A distinct set of Drosophila brain neurons required for neurofibromatosis type 1-dependent learning and memory

Abstract

Nonspecific cognitive impairments are one of the many manifestations of neurofibromatosis type 1 (NF1). A learning phenotype is also present in Drosophila melanogaster that lack a functional neurofibromin gene (nf1). Multiple studies have indicated that Nf1-dependent learning in Drosophila involves the cAMP pathway, including the demonstration of a genetic interaction between Nf1 and the rutabaga-encoded adenylyl cyclase (Rut-AC). Olfactory classical conditioning experiments have previously demonstrated a requirement for Rut-AC activity and downstream cAMP pathway signaling in neurons of the mushroom bodies. However, Nf1 expression in adult mushroom body neurons has not been observed. Here, we address this discrepancy by demonstrating (1) that Rut-AC is required for the acquisition and stability of olfactory memories, whereas Nf1 is only required for acquisition, (2) that expression of nf1 RNA can be detected in the cell bodies of mushroom body neurons, and (3) that expression of an nf1 transgene only in the alpha/beta subset of mushroom body neurons is sufficient to restore both protein synthesis-independent and protein synthesis-dependent memory. Our observations indicate that memory-related functions of Rut-AC are both Nf1-dependent and -independent, that Nf1 mediates the formation of two distinct memory components within a single neuron population, and that our understanding of Nf1 function in memory processes may be dissected from its role in other brain functions by specifically studying the alpha/beta mushroom body neurons.

Figure 1.

nf1 mutants exhibit size and memory phenotypes. A, Female and male adults were measured from the anterior tip of the antennae to the posterior tip of the abdomen. There was no significant difference between nf1^c00617 and control females, but nf1^c00617 males were significantly smaller than control males (*p < 0.005). Both genders of nf1^P2 and nf1^P1 were significantly smaller than both controls and nf1^c00617, and nf1^P2 flies were also significantly smaller than nf1^P1. For control, w(CS10) was used, n = 100, and for all nf1 mutants, n = 50. Means ± SEM are shown. *p < 0.005, **p < 0.0001. B, Learning and memory phenotype of nf1^c00617 homozygous mutants compared with other nf1 alleles. Performance was assayed 3 min, 3 h, and 24 h after training. All mutant groups showed impaired performance relative to controls. nf1^c00617 mutants performed significantly better than nf1^P1, nf1^P2, and rut²⁰⁸⁰ mutants when tested at 3 min after training but not when tested 3 or 24 h after training. A 5×-spaced protocol was used to elicit 24 h memory. For 3 min and 3 h memory, w(CS10) was used as a control. K33 was used as a control for 24 h memory. C, Expression of nf1 in all neurons rescues the 3 h memory phenotype of nf1^c00617 mutants. Both control and elav/uas–dnf1;nf1^c00617 flies performed significantly better than both elav/+;nf1^c00617 and nf1^c00617 flies, demonstrating a complete rescue of the nf1^c00617 memory phenotype. There was no significant difference between the performance of control and elav/uas–dnf1;nf1^c00617 flies or between elav/+;nf1^c00617 and nf1^c00617 flies. There was also no significant difference between the performance of nf1^c00617 and uas–dnf1/+;nf1^c00617 flies, as shown in Figures 5–7. Control flies were w(CS10). For 24 h memory, n = 14. For all other data, n = 6. Means ± SEM are shown. *p < 0.05, **p < 0.001 relative to controls.

Figure 2.

Neurofibromin is required for memory acquisition, not memory stability. A, nf1 mutants are defective in the acquisition of memories. All groups were trained with 1–15 trials (T), and performance was assayed immediately after conditioning. With one training trial exposure, all mutants performed significantly poorer than controls. When exposed to 15 training trials, all mutants performed at the same level as controls. For all other experiments, both nf1^P2 and rut²⁰⁸⁰ mutants performed significantly poorer than controls, whereas nf1^c00617 mutants did not. Across experiments, the performance of nf1^P2 and rut²⁰⁸⁰ mutants paralleled that of controls but was delayed. Control flies were w(CS10) for all experiments. For the five trial experiment, n = 9 for each group. For all other experiments, n = 6 for each group. Means ± SEM are shown. *p < 0.05. B, Memory stability is normal in nf1 mutants but defective in rut²⁰⁸⁰ mutants. Mutant performance at 3 min after training was normalized to control performance by varying the number of training trials, as indicated. Performance of nf1 homozygous mutants was not significantly different from control performance at the time points tested. Whereas rut²⁰⁸⁰ and control performances were indistinguishable 3 min after training, they were significantly different at both 30 and 60 min after training. Control flies were w(CS10) for all experiments. For 30 min decay of nf1^P2 performance, n = 11. For all other experiments, n = 6 for each group. Means ± SEM are shown. *p < 0.005.

Figure 3.

nf1 is endogenously expressed in mushroom body neurons. A–D, Series of frontal sections of w(CS10) adult fly heads arranged from anterior to posterior. A, Staining of an anterior section, at the level of the superior medial protocerebrum and antennal lobes. Intense staining was present in cell body regions, with no staining of the antennal nerve or neuropil. Scale bar, 100 μm. B, A more posterior section showing continued staining of cell body regions of the adult brain, with no staining of neuropil areas. C, Section is at the level of the central complex, showing staining of cell body regions, which surround the internal neuropil. D, Posterior section at the level of the calyx shows staining in cell body regions, including the cell body region of mushroom body neurons. E, Higher magnification of the region within the white box of D. Staining was visible in cells dorsal and lateral to the calyx neuropil, in the region known to contain cell bodies of the mushroom body neurons. Scale bar, 10 μm. F, In situ hybridization to nf1^P1 homozygous mutant shows no staining in cell body regions. Representative image was taken at the same magnification and at approximately the same level as B.

Figure 4.

nf1 expression in mushroom body neurons, only during adulthood, rescues nf1^c00617 memory. nf1 expression was restored in mushroom body neurons of homozygous nf1^c00617 mutants by expressing uas–dnf1 under the control of the Gene-Switch system. A, All groups were fed RU486 during development and adulthood, except as indicated, and performance was assayed 3 h after training. Expression of nf1 during development and adulthood resulted in complete rescue of the 3 h memory phenotype. For all groups, n = 6. Means ± SEM are shown. *p < 0.005. B, All groups were fed standard food during development and were transferred to food containing RU486 for 5 d after eclosion, except as indicated. Performance was assayed 3 h after training. Expression of nf1, only during adulthood, completely rescued the 3 h memory phenotype. For all groups, n = 6. Means ± SEM are shown. *p < 0.05.

Figure 5.

nf1 expression in α′/β′ or γ neurons does not restore 3 h memory. A, Expression of nf1 was restored in α′/β′ neurons of homozygous nf1^c00617 mutants by expressing a uas–dnf1 transgene under control of the c305a–gal4 driver. Performance was assayed 3 h after training. K33 was used as the control. All genotypes showed significant memory impairment relative to control flies. For all groups, n = 6. Means ± SEM are shown. *p < 0.0001. B, Expression of nf1 was restored in γ neurons of homozygous nf1^c00617 or nf1^P1 mutants by expressing uas–dnf1 under the control of the NP1131–gal4 driver. Performance was assayed 3 h after training. Control flies were w(CS10). All genotypes show significant memory impairment relative to control flies. For all groups, n = 6. Means ± SEM are shown. *p < 0.05.

Figure 6.

nf1 expression in α/β neurons rescues 3 h memory. Expression of nf1 was restored in α/β neurons of homozygous nf1^c00617 and nf1^P1 mutants by expressing a uas–dnf1 transgene under the control of either Mz1081–gal4 (A), NP9–gal4 (B), c739–gal4 (C), or 17d–gal4 (D). Performance was assayed 3 h after training. A–C, Expression of nf1 in α/β neurons resulted in complete 3 h memory rescue of both alleles. K33 was used as the control. All negative control groups showed significant memory impairment relative to control flies. D, Expression of nf1 under control of 17d–gal4 failed to rescue 3 h memory. Control flies were w(CS10). All genotypes showed significant memory impairment relative to control flies. For all experiments, n = 5–9. Means ± SEM are shown. *p < 0.05.

Figure 7.

nf1 expression in α/β neurons also rescues 3 min and 24 h memory but does not enhance control performance. A, Three minute memory was rescued by nf1 expression in α/β neurons. Expression of nf1 was restored in α/β neurons of homozygous nf1^P1 mutants by expressing a uas–dnf1 transgene under control of the c739–gal4 driver, and performance was assayed 3 min after training. Complete rescue of the phenotype was observed, consistent with experiments showing rescue of 3 h memory. Control flies were w(CS10). For all groups, n = 6. Means ± SEM are shown. *p < 0.005. B, nf1 expression in α/β neurons rescued 24 h memory. Expression of nf1 was restored in α/β neurons of homozygous nf1^P1 mutants by expressing a uas–dnf1 transgene under control of the c739–gal4 driver. Performance was assayed 24 h after 5×-spaced training, which produced long-lasting memory that was significantly different from that elicited by 5×-massed training. K33 was used as the control. For spaced versus massed, n = 12. For rescue, n = 15. Means ± SEM are shown. *p < 0.05. C, Control performance is not affected by neurofibromin overexpression in α/β neurons. Overexpression of uas–dnf1 in α/β neurons of heterozygous nf1^P1 flies was driven by c739–gal4, and performance was assayed 3 h after training. For all groups, n = 5. Means ± SEM are shown. *p < 0.005.

Figure 8.

Inhibiting nf1 expression in α/β neurons prevents memory rescue. A, Expression of nf1 was restored in α/β neurons of homozygous nf1^c00617 mutants by expressing a uas–dnf1 transgene under control of the c772–gal4 driver. No significant difference was observed between controls and c772/uas–dnf1;nf1^c00617 flies, but all other genotypes showed a significant memory impairment relative to controls. Control flies were w(CS10). Performance was assayed 3 h after training. For all groups, n = 15. Means ± SEM are shown. *p < 0.005. B, When MB{Gal80} was introduced to inhibit Gal4 activity in mushroom body neurons, expression of nf1 by c772–gal4 did not rescue the 3 h memory phenotype of nf1^c00617. Similar expression of uas–dnf1 in a heterozygous nf1^c00617 genetic background did not alter performance relative to control, but all other genotypes showed significant memory impairment by comparison. Performance was assayed 3 h after training. Control flies were w(CS10). For all groups, n = 8. Means ± SEM are shown. *p < 0.05.