BRAINS IN BRIEFS
Scroll down to see new briefs about recent scientific publications by neuroscience graduate students at the University of Pennsylvania. Or search for your interests by key terms below (i.e. sleep, Alzheimer’s, autism).
Why some people wake up under anesthesia and others don’t (Hint: It’s your Hormones)
or technically,
Hormonal basis of sex differences in anesthetic sensitivity
[See original abstract on Pubmed]
Dr. Andrzej (Andi) Wasilczuk was the lead author on this study. A recent graduate from Penn Bioengineering, Andi is captivated by the brain’s intricate ability to shift between states of consciousness. His research uses general anesthetics to uncover the neuronal circuits responsible for these shifts, aiming to understand how the brain sustains or disrupts consciousness. By identifying these critical networks, Andi is paving the way for personalized anesthesia and offering new insights into arousal state transitions.
or technically,
Hormonal basis of sex differences in anesthetic sensitivity
[See Original Abstract on Pubmed]
Authors of the study: Andrzej Z Wasilczuk, Cole Rinehart, Adeeti Aggarwal, Martha E Stone, George A Mashour, Michael S Avidan, Max B Kelz, Alex Proekt, ReCCognition Study Group
Waking up during surgery sounds like a nightmare, and did you know that females might be at higher risk for this than males? Through medication, general anesthesia makes a patient unconscious, which allows doctors to perform surgical procedures without the patient’s awareness or discomfort. General anesthesia puts the patient in a sleep-like state, and doing this is an involved process. Anesthesiologists must be highly trained to determine the best course of treatment. When creating a safe treatment plan, anesthesiologists take into account many factors, such as the patient’s body weight or pre-existing conditions. The sex of the patient, however, hasn’t been historically considered as an equally important factor in delivering a safe course of anesthesia.
Previous research about the link between sex and response to anesthesia was ambiguous and conflicting. Some early clinical trials suggested that females were more likely to wake up under anesthesia, while others found no significant difference between males and females. These clinical trials, however, had diverse patient populations and non-standardized anesthetic protocols, which would make it hard to directly compare anesthetic conditions between patients. Nevertheless, more recently, an analysis done on many of these studies has provided clear cut evidence that females are more resistant to the anesthetic state (Braithwaite et al., 2023). The question of how this sex difference arose, however, remained unanswered.
Dr. Andi Wasilczuk, a former Penn Bioengineering PhD student, and his team wanted to understand why females and males responded differently to anesthesia. To do this, they decided to focus on the hypothalamus, a structure in the brain heavily involved in both sleep-wake and anesthetic induced unconsciousness. The hypothalamus is regulated by hormones, which are the body’s chemical messengers—and the researchers knew that the levels of hormones typically differ between males and females. For example, males typically have a much higher level of the hormone testosterone, whereas females typically have higher levels of the hormone estrogen.
With these differences in mind, Dr. Wasilczuk wanted to know: Could hormonal differences across sexes alter the effectiveness of general anesthesia? He framed "effectiveness of general anesthesia" by using the idea of “anesthetic sensitivity.” Individuals who are more sensitive to anesthesia need less of the drug to fall and stay unconscious, and wake up smoothly after surgery. On the contrary, individuals with less anesthetic sensitivity, or have anesthetic resistance, require more anesthetic to fall and stay asleep, and wake up sooner once the anesthetic is removed.
Recognizing this gap in the research, Dr. Wasilczuk’s research group sought to test the influence of sex and sex hormones on anesthetic sensitivity in mice. First, the researchers compared the dosage of anesthetic required for the mice to be initially anesthetized (induction), and to wake up from anesthesia (emergence). They found that, across all four anesthetics the group tested, female mice required a much higher dose on induction, and were more likely to emerge at higher doses than males. Next, the researchers compared the time, given the same dosage, for female versus male mice to be induced and emerge from anesthesia. Female mice took significantly longer to be induced than males, and also were much quicker to emerge. These experiments indicated that female mice were indeed more resistant to anesthesia, compared to male mice.
Yet, the reason for these results remained unclear: Were these effects due to sex hormone differences? To find out, the researchers changed the mice's hormone levels by surgical removal of the testicles (castration) in male mice or ovaries (oophorectomy) in female mice post puberty. They repeated the experiments, this time using castrated males and oophorectomy females, then compared these mice to the untreated males and females tested before.
The results were striking. In both experiments, castrated males and oophorectomized females showed a similar resistance to anesthesia as untreated females. Oophorectomy did not change a female mouse’s anesthetic sensitivity. Castration, however, produced a female-like anesthetic sensitivity in males. Eliminating male sex hormones, therefore, seemed to remove the sex differences in response to anesthesia!
The researchers also directly measured the effect of testosterone. Under a steady dose of anesthetic, untreated males and castrated males were injected with testosterone, and continually tested for responsiveness using the righting reflex. Testosterone administration increased anesthetic sensitivity for both groups of mice in a dose-dependent manner. This finding could explain why males, who typically have higher testosterone, are more sensitive to general anesthetics, and therefore are at lower risk of waking up under anesthesia than females.
Intrigued, the researchers wondered: Can these sex differences be seen in brain activity? The conventional measure of anesthetic depth (how unconscious someone is in response to anesthesia) during surgery is the Electroencephalogram (EEG). EEG measures electrical brain activity through electrodes attached to the scalp. The researchers found that sex differences were not reflected in the EEG of the mice they tested. Similar conclusions were made when re-analyzing human data from another study. In this study, female volunteers displayed resistance to general anesthesia based on assessments of behavior and cognitive function, but not based on information gathered from the EEG.
Looking at the activity of individual neurons, however, clearly revealed sex differences. They looked for elevated levels of the protein c-Fos, an indicator of neuronal activity, throughout the whole brain. Compared to anesthetized male mice, anesthetized female mice had fewer neurons expressing c-Fos in sleep-promoting hypothalamic cells. In other words, anesthesia activates fewer sleep-promoting circuits in females than males, correlating with females’ greater resistance to anesthetics.
Compared to untreated male mice, castrated male mice also had reduced c-Fos expression in similar hypothalamic structures. Fewer sleep-promoting circuits were activated in castrated males (which displayed a similar aesthetic sensitivity to females) than untreated males. Thus, sex-dependent activity patterns, seen in hypothalamic structures, reflected anesthetic sensitivity trends!
Dr. Wasilczuk’s groundbreaking paper reveals why researching sex-dependence is incredibly important: females may need different anesthetic management than males due to their higher resistance to anesthesia. After years of standard general anesthesia administration to millions of patients, and using EEGs to measure anesthetic depth, Dr. Wasilczuk’s findings have huge clinical implications supporting personalized anesthetic care.
About the brief writer: Sydney Liu
Sydney is a guest writer for Brains in Briefs! She is a Penn undergraduate in Dr. Shinjae Chung’s lab researching what makes us sleep and the brain transitions between sleep states. She is a Junior majoring in neuroscience, and is interested in teaching. In her free time, she likes to draw!
Citations:
E. Braithwaite et al., Impact of female sex on anaesthetic awareness, depth, and emergence: A systematic review and meta-analysis. Br. J. Anaesth. 131, 510–522 (2023).
Interested in learning more about how anesthetic sensitivity is different in males and females? Check out Andi’s paper here!
Sex matters: Exploring sex differences in opioid withdrawal mechanisms
or technically,
Sex differences in VTA GABA transmission and plasticity during opioid withdrawal
[See original abstract on Pubmed]
Dan Kalamarides was the lead author on this study. Dan’s research interests are rooted in neuropsychopharmacology, that is, the intersection of brain physiology, drugs (both “good” and “bad”), and behavior. This interest has been applied in the context of preclinical models for several therapeutic areas including substance use disorders, pain, neuroinflammation, and depression. Dan is currently planning to transition to industry where he can leverage his neuroscience expertise in the pharmaceutical world to enhance treatment strategies for mental health disorders and brain diseases.
or technically,
Sex differences in VTA GABA transmission and plasticity during opioid withdrawal
[See Original Abstract on Pubmed]
Authors of the study: Daniel Kalamarides, Aditi Singh, Shannon Wolfmann, John Dani
Scientific research has long been biased on the basis of sex. From cells and tissues to animals and people, there is a long history of scientists including more male subjects in their studies. As a result, we don’t understand how female bodies respond differently to diseases or to treatments, and the quality of healthcare has suffered. The National Institutes of Health (NIH) and several scientific journals have started requiring researchers to consider sex in their science, but the progress towards equal representation of males and females has been slow.
Opioids - including heroin, fentanyl, morphine, and others - are one of many classes of drugs that affect men and women differently. For example, women are less responsive to the pain killing effects of opioids but more sensitive to affects the drugs have on respiration compared to men. This difference makes it a lot harder to safely and effectively treat women with opioids in the clinical setting, and it can make recreational opioid use more dangerous. Despite these differences in people, basic science research into the effects and mechanisms of opioids in females is still lacking compared to our understanding of the drugs in males.
One area of research on opioids that still has a lot of unanswered questions, related to sex differences and more generally, is opioid withdrawal. Scientists, including recent NGG graduate Daniel Kalamarides, want to better understand opioid withdrawal so that they can treat the withdrawal, help people feel better, and make it easier for people to stop using opioids. In his paper, Daniel and his fellow researchers wanted to learn more about how the brain changes during opioid withdrawal, while keeping in mind that these changes could look different in males and females. Specifically, he was curious about a brain region called the ventral tegmental area (VTA), which contains neurons responsible for releasing dopamine into another brain region (the striatum) involved in reward.
Previous studies have shown that the active effects of opioids (think the “high”) are in part caused by an increase in dopamine release from those neurons in the VTA. This happens because opioids remove a natural brake on the dopamine system. In an opioid-free brain, other inhibitory neurons in the VTA – known as GABAergic neurons because they release the neurotransmitter GABA – decrease the release of dopamine from the dopaminergic neurons. Opioids remove this brake by decreasing the activity of the inhibitory neurons. This makes the system go faster, or, more specifically, release more dopamine.
Your brain adapts if opioids are in the body for an extended period of time. In the VTA, this means that those inhibitory neurons amp up their control of the dopamine-releasing neurons so that, even in the presence of an opioid, a relatively normal amount of dopamine is released. This is fine until the opioids are removed. Now you have an overactive brake, and there’s not enough dopamine released into the reward-related brain regions.
Researchers have found, in male mice only, that the inhibitory neuron control of the dopamine-releasing neurons increases in withdrawal because the connections between them grow stronger. This increase in connectivity is known as long term potentiation (LTP) or plasticity, and it’s one of the primary mechanisms by which the brain changes depending on how it’s used and what it’s exposed to. Knowing that the effects of opioids can differ between males and females, Daniel explored whether a similar phenomenon occurs in female mice.
Daniel first induced opioid withdrawal in mice by giving them morphine for a week, then studied the properties of neurons in the VTA when the mice were in withdrawal. He used patch-clamp electrophysiology, a technique which allowed him to measure the electrical current flowing into or out of the neuron as he manipulated the voltage. By using this technique, he was able to learn about the strength of the connection between the inhibitory neurons and the dopaminergic neurons and compare that connection between male and female mice.
Daniel measured how likely the inhibitory neurons were to release GABA – and thus inhibit the dopamine-releasing neurons – spontaneously and when electrically stimulated. He found that, in male mice, morphine withdrawal increased the probability of GABA release (or increased the strength of that brake). This was a great result because previous studies had also found this phenomenon, which means that this science is replicable. When he looked at female mice, however, he didn’t see any difference between the morphine treated mice and the control mice. That’s a surprise!
Daniel also tried to experimentally force LTP to occur in the brains in morphine withdrawal so that he could learn more about how the probability of GABA release was changing. He stimulated the inhibitory neurons with a really high frequency of electrical current, which would cause LTP in a normal neuron. He found that he could cause plasticity in the female mice, but he couldn’t in the males. This result suggested that the increase in the probability of GABA release in males was due to LTP. The molecular components needed for LTP were all used up in the males, so Daniel couldn’t create more. The components were still available in the females, on the other hand, so Daniel was able to stimulate the neurons and cause LTP.
To be thorough, Daniel also asked if the male and female mice were experiencing a similar level of morphine withdrawal. If the female mice were going through less withdrawal, it could maybe explain the sex differences in plasticity in the VTA. Daniel measured the strength of withdrawal that the mice were experiencing by counting physical signs of morphine withdrawal, and he found that males and females displayed a similar number. After all of these experiments, we still don’t know for sure that the opioid withdrawal mechanisms in male and female mice are entirely different. If Daniel used a different dose of morphine or if he studied the brains at a different time into withdrawal, he might be able to observe the same plasticity in female mice that he saw in male mice. However, by running this control experiment, he was able to strengthen the argument that there is a true difference in how the male and female mouse brains changed in opioid withdrawal.
This research by Daniel and his fellow scientists reinforced the fact that opioids affect males and females differently, and they showed that we still don’t understand how female brains change in opioid withdrawal. Hopefully, this evidence will push other scientists to continue thinking about sex differences in opioid research and in neuroscience broadly. In the meantime, Daniel has led us a step closer towards developing treatments for opioid use disorder, and he’s contributed to reducing bias in science.
About the brief writer: Lyndsay Hastings
Lyndsay is a first year NGG PhD student broadly interested in the relationship between neurocircuitry and behavior.
Interested in learning more about how opioid withdrawal is different in males and females? Check out Daniel’s paper here!
A mouse model for autism and ADHD can mimic sex differences in sleep
or technically,
Hyperactivity and male-specific sleep deficits in the 16p11.2 deletion mouse model of autism.
[See Original Abstract on Pubmed]
or technically,
Hyperactivity and male-specific sleep deficits in the 16p11.2 deletion mouse model of autism.
[See Original Abstract on Pubmed]
Authors of the study: Angelakos CC, Watson AJ, O'Brien WT, Krainock KS, Nickl-Jockschat T, Abel T.
Patients with disorders like ASD/ADHD often have changes in the number of copies they have for a geneA unit of DNA that encodes a protein and tells a cell how to function. Typically, for each geneA unit of DNA that encodes a protein and tells a cell how to function there are two copies - one from each parent. Therefore, individuals with ASD/ADHD can have more copies, or fewer copies (also known as a deletion). One of these changes is a deletion in chromosomal region 16p11.2. People that have a deletion in this region are more likely to have ASD and ADHD. Previous research has shown that mice with a deletion in the 16p11.2 region show symptoms similar to ASD/ADHD like differences in brainThe brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. structure, cognitive ability, and communication. However, sleep problems remained largely unexplored, a problem that Christopher wanted to address.
Christopher observed that these animals were hyperactive, a behavior that is observed in individuals diagnosed with ADHD. He tracked all of the movements of the mice in their cages, observing an increase in activity in the 16p11.2 deletion mice throughout the day, and a robust increase during the dark (active) phase of their cycle. This led him to think that something may be altered in their circadian rhythms. To investigate this he monitored them for 24hrs and measured their sleep and activity to determine if it was normal.
He also examined their sleep cycles using polysomnography, which tracks brainThe brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. waves, eye movements, and limb movement during sleep. He wanted to know whether this was a problem of initiating sleep or maintaining sleep. He found that once the animals were asleep, they usually remained asleep for the same amount of time indicating that there was not a problem of staying asleep. On the other hand, once an animal was awake, it was usually awake for a longer period of time, indicating that it may have had trouble with initiating sleep. When Christopher further analyzed the data, he saw male mice with the 16p11.2 deletion spent a longer amount of time awake than regular mice. Coupled with his finding that these mice stay asleep as long as the regular mice, this suggests that they had a hard time falling asleep, rather than that they were waking up multiple times and having brief amounts of wakefulness. Interestingly, these disorders are more commonly found in males rather than females. Males are four times more likely to be diagnosed with ASD and three times more likely to be diagnosed with ADHD.
Issues with sleep in people that are diagnosed with autism or ADHD is a problem that needs to be addressed. Christopher asked whether we can use a mouse to model sleep problems in autism? He showed in his paper that the 16p11.2 deletion mouse can model sleep disturbances that are seen in humans. He is excited to see future work using this mouse model to uncover specific brainThe brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. circuits that may be involved and better treatment for sleep problems. Now that we have this experimental mode, we can determine if improving sleep quality will improve other psychiatric symptoms.
About the brief writer: Felicia Davatolhagh
Felicia is a PhD Candidate in Marc Fuccillo’s lab. She is a seventh year studying the impact of neuropsychiatric disease on synaptic connectivity and synaptic function.