lin W.-Y. Pyzocha L. Lin I.-R. Saitoe M. Tully T. Chiang A.-S. NMDA receptors mediate olfactory learning and memory in Drosophila.Curr. Biol. 2005; 15: 603-615Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar] report that their NMDA receptor antibodies did not preferentially label neurons in the mushroom bodies, bilateral structures in the insect brain known to be essential for olfactory conditioning [14Dubnau J. Tully T. Gene discovery in Drosophila: new insights for learning and memory.Annu. Rev. Neurosci. 1998; 21: 407-444Crossref PubMed Scopus (293) Google Scholar]; however, several interneurons that project to the mushroom bodies showed strong labeling. Second, extensive work with mutant flies has indicated that the cAMP signaling pathway is necessary for conditioning, and activation of this pathway is believed to occur via release of monoamines [14Dubnau J. Tully T. Gene discovery in Drosophila: new insights for learning and memory.Annu. Rev. Neurosci. 1998; 21: 407-444Crossref PubMed Scopus (293) Google Scholar]. Important outstanding issues, therefore, are the respective roles of NMDA receptor-dependent and cAMP-dependent pathways in Drosophila learning and memory, and the possible interaction of these pathways during conditioning. In summary, the findings of Xia et al. provide strong support for the idea that invertebrates learn (pace Professor X) using NMDA receptors [20Roberts A.C. Glanzman D.L. Learning in Aplysia: looking at synaptic plasticity from both sides.Trends Neurosci. 2003; 26: 662-670Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar]. This unexpected insight suggests the exciting prospect of a unified cellular model for associative learning, one that holds for both vertebrates and invertebrates. But it won't be simple.