Compromising Tyrosine Hydroxylase Function Extends and Blunts the Temporal Profile of Reinforcement by Dopamine Neurons in Drosophila

Abstract

For a proper representation of the causal structure of the world, it is adaptive to consider both evidence for and evidence against causality. To take punishment as an example, the causality of a stimulus is unlikely if there is a temporal gap before punishment is received, but causality is credible if the stimulus immediately precedes punishment. In contrast, causality can be ruled out if the punishment occurred first. At the behavioral level, this is reflected in the associative principle of timing-dependent valence reversal: aversive memories are formed when a stimulus occurs before the punishment, whereas memories of appetitive valence are formed when a stimulus is presented upon the relieving termination of punishment. We map the temporal profile of memories induced by optogenetic activation of the PPL1-01 neuron in the fly Drosophila melanogaster (of either sex) and find that compromising tyrosine hydroxylase function, either acutely by pharmacological methods or by cell-specific RNAi, extends and blunts this profile. Specifically, it (1) enhances learning with a time gap between the stimulus and PPL1-01 punishment (better trace conditioning), (2) impairs learning when the stimulus immediately precedes PPL1-01 punishment (worse delay conditioning), and (3) prevents learning about a stimulus presented after PPL1-01 punishment has ceased (worse relief conditioning). Under conditions of low dopamine, we furthermore observe a role for serotonin that is pronounced in trace conditioning, weaker in delay conditioning, and absent in relief conditioning. We discuss the psychiatric implications if related alterations in the temporal profile of reinforcement were to occur in humans.

Keywords: Drosophila; dopamine; punishment; reinforcement; schizophrenia; timing.

Figure 1.

Pharmacologically inhibiting the TH enzyme extends and blunts the temporal profile of reinforcement by PPL1-01. A, Schematics of a fly, its brain, and the mushroom bodies (top) and a highly simplified working hypothesis of association formation during punishment learning (bottom; for references see body text). The intrinsic neurons of the mushroom bodies represent odors in a sparse and combinatorial manner (mushroom body neurons in black). The dopaminergic PPL1-01 neuron (blue), which can be activated by, e.g., electric shock punishment, intersects the axons of the mushroom body neurons in what is called the γ1pedc compartment. Associative coincidence of odor activation and signaling from PPL1-01 (red shade within the compartment) induces associative presynaptic depression (stars) at the synapses from the odor-activated mushroom body neurons toward an approach-promoting output neuron of the compartment (purple). As no such depression takes place in a neighboring compartment in relation to its avoidance-promoting output neuron, this shifts the balance across the mushroom body output neurons to net avoidance as the learned behavior. In total, the mushroom body has 15 compartments, only two of which are sketched. The compartment depicted at the top represents the two compartments known to receive input from punishing stimuli (γ1pedc and γ2); the compartment depicted at the bottom represents compartments known to receive reward input (γ4, γ5). B, Procedure for presenting the reference odor (open clouds), the paired odor (grey clouds), and optogenetic activation of PPL1-01 (blue light bulb). The interval between the onset of the paired odor and the onset of PPL1-01 activation is called the interstimulus interval (ISI). For more details, see Extended Data Figure 1-1. C, Schematic of dopamine biosynthesis and of the inhibition of tyrosine hydroxylase (TH) by 3-iodo-ʟ-tyrosine (3IY). The dopamine precursor 3,4-dihydroxy-ʟ-phenylalanine (ʟ-DOPA) should compensate for the effects of 3IY on dopamine levels. DDC, dopamine decarboxylase. Drug feeding was performed by the tissue paper method. D, Relative to controls, punishment memory after odor → PPL1-01 training (ISI −15 s) is decreased upon feeding of 3IY (N = 25, 25). Relief memory after PPL1-01 → odor training (ISI 120 s) is unaffected (N = 25, 23). E, The decrease in punishment memory by 3IY can be rescued by additionally feeding ʟ-DOPA (ISI −15 s; N = 16, 16, 16). Relief memory is unaffected by 3IY, and by combining 3IY and ʟ-DOPA (ISI 120 s) (N = 16, 16, 15). F, Mapping out the effect of 3IY on the temporal profile of PPL1-01 reinforcement (N = 20, 20; 20, 20; 19, 20; 20, 20; 20, 20; 20, 20; 20, 20). 3IY decreases punishment memory (ISI −15 s, delay conditioning) and leaves relief memory with an ISI of 120 s unaffected. For a longer relief ISI of 240 s a decrease in relief memory is revealed. For a training procedure with a −40 s time gap between odor and PPL1-01 (ISI −100 s, trace conditioning), an increase in memory scores by 3IY is observed. G, The effects of 3IY on memory scores after trace, delay, and relief conditioning (ISIs of −100 s, −15 s, and 240 s, respectively) can be largely rescued, or even overcompensated, by ʟ-DOPA (N = 30, 31, 31; 30, 31, 31; 30, 31, 30). Plotted in D–G are the memory scores according to Equation 2, reflecting associative memory for the odor paired with optogenetic activation of PPL1-01; positive and negative memory scores reflect appetitive and aversive memory, respectively. Box plots represent the median as the middle line, 25%/75% quantiles as box boundaries, and 10%/90% quantiles as whiskers. Open box plots and circles refer to the control condition, and magenta and gray fill to groups fed with 3IY or with 3IY plus ʟ-DOPA, respectively. Flies were of the genotype PPL1-01 > ChR2-XXL. * and “ns” indicate significance and nonsignificance, respectively, in MW-U tests at an error rate of 5%, adjusted according to Bonferroni–Holm, except for E (ISI 120 s) where “ns” indicates nonsignificance in a KW test. In E (ISI −15 s), the exact p value is presented, which after Bonferroni–Holm correction is just about nonsignificant. Given that in all five other cases of comparison between these treatment groups, statistical significance is reached (Figs. 1D,F,G, 5B; Extended Data Fig. 1-2), our interpretation is that the narrow “miss” of significance in this case is a false negative. Data and statistical results are documented in Extended Data Tables and Table 1. Memory scores separated by sex are shown in Extended Data Figure 1-3. The anatomical image of the mushroom body in A is modified from Heisenberg and Gerber (2008).

Figure 2.

Pharmacological inhibition of the TH enzyme reduces brain levels of dopamine. Whole-brain levels of dopamine and serotonin after feeding 3-iodo-ʟ-tyrosine (3IY), an inhibitor of the tyrosine hydroxylase (TH) enzyme required for dopamine biosynthesis. Feeding of 3IY reduced dopamine levels, an effect that was restored by feeding the dopamine precursor 3,4-dihydroxy-ʟ-phenylalanine (ʟ-DOPA) in addition (N = 20, 20, 20). Drug feeding, performed by the tissue paper method, was without effect on serotonin levels (N = 20, 20, 20). Box plots represent the median as the middle line, 25%/75% quantiles as box boundaries, and 10%/90% quantiles as whiskers. Open box plots and circles refer to the control condition, and magenta and gray fill to groups fed with 3IY or with 3IY plus ʟ-DOPA, respectively. Flies were of the genotype PPL1-01 > ChR2-XXL. * indicates significance in MW-U tests at an error rate of 5%, adjusted according to Bonferroni–Holm. “ns” indicates nonsignificance in such a MW-U test (dopamine) or in a KW test (serotonin). Data and statistical results are documented in Extended Data Tables and Table 2. Data separated by sex are shown in Extended Data Figure 2-1.

Figure 3.

Local knockdown of the TH enzyme extends and blunts the temporal profile of reinforcement by PPL1-01. A, Schematic of dopamine biosynthesis and of the inhibition of tyrosine hydroxylase (TH) by RNA interference (RNAi). B, Mapping out the effect of TH-RNAi in the PPL1-01 neuron on the temporal profile of PPL1-01 reinforcement (N = 32, 32; 34, 34; 40, 40; 33, 34; 42, 42). Relative to controls, TH-RNAi promotes punishment memory upon trace conditioning (ISI −100 s) and decreases punishment memory upon delay conditioning (ISI −15 s). Relief memory is decreased (ISI 300 s). Control flies were of the genotype PPL1-01 > ChR2-XXL (open box plots and circles); flies for TH knockdown in the PPL1-01 neuron additionally carried the TH-RNAi construct (PPL1-01 > ChR2-XXL/TH-RNAi; box plots and circles with magenta fill). Other details are as in the legend of Figure 1. * indicates significance in MW-U tests at an error rate of 5%, adjusted according to Bonferroni–Holm, “ns” indicates nonsignificance in such tests. Data and statistical results are documented in Extended Data Tables and Table 3. Memory scores separated by sex are shown in Extended Data Figure 3-1.

Figure 4.

Compromising TH function extends and blunts the temporal profile of reinforcement by PPL1-01. A, Summary of the effects of compromising TH function on the temporal profile of PPL1-01 reinforcement, combined for 3IY and TH-RNAi, and across the present study. Shown are the memory scores of the respective control (open box plots) and TH-compromised cases (box plots and circles with magenta fill; N = 20, 20, 120, 122, 159, 165, 20, 20, 101, 99, 108, 113, 62, 62). B, Data for only the control cases shown in A, separated by sex. Neither for trace conditioning (ISI −100 s), nor for delay conditioning (ISI −15 s), nor for relief conditioning (ISIs 240 and 300 s) were sex-dependent differences observed (for a justification of why relief conditioning with ISIs of 80 and 120 s is not included in this grouping, see below; N = 120, 120, 159, 157, 169, 169). C, For the data in A, the difference in memory scores of the TH-compromised cases minus the scores in the controls is plotted, separately for female and male flies, to quantify how strongly compromising TH function affects memory scores, in either sex. For trace conditioning (ISI −100 s), the effect of compromising TH function was less pronounced in females than in males, whereas no such difference was observed for delay (ISI −15 s) and relief conditioning (N = 118, 119, 159, 157, 168, 167). For relief conditioning, data were considered only for those ISIs for which compromising TH function had an effect to begin with (A, 240 and 300 s). Other details are as in the legend of Figure 1. * indicates significance in MW-U tests at an error rate of 5%, adjusted according to Bonferroni–Holm, “ns” indicates nonsignificance in such tests. ^# indicates significance in OSS-tests at an error rate of 5%, adjusted according to Bonferroni–Holm. Data and statistical results are documented in Extended Data Tables and Table 4.

Figure 5.

Temporal profile of reinforcement by PPL1-01 upon pharmacologically inhibiting the TPH and the TH enzyme. A, Schematic of serotonin and dopamine biosynthesis, of the inhibition of tryptophan hydroxylase (TPH) by para-chlorophenylalanine (PCPA), and of the inhibition of tyrosine hydroxylase (TH) by 3-iodo-ʟ-tyrosine (3IY), respectively. 5-HTP, 5-hydroxytryptophan; ʟ-DOPA, 3,4-dihydroxy-ʟ-phenylalanine; DDC, dopamine decarboxylase. Drug feeding was performed by the food method. B, Relative to controls, punishment memory after odor → PPL1-01 trace conditioning (ISI −100 s) is increased upon feeding of 3IY, an effect that is fully reversed by an additional feeding of PCPA (N = 38, 39, 45). For delay conditioning (ISI −15 s), punishment memory is reduced by 3IY, an effect that is partially reversed by PCPA (N = 29, 33, 32). The reduction of relief memory (ISI 240 s) by 3IY was not reversed by PCPA (N = 25, 28, 29). C, PCPA alone has no effect on punishment memory after trace conditioning (ISI −100 s) (N = 39, 39) or delay conditioning (ISI −15 s) (N = 45, 48) and leaves relief memory intact, too (ISI 240 s; N = 44, 41). Open box plots and circles refer to the control condition, magenta and green fill to groups fed with 3IY or with 3IY plus PCPA, respectively. Flies were of the genotype PPL1-01 > ChR2-XXL. Other details are as in the legend of Figure 1. * indicates significance and “ns” nonsignificance in MW-U tests at an error rate of 5%, adjusted according to Bonferroni–Holm. Data and statistical results are documented in Extended Data Tables and Table 5. Memory scores separated by sex are shown in Extended Data Figure 5-1.