First paper to establish the relationship between NPF in C. elegans and sensitivity to the effects of ethanol.

First paper to establish the relationship between NPF in fruit flies and sensitivity to the effects of ethanol.

A comprehensive analysis of the various genes in the Drosophila that pertain to the vast majority of—if not all—neuropeptides and their receptors.

This was one of the first studies to establish the courtship conditioning paradigm for controlling the sexual experience of male flies that is now commonly used in Drosophila research.

There is a naturally occurring variability in rats' urge to consume alcohol that is correlated with the levels of NPY in their brain. In mice, artificially increasing the amount of NPY in the brain leads to higher consumption of alcohol and reduced sensitivity to the intoxicating effects of alcohol (as well as vice versa). This is very similar to what has also been shown in flies (see reference in previous paragraph).

Injection of NPY into specific parts of the mouse brain can trigger intense eating or inhibit sexual drive.

When the activity of the NPF circuit is reduced below normal, flies are less sensitive to the intoxicating effects of alcohol (i.e. they exhibited fewer signs of "drunkenness"). The vice versa has also been observed to be true.

You might be wondering why this step is necessary, especially for alcohol, since the researchers have already shown that flies consume more alcohol-laden food. However, in the alcohol/food consumption paradigm, it is impossible to completely dissociate the contribution of the hunger and the caloric contribution of alcohol from the objective enjoyment of the experience. Here, what is being tested for is the preference for odor (which is otherwise neutral) that has been paired in memory with the experience of alcohol consumption, independent of any confounding factors. Further, the actual test phase occurs 24 hours after the alcohol exposure, when the flies are fully sober. For these reasons, this type of odor-paired conditioning assay is a much better indicator of whether an experience of substance is objectively rewarding for a fly.

Here, the researchers put forth their theory for the mechanism driving all of the effects they have observed so far. This mechanism is based on the concept of homeostasis, which basically states that there is an "ideal state" for one's internal physiological make-up. Any time the body deviates from the ideal state, it will attempt to correct itself and return to this state. The researchers thus theorize that the amount of NPF in the brain is a marker for the "reward state."

If the fly mates repeatedly, it experiences something highly rewarding, leading to an increase in NPF beyond "normal" levels. The fly's body thus wants to return to a normal level of NPF. To do this, it must forgo alternate rewards such as alcohol. This would explain why mated flies do not have a preference for alcohol. Sexually deprived flies, on the other hand, experience a lack of reward, which leads to a decrease in NPF beyond normal levels. To compensate for this, when these flies are exposed to a second possible reward in the form of alcohol, they consume it excessively. This would explain why sexually deprived, virgin flies show an alcohol preference.

Overall, the researchers showed that when they artificially decreased NPF activity, flies preferred alcohol more, and when they artificially increased NPF activity, flies preferred alcohol less. Thus, they were able to establish a causal relationship between NPF and alcohol. Since the amount of NPF in a fly's brain is dependent on sexual history, this would explain their initial finding that sexually deprived flies prefer alcohol more than mated flies do.

In order to further confirm this causal relationship, the authors used the protein dTRPA1 (which activates specific neurons at a high temperature). Specifically, they inserted dTRPA1 into all of the same neurons that also contained NPF. Therefore, at a low temperature there would be no effect, but at a high temperature all of the neurons related to NPF would be activated. If the relationship between NPF and alcohol preference were indeed causal, then artificially increasing the activity of NPF-related neurons should reduce preference for alcohol.

An interfering RNA, also known as RNAi, is a molecule that can be tailored to specifically block the expression of a gene. Here, the authors used an NPFR-specific RNAi to prevent the cells from making NPF receptors.Without the receptors, NPF has no effect on the fly's brain.

Something that can have an altering effect on a particular phenomenon or behavior. For example, sleep might be considered a mediator of attentiveness in class, because the more soundly you sleep, the more likely you are to stay alert in school (and vice versa). Here, the researchers discuss the fact that the concentration of neuropeptide F in the brain has been known to change how organisms respond to sex.

Consistent with earlier results, the researchers found that the rejected subgroup maintained their preference for alcohol. On the other hand, the initially rejected but then mated subgroup demonstrated no alcohol preference. From this, the researchers concluded that it was the experience of sexual deprivation that was causing the rejected males to develop a preference for alcohol.

To test the possibility that cis-vaccenyl acetate (cVA) was the driving factor behind the alcohol preference amongst rejected males, the researchers conducted a follow up experiment in which one group of males was exposed to mated females, and the other was exposed to dead virgin females. Both of these groups thus experienced sexual rejection. However, the group exposed to the dead virgin females would not be exposed to the cVA cue, whereas the group exposed to mated females would. If there were a difference in alcohol preference between these two cohorts of male flies, then one could conclude that the preference for alcohol was not caused by sexual deprivation specifically, but instead due to exposure to cVA.

A calculated score, from –1 to 1, that indicates how much the flies preferred the alcohol-spiked food relative to the regular food. A number above zero means the flies ate more food with alcohol in it, and a number below zero means they ate more regular, nonalcoholic food.

Drosophila are typically fed a jellylike substance in the lab consisting largely of sugar. In this experiment, the flies were given a choice between just a regular tube of food or a tube that contained the food mixed with alcohol.

A behavioral model in which an organism is offered a choice between two alternatives. Typically, the organism is allowed to freely choose amongst these alternatives, and its choices are used to define whether or not the organism has a preference for one alternative over another.

Not enjoyable to eat, most often due to a bitter taste.

Fruit flies have taste receptors just like humans, but they aren't just limited to the tongue! Taste receptors in fruit flies are mounted all over the body, including leg bristles and wings.

Read more from University of California, Berkeley: https://www.berkeley.edu/news/media/releases/2004/06/25_flies.shtml

The authors will argue in this paper that they have discovered an explanation for how addictive substances/experiences can affect a fly's behavior and neurobiology. Understanding the effect of reward on an organism is important because it can lead to a better understanding of addiction (which can be seen as an abnormal response to a rewarding substance).

The rewards the authors use here are mating (sex) and ethanol (alcohol). The authors cite previous research to show that flies find alcohol to be rewarding and that they demonstrate addictionlike behaviors in pursuit of alcohol. These distinctions are important to demonstrate the similarities between fly and human responses to alcohol. They then build upon this previous research for their own experiments.

How do animals react to alcohol? Do they experience the same feeling of drunkenness that humans do?

Read more in National Geographic: https://news.nationalgeographic.com/2015/11/151121-animals-science-drunk-insects-mammals-drinking/

Neuropeptide F (NPF) is a specific type of neuropeptide found in flies. Human beings produce a similar type of neuropeptide called neuropeptide Y. NPF is believed to play an important role in regulating reward-seeking behaviors.

Neurons in the brain can communicate with one another in one of two ways—electrically or chemically. Neuropeptides are proteinlike chemical substances that a neuron is capable of secreting in order to initiate a somewhat long-lasting chemical communication with its neighboring neurons.

In all neurons.

When the amount of NPF receptor present in the brains of baby fruit flies (still in the larval stage) is artificially increased, these flies become more likely to consume noxious—that is, bitter or unpleasant—food.

In order to confirm that NPF was causally related to the observed changes in alcohol preference, the researchers manually reduced the amount of NPF receptor the flies could produce in all neurons. They did this by using an RNAi, which can specifically prevent the formation of molecules coded by genes. Since the researchers had previously shown that mated flies have higher levels of NPF than virgin flies, and that this was correlated with a reduced preference for alcohol, they hypothesized that if they manually inhibited NPF's ability to affect the fly, then mated flies would show an increase in their preference for alcohol.

The researchers found that genetically reducing the number of NPF receptors in the brain for mated flies (who would have had elevated levels of the NPF molecule) caused these flies to develop a preference for alcohol. On the other hand, virgin flies (who would have had average levels of NPF molecules in their brains) were unaffected by this. This points to a causal relationship between the function of NPF and its receptor, and preference for alcohol.

The authors of this paper have made good on this assertion. In a follow-up study published recently (2018)30368-3), researchers have shown that the mere act of ejaculation, even if triggered artificially in the lab, can affect how much male flies drink. This more recent research is summarized in the following article: https://www.sciencenews.org/article/male-fruit-flies-enjoy-ejaculation

We know that memories associated with food (for example where food is located, how good/bad the food is, etc.) are modulated by hunger levels. Typically, flies that are hungrier tend to form more robust food-associated memories, and vice versa. In the study referenced here, the researchers showed that a similar behavior can be elicited using NPF—artificially activating NPF neurons promotes better formation of food-associated memories.

When the NPF circuit in Drosophila is artificially activated, flies do not seem bothered by stressful things in the environment that under normal conditions would have provoked some sort of avoidance behavior.

Extremely young Drosophila spend the majority of their time eating, whereas older larvae start to engage in behaviors such as moving around and cooperating with fellow larvae. This change in behavior from young to old larvae is correlated with a reduction in the amount of NPF in the brain, suggesting that this molecule regulates feeding behaviors in the fly.

Chemicals with distinct smells.

This sentence describes how the researchers used the conditioning assay to test whether mating is rewarding. The two odors they used came from the chemicals ethyl acetate (EA) and isoamyl alcohol (IAA). The specifics of these two chemicals are not important—any two clearly distinct smelling chemicals could have been used. Male flies were exposed to one of the two odors in the presence of sexually receptive females (who the males presumably mated with), and were exposed to the remaining odor in the absence of females. They then tested to see whether males preferred the females/mating-associated odor over the other neutral odor.

An experimental method used to determine if and how much an animal enjoys one thing over another. The concept behind this is similar to a two-choice preference assay (described earlier). In this particular experiment, the researchers used a Y maze.

As shown in the diagram, the animal starts in one prong of the maze (the picture here depicts a rat, but the researchers in the current study used flies). The other two prongs of the maze are each loaded with one of the two options being presented to the animal. This could be a food reward, a physical object, an odor, etc. Whichever of the two prongs the animal opts to spend the most time in is considered its preferred choice.

Here, the researchers are aiming to verify their theory about how mating, ethanol, and activity of the NPF circuit are connected (for more on this, check out the previous paragraph) by proving that each of these three things are inherently rewarding.

An example of how they could do this would be to repeatedly expose the flies to alcohol while simultaneously also exposing them to a particular odor cue—let's call it odor A. After repeated exposure to alcohol and odor A in conjunction, the flies will learn that if they smell odor A, alcohol must be near.

Once this association has formed, the researchers could put the flies in a Y maze, with one prong containing odor A, and the other prong containing a random odor—say odor B. Since odor A predicts alcohol, if the flies spend the majority of their time in the prong containing odor A, it would indicate that they are seeking out alcohol, presumably because it is rewarding.

This type of manipulation is exactly what the researchers did here, except that they also investigated mating and manual activation of the NPF circuit (in addition to alcohol) as possible rewarding elements.

The researchers found that when they manually activated NPF-related neurons (recall that when using dTRPA1, this occurs at a higher temperature), preference for alcohol was significantly diminished, even amongst virgin flies.

A genetic manipulation wherein the expression of a gene is reduced. In this case, the authors want to experimentally reduce the amount of NPF or NPF receptors (NPFR) that the flies are producing in order to inhibit the function of the NPF molecule. This is accomplished by down-regulating the genes that cause NPF or NPFR to be made.

Think of a gene as a radio with a volume knob. The cell is always making the molecules that the gene dictates (aka playing music on the radio), but a scientist can turn the knob to reduce the amount of the molecule that is produced (turn down the volume).

A technique for detecting the presence of particular substance in cells. Typically, this is done by taking the cells of interest and pouring a specialized, colored dye over them. This dye is selectively attracted to the substance that you are interested in—wherever the substance is present, the dye will stick. If the molecule you are searching for is not present, the dye will simply wash off. This technique can therefore be used to detect where in a group of cells a molecule might be present, as well as how much of that molecule is present in that location. In this experiment, the researchers poured a dye that sticks to NPF over sections of the flies' brains in order to detect how much NPF was present.

An experimental method for causing rodents to experience stress. Specifically, restraint stress involves placing a mouse in a small plastic tube. This tube is tight enough to prevent the mouse from moving too much. This inability to move causes the mice to experience intense discomfort and stress.

A species of worm that is often used to study the effects of various genes. C. elegans are used because all of their genes are known, they are relatively easy to genetically manipulate, and they have a number of genetic similarities to human beings and other mammals.

Researchers have found a number of genes in Drosophila flies that are remarkably similar in composition to genes that are found in people. Often, these highly similar genes between the two species serve overlapping functions as well. Such similar (but not completely identical) genes that exist across species are referred to as homologs of one another. As an analogy, if you think of an iPhone as a gene, then an iPhone 6 and an iPhone 8 might be considered homologs of one another in that they are extremely similar in a number of ways, but not quite identical.

The outermost layer of the Drosophila's body. Often, chemical cues used for communication between flies are found on the cuticle layer.

The researchers found that even when the male virgin flies were housed in large groups, their preference for alcohol persisted, and that mated flies with similar housing conditions did not prefer alcohol. This indicates that social isolation is not the reason for the observed alcohol preference in virgin flies.

The group of male flies that were exposed only to females who had already mated and were thus no longer sexually receptive. These male flies experience sexual rejection.

Unpleasant, often used to describe stimuli in experiments.

Another word for sex.

Here is a video showing the distinct experiences of the males in the two groups. In the first portion of the video, you will see a male successfully mate with a receptive female. You will then see a male unsuccessfully attempt to mate with an unreceptive female.

https://www.youtube.com/watch?v=sH9Xjk28cZ0

Female flies, who have never mated before, that accept the sexual advances of males, and will often mate with them. For this reason, they are considered "receptive" to the act of mating.

Refers to a type of commonly employed behavioral model in Drosophila research. In this model, a male is exposed to a female who has already mated with a different fly. The male Drosophila will attempt to court the female by carrying out a series of behaviors such as tapping her with his forelegs, or singing her a song. However, since the female has already mated, she will not be receptive to these attempts, and the male fly will experience sexual rejection. This leads to an overall reduction in the male's courtship attempts.

Fruit flies are a good model organism for this kind of research; however, there is a danger of applying these findings too heavily to human behavior which is much more complex.

Read more: https://mappingignorance.org/2016/11/23/sex-alcohol-flies/

In behavioral neuroscience, cues refer to elements in one's environment that predict an upcoming event. For example, the ringing of the school bell at noon might be considered a cue for lunch. Cues are important because they often take on the ability to motivate organisms to do things, even though cues themselves have no inherent value.

Something that is tractable is easy to control or influence. The genetics of the Drosophila fruit fly are considered tractable because we are aware of every single gene in this species, and it is easy to engineer flies that possess or lack specific genes. This ability to insert or remove specific genes from a fly's DNA allows scientists to understand the roles that these genes play.

In neuropsychology, addictive behaviors are a specific class of behaviors that one might exhibit while suffering from addiction. An example of an addictive behavior would be compulsive pursuit of a specific substance/activity, in spite of negative consequences associated with doing so.

Refers to a set of interconnected brain areas that are involved in the appraisal and pursuit of reward. Examples of such areas include the ventral tegmental area and the nucleus accumbens. These brain regions rely heavily on the use of a chemical called dopamine to communicate with one another. Source of image: http://thebrain.mcgill.ca/flash/d/d_03/d_03_cr/d_03_cr_que/d_03_cr_que.html

The common chemical name for alcohol. In a beverage such as wine, for instance, the sugar in the grapes gets converted into ethanol during the fermentation process. This is what makes wine alcoholic.

Refers to a set of neurons in the brain that work together towards some common goal. This goal can be very tangible, such as regulating a certain behavior. Often, these cells are connected to one another in a way that allows them to communicate, somewhat similar to a circuit of wires that one may expect to find in an electronic appliance.

The scientific name for the common fruit fly. These flies are often used in neuroscience research because their nervous systems are relatively simple, and they share a number of genes in common with humans.

A term for drugs that can become addictive and lead people to use them improperly due to their rewarding properties. This includes illegal drugs such as cocaine, but also legal ones such as Oxycontin.

When NPF cells are activated in tandem with the ethanol vapour and odor exposure, the preference for the ethanol paired odor (that develops for controls) is completely absent. This indicates that the activity of the NPF cells, which the previous experiment showed was inherently rewarding, interferes with the experience of the ethanol.

This paper established the odor-ethanol pairing method for probing motivation and rewarding properties that is used in the current study. It is by the same authors.

First paper to establish the relationship between NPY in mice and ethanol consumption/sensitivity.

The first paper to establish the two-choice preference assay for flies using food, including a method to precisely measure how much food was being consumed at any given time.

A review paper highlighting various past studies that have shown that it is possible to establish direct causal links between genetics and behavior.

A landmark review paper outlining how and why addiction occurs. This paper has been cited over 750 times.

A BBC article briefly summarizing the findings from this study:

https://www.bbc.com/news/science-environment-17357560

The researchers finally conclude that their findings are consistent with past research on NPY and NPF. Moreover, this research introduces a new dimension to our understanding of this molecule by demonstrating it to be a common substrate regulating both sex and alcohol consumption in complementary ways.

Injection of NPY can have either sleep or wakefulness promoting effects on a mouse, depending on where exactly in the brain the molecule is injected, as well as context.

Similar to work done in flies on the role of NPF in regulating responses to physical stressors (see previous paragraph), studies in mice have also shown that when the activity of NPY is increased, stress responses are decreased. The vice versa has also been observed to be true.

When mice experience fasting, neurons in their brain containing NPY become more active, promoting feeding behavior.

Females who had already mated with a different group of male flies.

From this, the researchers conclude that the NPF system (circuit activity and amount of the molecule present) is affected by and reflective of rewarding experiences, including but not limited to mating and consumption of alcohol.

The researchers found that shortly after exposure to alcohol, NPF levels were indeed elevated, but that these levels eventually returned back to normal by the next day. Exposure to air, on the other hand, had no impact whatsoever on the amount of NPF in the brain.

The final thing the researchers wanted to test was whether artificial activation of the NPF circuit was inherently rewarding. To do this, they once again relied on the dTRPA1 proteins, which can activate neurons at high temperatures. The researchers inserted this protein into neurons that were part of the NPF circuit. They then exposed the flies to one smell while keeping a high temperature (thus activating the NPF neurons). This smell became associated with a state of high NPF circuit activity. Conversely, they exposed the flies to the second smell while keeping a low temperature. This smell became associated with a state of normal/low NPF circuit activity. Finally, they placed the flies in a Y maze (as before) and checked to see if the flies would prefer the prong containing the NPF-activity paired odor, or the unpaired odor.

The researchers found that flies preferred the odor that was associated with mating, as well as with exposure to alcohol. This indicates that both of these things are "rewarding" experiences to the fly.

The researchers used time spent in the two prongs of the Y maze to develop a preference score. Per this score, a positive value would indicate that the flies preferred the odor that was associated with one of the three activities (mating, alcohol exposure, or activation of the NPF circuit). Conversely, a negative score would indicate that the flies preferred the odor that was not associated with these activities. A score of zero would indicate no obvious preference between the two.

In neuroscience, a pathway or a circuit refers to a set of neurons that function together to accomplish the same function. Often, these neurons all release the same molecules. Here, the researchers draw our attention to sets of neurons releasing NPF, referring to this as the NPF-NPFR pathway.

So far, the researchers have shown that changes in NPF levels correlate with both sexual experience and alcohol preference. Specifically, a more active sexual history is correlated with an increase in NPF levels, which is correlated with a decrease in preference for alcohol (and vice versa). However, one of the most important rules to remember in science is that correlation does not equal causation. The researchers thus decided to follow up with a direct genetic manipulation of NPF in order to establish a causal relationship.

There are a number of steps that must occur in order for neuropeptide F to be made in the brain. The first step is for the gene for NPF to be read by the neuron. As the neuron reads this gene, it makes a local copy of it known as a transcript. These transcripts are then recruited by other processes in the cell that use the transcript as an instruction manual about how to build the NPF molecule. Each time a new NPF molecule is made, a new NPF transcript must be created first. For this reason, it is possible to measure the amount of NPF transcripts in the brain in order to determine how much NPF was being produced.

Another experimental method for causing rodents to experience stress in which newly born offspring are taken away from their mothers and raised separately. The lack of a mother present to raise the babies causes them to experience intense stress.

As a final follow-up experiment, the researchers took a group of sexually rejected males and split them up into two. One subgroup was left alone, and then tested for alcohol preference. The other subgroup was exposed to virgin females just before testing. As such, these males did in fact get an opportunity to mate before undergoing the alcohol preference test. If the first subgroup continued to prefer alcohol, but this preference were to disappear for the second group (who initially experienced rejection, but then were able to mate just before testing), it would indicate that the main cause for the alcohol preference was indeed sexual deprivation.

The researchers found that both males exposed to mated females (and by extension cVA) as well as males exposed to decapitated virgin females (who lacked cVA) demonstrated a preference for alcohol. To confirm this finding, they also simply exposed a third group of males to the chemical by itself without a female present before testing their preference for alcohol. This third group too maintained a preference for alcohol. Based on this, they concluded that cVA could not be the driving cause for the observed alcohol preference, and that the effect was likely due to the actual sexual deprivation involved.

The researchers found that even these males who did not experience the social aspect of rejection by a female maintained a preference for alcohol. From this, they concluded that social rejection was not the main cause for the observed alcohol preference amongst virgin male flies.

For the second follow-up experiment, the researchers wanted to investigate the possibility that it was not actually the lack of sex, but rather the experience of rejection by a female that was driving the virgin males to prefer alcohol.

Flies do not have language, but they do communicate with one another in other ways. One such way a fly may communicate is by releasing chemical compounds that other flies can sense, and accordingly respond to. These are known as chemosensory cues. In this case, the authors are referring to chemicals released by the mated female that signal to the male that she is no longer receptive to mating.