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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2917756
NIHMSID: NIHMS224971

A DISTINCT SET OF DROSOPHILA BRAIN NEURONS REQUIRED FOR NF1-DEPENDENT LEARNING AND MEMORY

Abstract

Non-specific 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 α/β 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 α/β mushroom body neurons.

Keywords: olfactory learning, Drosophila, Neurofibromatosis Type 1, mushroom bodies, rutabaga, adenylyl cyclase, neurofibromin

INTRODUCTION

Neurofibromatosis Type 1 (NF1) is an autosomal, dominant genetic disorder that afflicts approximately one in every 3500 individuals. Like other clinical manifestations of NF1, expression and penetrance of cognitive phenotypes varies and may include deficiencies of visual-spatial processing, executive function, and attention (for review, Acosta et al. 2006; North 2000; Ozonoff 1999). Homologs of human Nf1 in mouse and Drosophila melanogaster share significant identity at the protein level, and animal models in both species were developed shortly after the human Nf1 gene was cloned (The et al., 1997; Jacks et al., 1994; Bernards et al., 1993). Both models demonstrate cognitive phenotypes, and insights gained through animal studies have shed light on the genetic and biochemical basis of these defects.

Drosophila has been utilized extensively for expanding our basic understanding of memory, making it ideal for investigating NF1 cognitive deficits. After olfactory classical conditioning, Drosophila form protein synthesis-independent early memories (PSI-EM), comprised of short-term memory (STM) tested at 3 minutes after training, middle-term memory (MTM) often tested at 3 hours after training, and protein synthesis-dependent long-term memory (PSD-LTM), tested at 24 hours after conditioning. The nf1 mutant flies demonstrate deficiencies in PSI-EM and PSD-LTM (Ho et al., 2007; Guo et al., 2000). A current model postulates that Nf1 contributes to PSI-EM through stimulation of the rutabaga-encoded adenylyl cyclase (Rut-AC, Guo et al., 2000). Stimulation of Gαs-dependent AC activity requires only the Nf1 C-terminal domain (Hannan et al., 2006). The PSI-EM phenotypes of nf1, rut-AC, and nf1/rut-AC mutants are similar (Guo et al., 2000), both genes are required at the time of learning (Mao et al., 2004; McGuire et al., 2003; Guo et al., 2000), and either ubiquitous expression of a constitutively active protein kinase A (hsPKA*) transgene (Guo et al., 2000) or neuronal expression of a Nf1 C-terminal domain transgene (Ho et al., 2007) rescues the nf1 phenotype. Furthermore, the current model also postulates that Nf1 contributes to PSD-LTM through regulation of Ras via its GAP-related domain (GRD; Ho et al., 2007; Hannan et al., 2006). Stimulation of Ras-dependent AC activity is absent in nf1 mutants, but transgenic expression of the Nf1-GRD restores this activity (Hannan et al., 2006) and improves the PSD-LTM phenotype of nf1 mutants (Ho et al., 2007).

It is surprising that endogenous Nf1 expression has not been observed in adult mushroom body (MB) neurons (Walker et al., 2006). MB neurons are essential for olfactory memory formation (Davis, 2005; McGuire et al., 2001), and Rut-AC is preferentially expressed in these neurons (Han et al., 1992). Rescue experiments demonstrated that transgenic expression of rut-AC in α/β and γ MB neurons restores normal memory in homozygous mutants (Blum et al., 2009; Akalal et al., 2006; Mao et al., 2004; McGuire et al., 2003; Zars et al., 2000). If Nf1 indeed stimulates Rut-AC activity during learning, it is probably expressed, and required, in MB neurons.

Here, we explore whether Nf1 and Rut-AC are involved in the same operational phase of learning, whether they are expressed in the same neurons, whether both are required in the same neurons for rescue of PSI-EM, and whether the Ras-mediated function of Nf1 is required in overlapping neurons. We report a role for Rut-AC in memory stability that is Nf1-independent, observe nf1 expression in MB neurons, and demonstrate a requirement for nf1 expression in α/β MB neurons for both PSI-EM and PSD-LTM.

METHODS

Fly culture and genetics

Flies were reared on standard cornmeal medium, at 25°C, 60% relative humidity, and a 12-hour light/dark cycle. For Gene-Switch experiments, an appropriate volume of RU486 stock solution was mixed into molten standard medium at 65°C, along with food coloring, to a final concentration of 200 µM. Flies were reared and maintained on this altered food source throughout development and adulthood or were transferred from standard food to food containing RU486 at eclosion, and were trained at 5 days post-eclosion. A. Bernards (Massachusetts General Hospital, Boston, MA, USA), A. Sehgal (University of Pennsylvania, PA, USA), and M. Stern (Rice University, TX, USA) provided fly stocks. All stocks used in this study were outcrossed to our w(CS10) stock (w1118 flies outcrossed to Canton-S for ten generations) for six generations, except for the rut2080 stock, which contains a P-element insertion bearing the rosy gene. As a result, rut2080 is maintained in a ry background. K33 flies contain a P-element in the E(spl) complex, which was mobilized to produce the nf1P2 allele. Subsequently, an imprecise excision of the nf1P2 P-element produced the nf1P1 allele (The et al., 1997). K33 flies do not show a behavioral phenotype relative to w(CS10) flies, so both of these lines were used interchangeably as controls (labeled as “control” in figures). PBac{PB}Nf1c00617 (nf1c00617) flies (Thibault et al., 2004) were obtained from the Drosophila Stock Center (Bloomington, IA, USA). We confirmed the insertion site of nf1c00617 via iPCR using primer sequences generated and made publicly available by Exelixis, Inc. through the Bloomington Drosophila Stock Center. As reported on the FlyBase website, the stock nf1c00617 contains an insertion of the PBac{PB} transposon in the 7th intron of the nf1 genomic sequence, at position 21811820 of the D. melanogaster Genome (R5.5).

Behavioral assays

Olfactory learning and memory experiments were conducted using an olfactory classical conditioning paradigm (Beck et al., 2000). Standard training procedures were used for all 3-minute and 3-hour memory experiments. Two-to-seven day-old flies were exposed to a single training trial, in which they were sequentially presented with methylcyclohexanol (MCH) and benzaldehyde (BA) odors for one minute each. During presentation of the first odor (CS+), flies were simultaneously exposed to 12-1.25s pulses of 90V electric shock. Following a 30-second delay, the second odor (CS−) was presented in the absence of electric shock. Flies were then transferred to a T-maze and allowed to choose between the two trained odors, each contained within one arm of the maze. Avoidance of the CS+ during testing was calculated as a Performance Index (P.I.), defined as the fraction of flies preferring the CS- minus the fraction of flies preferring the CS+. A P.I. of 1.0 indicates that all flies avoided the CS+, and a P.I. of 0.0 indicates a 50:50 distribution between the T-maze arms and therefore no learning. To control for possible naïve odor bias, each trial was comprised of two groups in which the first group was trained with MCH as the CS+ and the second group was trained with BA as the CS+. To control for visual distraction, all experiments were performed in a darkroom illuminated with dark red light. Environment within the darkroom was also controlled at 23–25°C and 65–75% humidity. For 24-hour memory assay, flies were 1–4 days old and either a 5X–massed or 5X–spaced protocol was followed. During massed training, flies were presented with 5 training cycles, as described above, with a 30-second intertrial interval. During spaced training, flies were presented with 5 training cycles with a 15-minute intertrial interval, to elicit PSD-LTM (Yu et al., 2006). For memory acquisition and stability assays, a modified training paradigm known as the short program was used (Beck et al., 2000). In this schedule, odor exposures were reduced to 10 seconds with a single 1.25s electric shock presented at the 8th second, and a 30-second delay between odor presentations. When multiple training trials were presented, there was a 30-second intertrial interval.

RNA in situ hybridization

Probe template containing an 875 base-pair region, including exons 6 and 7, was amplified from NF1mini plasmid (A. Bernards, Massachusetts General Hospital, Boston, MA, USA) by PCR and TA cloned into pCR®II-TOPO® plasmid (Invitrogen, CA, USA). DIG-labeled RNA probes were transcribed from linearized plasmid in the anti-sense orientation, using the DIG RNA Labeling Kit (SP6/T7) (Roche, IN, USA). Both w(CS10) and nf1P1 fly heads were cryosectioned (15µm) and fixed in 4% paraformaldehyde. Sections were denatured with 0.2N HCl, treated with Proteinase K, post-fixed in 4% paraformaldehyde, and acetylated. Following a one-hour prehybridization at 50°C, denatured DIG-labeled probes were hybridized to sections at 50°C for 16–24 hours. Hybridization buffer contained 50% formamide, 5X SSC, 5X Denhardt’s, 250µg/mL yeast tRNA, 500µg/mL salmon sperm DNA, 50µg/mL heparin, 2.5mM EDTA, 0.1% TWEEN-20, 0.25% CHAPS. Following a number of washes to decrease salt concentration, slides were incubated with α-DIG-AP (Roche, IN, USA) and visualized using NBT/BCIP (Roche, IN, USA) solution. When staining was complete, slides were washed in PBST and mounted in Glycergel (Dako, CA, USA).

RESULTS

nf1c00617 mutants exhibit size and cognitive phenotypes

We first explored whether the putative allele, nf1c00617, conferred the small size and learning phenotypes typical of nf1P1 and nf1P2 mutants (Guo et al., 2000; The et al., 1997). To measure body size, female and male adult flies were measured from the anterior tip of the antennae to the posterior tip of the abdomen. As expected, both nf1P1 and nf1P2 males and females were significantly smaller than control males and females, respectively (The et al, 1997). In contrast, only nf1c00617 males were smaller than control males, but the nf1c00617 males were still significantly larger than nf1P1 and nf1P2 males. There was no significant size difference between nf1c00617 and control females (Figure 1A).

Figure 1
nf1mutants exhibit size and memory phenotypes

In addition to a mild size phenotype, nf1c00617 homozygous mutant flies also exhibited 3-minute, 3-hour, and 24-hour memory phenotypes (Figure 1B). nf1c00617 performance at all time points was significantly poorer than the control, but when tested 3 minutes after training these flies performed significantly better than nf1P1, nf1P2, and rut2080 homozygous mutants. We also sought to confirm that the cognitive phenotype of nf1c00617 mutants is due to a disruption of Nf1 function. We expressed a transgene containing full-length Drosophila nf1 (uas-dnf1) in the nf1c00617 homozygous mutant background using the elav-gal4 driver, which promotes gene expression in all neurons. Restoration of nf1 expression in all neurons fully rescued the 3-hour memory phenotype of nf1c00617 homozygous mutants, confirming that the nf1c00617 insertion does indeed disrupt Nf1-dependent memory (Figure 1C).

nf1 mutants are defective in memory acquisition, but not memory stability

While a requirement for neurofibromin during olfactory conditioning was established previously (Guo et al., 2000), former experiments did not address which operational phase of learning is impaired. Any deficit in olfactory memory may represent a failure to associate the odor and shock stimuli, an increased rate of memory decay, or failure of memory retrieval (Cheng et al., 2001). Memory acquisition and stability were assayed for nf1c00617, nf1P2, and rut2080 homozygous null mutants. Flies were presented 1–15 training trials with a 30-second intertrial interval and performance was assayed immediately following the last training trial (Figure 2A). Control performance improved with increasing number of training trials, and reached a plateau at a ceiling level with as few as 5 training trials. After a single training trial, all mutants performed poorly relative to the control, but nf1c00617 flies did not continue to display a learning deficit when presented with additional training trials. In contrast, nf1P2 and rut2080 flies performed poorly until after 15 training trials, at which point their performance was not significantly different from the control. Our results suggest that both Nf1 and Rut-AC are required for the acquisition of olfactory memory.

Figure 2
Neurofibromin is required for memory acquisition, not memory stability

Immediate performance of each mutant was then normalized relative to the control so that memory decay could be compared (Figure 2B). When nf1c00617 mutants and controls were both trained with 3 trials, immediate memory was identical. Likewise, memory tested at 2 subsequent time points was indistinguishable. Normalization of nf1P2 performance required training with 7 trials when the control was trained with 3 trials. Memory of nf1P2 mutants, when tested at 2 subsequent time points, was also indistinguishable from performance of controls, suggesting that Nf1 is not required to maintain olfactory memories over time. Finally, the immediate performance of rut2080 and control flies was normalized by 10 and 3 training trials, respectively. As early as 30 minutes after training, the memory of rut2080 flies was abolished, suggesting that Rut-AC plays an important role in maintaining memories over time. Furthermore, our results suggest that the role of Rut-AC in memory stability is Nf1-independent.

nf1 is expressed broadly in the brain and in mushroom body neurons

Walker et al. (2006) reported widespread expression of Nf1 in the central nervous system (CNS) of Drosophila larvae, but a notable absence of expression in third instar mushroom body neurons. Additionally, the authors also indicate an absence of Nf1 expression in the adult mushroom body neurons but expression elsewhere in the adult CNS. However, the putative association of Nf1 with Rut-AC in learning (Guo et al., 2000), the preferential expression of Rut-AC in mushroom body neurons (Han et al., 1992), and the clear evidence indicating that Rut-AC is required in adult mushroom body neurons for olfactory learning (Blum et al., 2009; Akalal et al., 2006; Mao et al., 2004; McGuire et al., 2003; Zars et al., 2000) necessitates Nf1 expression in adult mushroom body neurons.

We therefore probed nf1 gene expression by RNA in situ hybridization on w(CS10) brain sections. Figure 3 illustrates that nf1 is expressed in many, or all, cell body regions of the central brain. Expression of nf1 was apparent in cell body regions surrounding the antennal lobes, protocerebrum, lateral horn, and mushroom body calyces. Further examination revealed nf1 RNA expression in many mushroom body cell bodies (Figure 3E). Hybridization of the same probe with nf1P1 homozygous mutant brains did not result in staining, confirming the specificity of the anti-sense probe. Our results indicate that the nf1 gene is broadly expressed in the adult CNS, including the mushroom body neurons.

Figure 3
nf1 is endogenously expressed in mushroom body neurons

Considering the robust expression of nf1 RNA in mushroom body cell bodies, it is surprising that attempts to visualize the Nf1 protein in adult mushroom body neurons, with the same antibodies used for larval immunohistochemistry, have not been successful (Walker et al., 2006; Buchanan and Davis, unpublished). However, three different Nf1 isoforms exist, which encode peptides of 2746 (Nf1-RC), 2764 (Nf1-RD), and 2802 (Nf1-RB) amino acids. It is not known which of these is expressed in the adult CNS. We have developed RNA probes against a sequence region that is common among all transcripts. Walker et al. (2006) used monoclonal antibodies (DNF1–21) generated against the C-terminal 450 amino acids of Nf1-RB (The et al., 1997). Only 195 of these residues is present in Nf1-RC, and these are followed by 199 resides that are not in common with Nf1-RB. Furthermore, only 411 out of the 450 amino acids in Nf1-RB are present in Nf1-RD, and this is followed by 1 residue that is not in common with Nf1-RB. It seems probable that the DNF1–21 does not react with the isoform that is expressed in mushroom body neurons.

Many other genes, required for learning and memory, are preferentially expressed in mushroom body neurons (Davis, 2005; Cheng et al., 2001; Skoulakis et al., 1993; Han et al., 1992; Nighorn et al., 1991), so expression of Nf1 in the adult mushroom bodies makes sense. Additionally, the broad nature of nf1 expression in the central brain is not surprising due to its role in other behavioral outputs, i.e., circadian rhythm (Williams et al., 2001) and escape latency (The et al., 1997), both of which may require Nf1 expression in distinct neurons of the adult brain.

nf1 expression in mushroom body neurons, only during adulthood, rescues 3-hour memory

Previous work using a ubiquitously-expressed, heat shock-inducible, wild-type transgene (hsnf1) demonstrated that nf1 is required in adulthood for normal olfactory associative learning (Guo et al., 2000). Although these experiments identified a physiological role for Nf1 in memory formation, they did not identify the neurons that require Nf1 for normal olfactory learning. After this work was completed, the Gene-Switch system was developed in our lab for simultaneous temporal and spatial control of gene expression (Mao et al., 2004). Gene-Switch is a Gal4-based, RU486-inducible regulator of uas transgene expression. With this system, we have been able to simultaneously identify time and space requirements of nf1 expression for normal olfactory learning.

Expression of uas-dnf1 was first induced by Gene-Switch line 12-1 during both development and adulthood, to confirm that this system could induce sufficient nf1 expression to rescue the nf1c00617 3-hour memory phenotype. Gene-Switch line 12-1 utilizes a mushroom body enhancer that induces expression in both α/β and γ mushroom body neurons (Mao et al., 2004), and performance of this line does not differ from w(CS10) controls (data not shown). Animals were reared on food laced with RU486 from embryogenesis, through training and testing as adults. Expression of uas-dnf1 in mushroom body neurons during development and adulthood resulted in complete rescue of the 3-hour memory phenotype (Figure 4A). Next, flies were reared on normal food and transferred to food containing RU486 at eclosion. Our results indicate that expression of nf1, only in adult mushroom body neurons, is sufficient for complete rescue of the nf1 3-hour memory phenotype (Figure 4B).

Figure 4
nf1expression in mushroom body neurons, only during adulthood, rescuesnf1c00617memory

nf1 expression in α’/β’ or γ neurons does not restore 3-hour memory

Recent efforts, in our lab and others, have begun to distinguish the specific roles of individual mushroom body neuron subtypes in learning and memory (Krashes et al., 2007; Akalal et al., 2006; Yu et al., 2006; Yu et al., 2005). Some evidence suggests that synaptic output from α’/β’ neurons is required during acquisition and consolidation for the formation of stable memories (Wang et al., 2008; Krashes et al., 2007). Because our data implies a role for neurofibromin in memory acquisition, we decided to see if nf1 expression in α’/β’ neurons would be sufficient to rescue the 3-hour memory phenotype.

nf1 was expressed in α’/β’ neurons of nf1c00617 flies with the c305a–gal4 driver (Aso et al., 2009; Krashes et al., 2007). It was not possible to test for rescue of the nf1P1 phenotype, because w;c305a;nf1P1 animals have severely reduced fecundity. However, expression of nf1 in this subset of mushroom body neurons was not sufficient to rescue the 3-hour memory phenotype of nf1c00617 homozygous mutants (Figure 5A). This result suggests that Nf1-dependent signaling pathways are not required in α’/β’ neurons for acquisition and early memory formation.

Figure 5
nf1 expression in α’/β’ or γ neurons does not restore 3-hour memory

A close association between nf1 and rut-AC during learning has been well established (Ho et al., 2007; Guo et al., 2000), and expression of rut-AC in γ neurons of the mushroom bodies partially rescues the learning phenotype of rut mutants (Blum et al., 2009; Akalal et al., 2006; Mao et al., 2004; McGuire et al., 2003; Zars et al., 2000). Therefore, we examined whether uas-dnf1 expression in γ neurons might rescue the phenotype of nf1 mutants. Driver NP1131-gal4 was used to express uas-dnf1 in γ neurons (Aso et al., 2009; Akalal et al., 2006). However, expression of nf1 in this subset of mushroom body neurons was not sufficient to rescue the 3-hour memory phenotype of either mutant allele (Figure 5B). This result suggests that Nf1-dependent signaling pathways are not required in γ neurons during memory acquisition and early consolidation. Furthermore, any role of Rut-AC in γ neurons, during PSI-EM processing, is Nf1-independent.

nf1 expression in α/β neurons restores protein synthesis-independent memory

Because expression of rut-AC in α/β neurons partially rescues the learning phenotype of rut mutants (Blum et al., 2009; Akalal et al., 2006; Mao et al., 2004; McGuire et al., 2003; Zars et al., 2000), we next expressed uas-dnf1 in both nf1P1 and nf1c00617 homozygous mutant backgrounds using the Mz1081-gal4 driver. With this driver, transgene expression is promoted throughout the central brain, including antennal lobe and α/β neurons. Expression of uas-dnf1 in these neurons was sufficient to rescue the 3-hour memory phenotype of both nf1 homozygous mutant alleles (Figure 6A). Next, we used the NP9-gal4 driver, which shows a more limited expression pattern relative to Mz1081-gal4. NP9 is also broadly expressed in the central brain, including antennal lobe and α/β neurons. Expression of uas-dnf1 with this driver was also able to fully rescue the 3-hour memory phenotype of both nf1P1 and nf1c00617 homozygous mutant alleles (Figure 6B). We next expressed uas-dnf1 using the c739-gal4 driver, which promotes robust transgene expression in the α/β mushroom body neurons and weak transgene expression in other neurons of the central brain (Aso et al., 2009). Once again, we saw complete rescue of the 3-hour memory phenotype, providing more substantial evidence that the expression of nf1 only in α/β neurons is sufficient for restoring normal memory in nf1 mutants (Figure 6C). Finally, we expressed uas-dnf1 using 17d–gal4, which promotes transgene expression in the central core of α/β mushroom body neurons (Aso et al., 2009). Expression with this driver did not restore normal 3-hour memory of either mutant allele (Figure 6D), suggesting that either the expression level induced by this driver is not sufficient or that it does not promote expression in a sufficient number of α/β neurons to support learning.

Figure 6
nf1expression in α/β neurons rescues 3-hour memory

We also tested whether uas-dnf1 expression in α/β neurons would rescue 3-minute memory, the earliest testable form of PSI-EM. We used the c739-gal4 driver to promote expression of uas-dnf1 in α/β neurons of nf1P1 homozygous mutant flies. We observed full rescue of the nf1 phenotype at 3 minutes after training (Figure 7A). Additionally, we tested the effect of over expressing nf1 in normally performing flies using c739-gal4. Consistent with a previous report (Guo et al., 2000), we observed no enhancement of normal performance when nf1 was over expressed in α/β neurons (Figure 7B).

Figure 7
nf1expression in α/β neurons also rescues 3-minute and 24-hour memory, but does not enhance control performance

Because Mz1081-gal4, NP9-gal4, and c739-gal4 drivers all promote some expression outside of the α/β neurons, i.e., in antennal lobe neurons, we wanted to further demonstrate that expression of nf1 in α/β neurons, rather than these other sites of expression, rescued the memory phenotype. Our lab previously introduced a MB{Gal80} transgene into the chromosome carrying c772-gal4, by recombination (Liu et al., 2007). The MB{Gal80} transgene contains the gal80 repressor gene for Gal4 (Lee and Luo, 1999) downstream of a mushroom body enhancer, which drives expression predominantly in the mushroom body neurons (Mao et al., 2004; Zars et al., 2000). Combining the MB{Gal80} transgene with c772-gal4 specifically suppresses uas-transgene expression in the mushroom body neurons, while expression in antennal lobe neurons is unaffected (Liu et al., 2007). Expression promoted by c772-gal4 by itself is strongest in α/β and γ mushroom body neurons, moderate in antennal lobe neurons, and weak in α’/β’ mushroom body neurons (Aso et al., 2009; Liu et al., 2007).

As expected, the nf1c00617 3-hour memory phenotype was fully rescued when c772-gal4 was used to express uas-dnf1 (Figure 8A). However, flies carrying the combined c772-gal4; MB{Gal80} driver along with uas-dnf1, exhibited a performance score that was indistinguishable from nf1c00617 homozygous mutants (Figure 8B). Therefore, rescue of the 3-hour memory phenotype of nf1 mutants requires expression in α/β mushroom body neurons.

Figure 8
Inhibiting nf1 expression in α/β neurons prevents memory rescue

nf1 expression in α/β neurons restores protein synthesis-dependent long-term memory

Recent work in our lab suggests that a memory trace can be visualized in the α/β neurons of mushroom bodies 24 hours after adult flies are subjected to a 5X–spaced training procedure (see methods) that produces PSD-LTM (Yu et al., 2006). In contrast, a 5X–massed training procedure produces neither PSD-LTM, nor a memory trace in α/β neurons. A deficit in LTM has been reported for nf1 mutants trained with a spaced protocol (Ho et al., 2007). It is possible that this phenotype is due to the requirement for nf1 in α/β neurons. We therefore used c739-gal4 to promote expression of uas-dnf1 in α/β neurons of nf1P1 homozygous mutant flies, and tested for rescue 24 hours after 5X–spaced training. We observed full rescue of the nf1 phenotype at 24 hours (Figure 7C). As expression of nf1 in α/β neurons can restore both PSI-EM and PSD-LTM, our results suggest that Nf1 mediates multiple types of memory processing, through at least two different biochemical pathways, within the same population of neurons.

DISCUSSION

Regardless of species being studied, neurofibromin is involved in many different brain activities including, but not limited to, cognitive processes, circadian rhythms, cortical development, and glial development. Even within the cognitive realm, Nf1 function depends on the context of specific training conditions. Protein synthesis-independent short- and middle-term memories appear to require an activation of Rut-AC by Nf1, whereas protein synthesis-dependent long-term memory requires an additional modulation of Ras activity (Ho et al., 2007; Hannan et al., 2006). By rescuing the performance of homozygous mutants, we have demonstrated that expression of Nf1 in adult α/β mushroom body neurons is sufficient to support all forms of Nf1-dependent memory. We have also revealed a requirement for Nf1 during acquisition. Together, our observations expand our current understanding of Nf1 and Rut-AC functions and challenge current models of mushroom body neuron activity in olfactory memory formation.

Rut-AC is required in both α/β and γ mushroom body neurons for complete rescue of rut STM deficits (Akalal et al., 2006), yet we have shown that Nf1 is required only in the α/β mushroom body neurons. It is unclear why Rut-AC activation would only require Nf1 in one subset of neurons. One possibility is that the Nf1 stimulation of Rut-AC in α/β neurons during acquisition may indirectly facilitate, through unknown signals, Rut-AC activity in the γ neurons. Prior results have been interpreted to suggest that there are communication loops that exist between certain types of mushroom body neurons, and with extrinsic mushroom body neurons for normal learning and consolidation (Yu et al., 2006; Krashes et al., 2007). A similar process could allow Rut-AC activation in γ neurons to be indirectly dependent upon Nf1 in α/β mushroom body neurons. Alternatively, it could be that the Rut-AC is dependent upon Nf1 in the α/β mushroom body neurons for its role in learning but Nf1-independent in γ mushroom body neurons.

For rescue of PSD-LTM, both Rut-AC (Blum et al., 2009) and Nf1 expression are required only in α/β neurons, suggesting that their interaction is necessary to support this form of memory as well. A recent study concluded that the Nf1-GRD, which has been shown to mediate an adenylyl cyclase activity (Hannan et al., 2006), is necessary and sufficient for Nf1-dependent LTM (Ho et al., 2007). In contrast to Rut-AC, this adenylyl cyclase activity is stimulated by Ras and is Gαs-independent. It is important to note, however, that the Nf1-GRD domain only partially rescued the LTM phenotype of nf1 mutants. Full rescue of LTM required a full-length nf1 transgene. Together, these data and ours suggest that Nf1 simultaneously mediates the activation of both AC signaling pathways in α/β neurons to facilitate new protein synthesis and the formation of long-lasting memory.

Early work on the role of Rut-AC in olfactory associative memory suggested that this adenylyl cyclase plays a role in behavioral acquisition (Dudai et al., 1988; Tully and Quinn 1985). Using an olfactory avoidance assay, it was suggested that rutabaga mutants could obtain normal performance with more intense training (Dudai et al., 1984). We have also observed a delay in the acquisition of olfactory memory in rutabaga mutants, which require 3 times the amount of training as controls to overcome. A similar delay in acquisition was discovered for nf1 mutants, consistent with the hypothesis that Nf1 is required for G-protein activation of Rut-AC during learning (Ho et al., 2007; Guo et al., 2000). Our results also demonstrate that Rut-AC is essential for the stability of olfactory memory. However, this function is independent of an interaction with Nf1. We believe that the association of Nf1 and Rut-AC may be transient, only required for the initial activation of Rut-AC in its role as a molecular coincidence detector in α/β neurons (Tomchik and Davis, 2009). If this model were true, memory stability would therefore require continued stimulation of Rut-AC molecules via an independent and perhaps spatially distinct mechanism that does not require Nf1.

Recent efforts, in our lab and others, have attempted to assign temporal and operational phases of olfactory memory processing to distinct regions within the adult olfactory system. Upon pairing of odor and electric shock, new projection neuron synapses are recruited to the odor representation (Yu et al., 2004). Pairing dopamine application with neuronal depolarization in adult brain preparations results in a Rut-AC-dependent synergistic increase of cAMP in both α and α’ lobes (Tomchik and Davis, 2009), and memory acquisition requires synaptic transmission from α’/β’ neurons (Krashes et al., 2007). Although we have demonstrated that both Nf1 and Rut-AC are required for memory acquisition, neither of these need be expressed in α’/β’ neurons (Blum et al., 2009; Akalal et al., 2006; Mao et al., 2004; McGuire et al., 2003; Zars et al., 2000). We therefore propose that memory acquisition cannot be thought of as a specific event involving a distinct neuronal subset. Rather, we envision a model in which the pairing of odor and electric shock induces a change on the neuronal systems level. Each individual neuron subset may register this change in a different way, but every change is in some way necessary for memory acquisition as a whole. Additional work will be required to determine whether memory consolidation, retrieval, or processing of longer-term memories also require plasticity throughout the entire olfactory system.

It is clear from the data herein that Nf1 function is required in the adult brain, in α/β neurons defined by the c739-gal4 driver, for PSI-EM formation and for PSD-LTM formation. By identifying a minimal region in which Nf1 expression is required, we are now able to isolate its role in memory formation from others that may occur in the brain. This mapping promises a more accurate analysis of Nf1-dependent memory and insights into both memory processing as a whole and into the cognitive deficits associated with Neurofibromatosis Type 1.

Acknowledgements

We thank Curtis Wilson for technical assistance. These studies were supported by NIH grant NS19904 to RLD. MEB was supported by a Children’s Tumor Foundation Young Investigator Award and by Grant Number T32GM008307 from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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