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Brandeis University, Department of Biology and Volen Center for Complex Systems, Waltham, Massachusetts 02454-9110, USA
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ABSTRACT |
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 TOP ABSTRACT Results and DiscussionMaterials and MethodsReferences |
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).
In Drosophila courtship conditioning, CaMKII is important for behavioral changes that occur during the training period and for formation of associative memory (Griffith et al. 1993; Joiner and Griffith 1997, 1999). In this behavioral paradigm, a male is exposed to a previously mated female for 1 h, and the effects of training are assessed by measuring courtship of a subsequently presented virgin female. Naive males exposed to virgin females sense female-specific stimulatory pheromones and rapidly initiate courtship, usually copulating within 15 min. In comparison, trained males show reduced courtship of virgins, and this is believed to be the result of association of stimulatory pheromones with an aversive substance given off by mated females (Tompkins et al. 1983). Memory formation can be blocked by inhibition of CaMKII in mushroom bodies, central complex, and parts of the lateral protocerebrum (Joiner and Griffith 1999). The behavior of males during training is also plastic; they initiate vigorous courtship, but over the course of training they decrease their intensity of courtship and rarely copulate. The suppression of courtship during training is driven by a neuronal circuit distinct from that mediating associative memory formation, involving neurons in the antennal lobes and lateral protocerebrum (Joiner and Griffith 1999).
The requirement for CaMKII activity was determined using spatially restricted expression of a peptide, which inhibits both the calcium-dependent and the calcium-independent (autophosphorylated at T287) forms of the kinase. The role of these two forms of CaMKII was dissected by spatially and temporally regulated overexpression of T287A and T287D CaMKII point mutants (Mehren and Griffith 2004). Expression of the constitutively active T287D CaMKII enhanced the suppression of courtship during training but had no effect on memory formation. Addition of T287A CaMKII, which is incapable of becoming calcium independent, had no behavioral effects. The effect of T287D CaMKII on courtship suppression was only seen in animals that expressed T287D in subsets of the adult brain defined by the 30Y-GAL4 pattern, which includes the mushroom bodies, lateral protocerebrum, antennal lobes, subesophageal ganglion, and optic lobes. These results suggested that the decision to court was not sensitive to the total amount of calcium-stimulable kinase but rather to some threshold level of constitutively active CaMKII.
In this study, we demonstrate that both initial and training-dependent changes in courtship respond to the level of calcium-independent CaMKII in a dose-dependent manner. We further show that initial courtship levels can be modulated by noncholinergic neurons, whereas the effects of T287D on training-dependent changes in courtship are mediated by cholinergic antennal lobe neurons.
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Results and Discussion |
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TOPABSTRACTResults and DiscussionMaterials and MethodsReferences |
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). This system allows for both spatial and temporal control of the level of transgene expression. The tetracycline transactivator (UAS-tTA) is driven by GAL4 (a yeast transcription factor that binds to the UAS element and drives transcription of UAS-linked genes) in a spatially restricted pattern. The spatial restriction of GAL4 is achieved by expressing it under control of particular defined or endogenous promoter sequences. The effector transgene (tetO-T287D) in this scheme is expressed in the GAL4 pattern, but its levels can be controlled by feeding the animal tetracycline, which inhibits transcription by tTA. This system allows complete suppression of toxic transgenes during development by growing larvae on 10 µg/mL tetracycline in the food agar. In adults, complete suppression can be achieved by feeding 100 µg/mL tetracycline in sucrose. Removal or reduction of the drug leads to induction of the transgene.
Previous work had shown that expressing T287D with tetracycline-controlled 30Y-GAL4 and allowing maximal adult induction by placing newly eclosed males on food containing no tetracycline was able to cause both a lowered level of initial courtship and an enhancement of trainer-dependent suppression (Mehren and Griffith 2004). If the mechanism of calcium-independent CaMKII’s actions was dependent on the level of calcium-independent CaMKII, we would expect that manipulating the amount of induction by placing newly eclosed males on a range of drug concentrations from 100 to 0 µg/mL should produce a dose-dependent range of levels of courtship suppression. Figure 1 shows that after rearing on a maximally suppressing dose of tetracycline, suppression scales with the adult level of tetracycline. Initial courtship (Fig. 1A) is decreased with increasing expression of T287D and the animals fall into two groups that are statistically different: Untreated wild-type males, fully suppressed (100 µg/mL tetracycline) T287D males, and T287D males maintained on 10 µg/mL tetracycline all show high initial courtship, whereas animals on 1 or 0 µg/mL tetracycline have a similar, low level of courtship. This suggests that there is a bimodal relationship between the level of constitutive CaMKII and initiation of courtship.
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). But in contrast to the effects of T287D dosage on initial courtship, the effects on the trainer-specific component are more graded over the range of doses administered. Animals expressing the maximum level of T287D (fed 0 µg/mL tetracycline as adults) suppress courtship toward the mated female (CIf/CIin = 0.04 ± 0.04, data presented as mean ± SEM) 12-fold better than animals fed 10 µg/mL tetracycline (CIf/CIin = 0.47 ± 0.14), and 18-fold better than fully suppressed animals (fed 100 µg/mL tetracycline [CIf/CIin = 0.70 ± 0.09]). The difference in the effects of particular doses of T287D on initial and trainer-dependent suppression suggests that these effects may be mediated by distinct sets of cells that innervate the antennal lobe.
To refine the mapping of these two types of courtship suppression, we utilized GAL4 lines that are restricted to neurons expressing particular neurotransmitters. The antennal lobe is known to contain synapses made by cholinergic olfactory receptor neurons (Barber et al. 1989), cholinergic projection neurons (Yasuyama et al. 2003), GABAergic interneurons (Ng et al. 2002; Wilson and Laurent 2005) and perhaps other neurons that release modulatory peptides or amines (Python and Stocker 2002). By restricting T287D expression to adult neurons of particular neurochemical classes, we can subdivide the antennal lobe circuit. Figure 2A shows the expression patterns of Cha-GAL4 (restricted to cholinergic neurons), TH-GAL4 (restricted to dopaminergic neurons), GAD-GAL4 (restricted to GABAergic neurons) as assessed by mCD8–GFP expression. All of these neurochemical GAL4s show antennal lobe expression.
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). GAL80 is a yeast protein that antagonizes GAL4 action. Expression of GAL80 under control of the Cha promoter will block GAL4 action exclusively in the cholinergic subpopulation of a particular GAL4 pattern (Kitamoto 2002). Figure 2B shows the effect of adding Cha-GAL80 to 30Y-GAL4. All antennal lobe expression of UAS-mCD8-GFP is lost, but substantial expression remains in the mushroom body, pars intercerebralis, and extrinsic mushroom body neurons, indicating that these neurons are not cholinergic. To assess the contribution of these neurons to the T287D-dependent decrease in initial courtship, we examined the ratio of courtship during the first 10 min of exposure to a mated female (CIin) of males with adult expression of constitutively active CaMKII in neurochemical subsets of cells to the mean of their GAL4 driver control (Fig. 3A). CIins for effector transgene controls (tetO-T287D/+; UAS-tTA/+, 0.51 ± 0.05; UAS-tTA/+; Cha-GAL80/+, 0.68 ± 0.05) did not differ significantly from those of wild type (0.60 ± 0.05) (data presented as mean ± SEM and analyzed by ANOVA F[2,64] = 2.78, P = 0.07). A ratio of less than one indicates a T287D-specific decrease in initial courtship and controls for nonspecific effects of individual GAL4 drivers. Consistent with our observations in Figure 1A, initial levels of courtship in animals expressing T287D under control of 30Y-GAL4 are significantly lower than the GAL4 control. Interestingly, TH-GAL4, Cha-GAL4, and GAD-GAL4 drivers cannot cause a T287D-specific decrease in initial courtship. In agreement with this, removal of the cholinergic component of the 30Y pattern by coexpression of Cha-GAL80 does not alter the effects of T287D, indicating that the cells marked by 30Y that are controlling initial courtship are noncholinergic.
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Our data suggest that the ability of constitutively active CaMKII to promote suppression of courtship derives from its actions in two separable populations of cells, both likely innervating the antennal lobes. Initial courtship levels are modulated by CaMKII activity in a population of noncholinergic neurons. These neurons are very sensitive to the levels of calcium-independent CaMKII.
Plasticity of courtship during exposure to a mated female is responsive to CaMKII levels in a different set of cells. Expression of T287D in cholinergic inputs into the antennal lobes driven by either 30Y-GAL4 or Cha-GAL4 is capable of accelerating the trainer-dependent suppression that occurs in response to pheromonal signals that mated females emit. These cells are sensitive to graded increases in constitutive CaMKII activity. Both olfactory sensory neurons and projection neurons are cholinergic and innervate the antennal glomeruli. Since T287D expression in primary sensory neurons does not produce an enhancement of training (Mehren and Griffith 2004), the most likely antennal cholinergic neurons to be responsible for this plasticity effect are the projection neurons. We have also previously shown that 30Y-GAL4 does not express in antennae (Mehren and Griffith 2004) where primary olfactory neurons reside, supporting the idea that the CaMKII-sensitive cholinergic neurons of the 30Y-GAL4 pattern are projection neurons. This makes it likely that projection neurons are active participants in CaMKII-dependent short-term plasticity of courtship behavior.
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Materials and Methods |
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TOPABSTRACTResults and DiscussionMaterials and MethodsReferences |
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Fly stocks and crosses were maintained on cornmeal, yeast, dextrose, and agar medium and kept at 25°C in a 12-h light/12-h dark cycle. Tetracycline feeding was performed by growing larvae on medium containing tetracycline at 10 µg/mL food or by feeding tetracycline to adults at a concentration of 0, 1, 10, or 100 µg/mL in a 4% sucrose solution, as described in Mehren and Griffith (2004). Canton S flies were used as wild type, and all transgenic lines were in a Canton S, white background. UAS-tTA was a gift from Bruno Bello (University of Basel, Basel, Switzerland). The tetO-CaMKII-T287D line was described in Mehren and Griffith (2004). The lines 30Y-GAL4 (Yang et al. 1995), Cha-GAL4 (Salvaterra and Kitamoto 2001), Cha-GAL80 (Kitamoto 2002) and TH4B-GAL4 (Friggi-Grelin et al. 2003) have been previously described. GAD2B-GAL4 was a generous gift of Toshihiro Kitamoto (University of Iowa, Iowa City, IA).
Immunohistochemistry
Brains were dissected in PBS from 5- or 6-d-old adult flies, fixed in 4% paraformaldehyde for 20–30 min, washed in PBS, and mounted in glycerol and Vectashield (Vector Laboratories Inc.). Images were acquired using the 40x objective on a Leica TCS SP2 confocal scanning microscope.
Behavior
All behavioral observations were performed at 25°C and 70% relative humidity in a Harris Environmental Room, in dim red light, as described in Mehren and Griffith (2004). Courtship conditioning was carried out by placing individual males with fertilized females (mated the day before) in Plexiglas mating chambers (8 mm diameter, 3 mm high) for 1 h. Courtship indices (CIs) were measured for 3 time intervals during training (0–10 min [CIin], 20–30 min, and 50–60 min [CIf]). A training index was taken as the fraction of the CIf over the CIin. Flies who had CIins < 0.1 were considered "nonbehaving" and therefore were excluded from analysis. Each CI was subjected to arcsine or arcsine square root transformation to approximate normal distributions. Transformed CIs or training indices were subjected to a one-way ANOVA with genotype as the main effect, using JMP (version 5.0.1.2
[EC]
; SAS Institute), and planned comparisons of means were performed with Student’s t-test (adjusted for experiment-wise error).
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Acknowledgments |
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FOOTNOTES |
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E-mail griffith{at}brandeis.edu; fax (781) 736-3107. 
1 Present address: Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, New York 10021-6399, USA.
Article published online before print. Article and publication date are at http://www.learnmem.org/cgi/doi/10.1101/lm.317806
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References |
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TOPABSTRACTResults and DiscussionMaterials and MethodsReferences |
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= 0.013). (B) Dose dependence of the time course of courtship suppression for T287D CaMKII flies. n = 10–24.
