Highly sensitive in vivo detection of dynamic changes in enkephalins following acute stress.

Enkephalins are opioid peptides that modulate analgesia, reward, and stress. In vivo detection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. To begin to address this we previously developed a system combining in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents (Al-Hasani, 2018, eLife). Here not only do we show improved detection resolution but also a critical discovery in the stabilization of enkephalin detection, which together allowed us to investigate enkephalin release during acute stress. We present an analytical method for Met- and Leu-Enkephalin (Met-Enk & Leu-Enk) detection in the mouse Nucleus Accumbens shell (NAcSh) after acute stress. We confirm that acute stress activates enkephalinergic neurons in the NAcSh using fiber photometry and that this leads to the release of Met- and Leu-Enk. We also demonstrate the dynamics of Met- and Leu-Enk release as well as how they correlate to one another in the ventral NAc shell, which was previously difficult due to the use of approaches that relied on mRNA transcript levels rather than post-translational products. This approach increases spatiotemporal resolution, optimizes the detection of Met-Enkephalin through methionine oxidation, and provides novel insight into the relationship between Met- and Leu-Enkephalin following stress.

An electrochemical approach for rapid, sensitive, and selective detection of dynorphin.

The endogenous opioid peptide systems are critical for analgesia, reward processing, and affect, but research on their release dynamics and function has been challenging. Here, we have developed microimmunoelectrodes (MIEs) for the electrochemical detection of opioid peptides using square-wave voltammetry. Briefly, a voltage is applied to the electrode to cause oxidation of the tyrosine residue on the opioid peptide of interest, which is detected as current. To provide selectivity to these voltammetric measurements, the carbon fiber surface of the MIE is coated with an antiserum selective to the opioid peptide of interest. To test the sensitivity of the MIEs, electrodes are immersed in solutions containing different concentrations of opioid peptides, and peak oxidative current is measured. We show that dynorphin antiserum-coated electrodes are sensitive to increasing concentrations of dynorphin in the attomolar range. To confirm selectivity, we also measured the oxidative current from exposure to tyrosine and other opioid peptides in solution. Our data show that dynorphin antiserum-coated MIEs are sensitive and selective for dynorphin with little to no oxidative current observed in met-enkephalin and tyrosine solutions. Additionally, we demonstrate the utility of these MIEs in an in vitro brain slice preparation using bath application of dynorphin as well as optogenetic activation of dynorphin release. Future work aims to use MIEs in vivo for real-time, rapid detection of endogenous opioid peptide release in awake, behaving animals.

5-HT(2A) receptor blockade and 5-HT(2C) receptor activation interact to reduce cocaine hyperlocomotion and Fos protein expression in the caudate-putamen.

Both the 5-HT(2A) receptor (R) antagonist M100907 and the 5-HT(2C) R agonist MK212 attenuate cocaine-induced dopamine release and hyperlocomotion. This study examined whether these drugs interact to reduce cocaine hyperlocomotion and Fos expression in the striatum and prefrontal cortex. We first determined from dose-effect functions a low dose of both M100907 and MK212 that failed to alter cocaine (15 mg/kg, i.p.) hyperlocomotion. Subsequently, we examined whether these subthreshold doses given together would attenuate cocaine hyperlocomotion, consistent with a 5-HT(2A)/5-HT(2C) R interaction. Separate groups of rats received two sequential drug injections 5 min apart immediately before a 1-h locomotion test as follows: (1) saline + saline, (2) saline + cocaine, (3) 0.025 mg/kg M100907 + cocaine, (4) 0.125 mg/kg MK212 + cocaine, or (5) cocktail combination of 0.025 mg/kg M100907 and 0.125 mg/kg MK212 + cocaine. Brains were extracted for Fos immunohistochemistry 90 min after the second injection. We next examined the effects of 0.025 mg/kg M100907 and 0.125 mg/kg MK212, alone and in combination, on spontaneous locomotor activity. While neither drug given alone produced any effects, the M100907/MK212 cocktail attenuated cocaine hyperlocomotion as well as cocaine-induced Fos expression in the dorsolateral caudate-putamen (CPu), but had no effect on spontaneous locomotion. The findings suggest that 5-HT(2A) Rs and 5-HT(2C) Rs interact to attenuate cocaine hyperlocomotion and Fos expression in the CPu, and that the CPu is a potential locus of the interactive effects between these 5-HT(2) R subtypes on behavior. Further research investigating combined 5-HT(2A) R antagonism and 5-HT(2C) R agonism as a treatment for cocaine dependence is warranted.

Challenges and new opportunities for detecting endogenous opioid peptides in reward.

The endogenous opioid peptide system, comprised of enkephalins, endorphins, dynorphins, and nociceptin, is a highly complex neurobiological system. Opioid peptides are derived from four precursor molecules and undergo several processing events yielding over 20 unique opioid peptides. This diversity together with low in vivo concentration and complex processing and release dynamics has challenged research into each peptide's unique function. Despite the subsequent challenges in detecting and quantifying opioid peptides in vivo, researchers have pioneered several techniques to directly or indirectly assay the roles of opioid peptides during behavioral manipulations. In this review, we describe the limitations of the traditional techniques used to study the role of endogenous opioid peptides in food and drug reward and bring focus to the wealth of new techniques to measure endogenous opioid peptides in reward processing.

Perinatal oxycodone exposure causes long-term sex-dependent changes in weight trajectory and sensory processing in adult mice.

RATIONALE: In utero opioid exposure is associated with lower weight and a neonatal opioid withdrawal syndrome (NOWS) at birth, along with longer-term adverse neurodevelopmental outcomes and mood disorders. While NOWS is sometimes treated with continued opioids, clinical studies have not addressed if long-term neurobehavioral outcomes are worsened with continued postnatal exposure to opioids. In addition, pre-clinical studies comparing in utero only opioid exposure to continued post-natal opioid administration for withdrawal mitigation are lacking. OBJECTIVES: Here, we sought to understand the impact of continued postnatal opioid exposure on long term behavioral consequences. METHODS: We implemented a rodent perinatal opioid exposure model of oxycodone (Oxy) exposure that included Oxy exposure until birth (short Oxy) and continued postnatal opioid exposure (long Oxy) spanning gestation through birth and lactation. RESULTS: Short Oxy exposure was associated with a sex-specific increase in weight gain trajectory in adult male mice. Long Oxy exposure caused an increased weight gain trajectory in adult males and alterations in nociceptive processing in females. Importantly, there was no evidence of long-term social behavioral deficits, anxiety, hyperactivity, or memory deficits following short or long Oxy exposure. CONCLUSIONS: Our findings suggest that offspring with prolonged opioid exposure experienced some long-term sequelae compared to pups with opioid cessation at birth. These results highlight the potential long-term consequences of opioid administration as a mitigation strategy for clinical NOWS symptomology and suggest alternatives should be explored.

An open-source device for measuring food intake and operant behavior in rodent home-cages.

Feeding is critical for survival, and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa. It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders. Operant behavioral testing can be used to measure the motivational component to feeding, but most food intake monitoring systems do not measure operant behavior. Here, we present a new solution for monitoring both food intake and motivation in rodent home-cages: the Feeding Experimentation Device version 3 (FED3). FED3 measures food intake and operant behavior in rodent home-cages, enabling longitudinal studies of feeding behavior with minimal experimenter intervention. It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings. Finally, FED3 design files are open-source and freely available, allowing researchers to modify FED3 to suit their needs.

Obesity and anorexia nervosa are two health conditions related to food intake. Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is, why animals seek and consume food. Measuring an animal's food intake is usually done by weighing food containers. However, this can be inaccurate due to the small amount of food that rodents eat. As for studying feeding motivation, this can involve calculating the number of times an animal presses a lever to receive a food pellet. These tests are typically conducted in hour-long sessions in temporary testing cages, called operant boxes. Yet, these tests only measure a brief period of a rodent's life. In addition, it takes rodents time to adjust to these foreign environments, which can introduce stress and may alter their feeding behaviour. To address this, Matikainen-Ankney, Earnest, Ali et al. developed a device for monitoring food intake and feeding behaviours around the clock in rodent home cages with minimal experimenter intervention. This 'Feeding Experimentation Device' (FED3) features a pellet dispenser and two 'nose-poke' sensors to measure total food intake, as well as motivation for and learning about food rewards. The battery-powered, wire-free device fits in standard home cages, enabling long-term studies of feeding behaviour with minimal intervention from investigators and less stress on the animals. This means researchers can relate data to circadian rhythms and meal patterns, as Matikainen-Ankney did here. Moreover, the device software is open-source so researchers can customise it to suit their experimental needs. It can also be programmed to synchronise with other instruments used in animal experiments, or across labs running the same behavioural tasks for multi-site studies. Used in this way, it could help improve reproducibility and reliability of results from such studies. In summary, Matikainen-Ankney et al. have presented a new practical solution for studying food-related behaviours in mice and rats. Not only could the device be useful to researchers, it may also be suitable to use in educational settings such as teaching labs and classrooms.

Central leptin signaling transmits positive valence.

Hunger resulting from food deprivation is associated with negative affect. This is supported by recent evidence showing that hunger-sensitive neurons drive feeding through a negative valence teaching signal. However, the complementary hypothesis that hormonal signals of energy surfeit counteract this negative valence, or even transmit positive valence, has received less attention. The adipose-derived hormone leptin signals in proportion to fat mass, is an indicator of energy surplus, and reduces food intake. Here, we showed that centrally-delivered leptin reduced food intake and conditioned a place preference in food-restricted as well as ad libitum fed rats. In contrast, leptin did not reduce food intake nor condition a place preference in obese rats, likely due to leptin resistance. Despite a well-known role for hindbrain leptin receptor signaling in energy balance control, hindbrain leptin delivery did not condition a place preference in food-restricted rats, suggesting that leptin acting in midbrain or forebrain sites mediates place preference conditioning. Supporting the hypothesis that leptin signaling induces a positive affective state, leptin also decreased the threshold for ventral tegmental area brain stimulation reward. Together, these data suggest that leptin signaling is intrinsically preferred, and support the view that signals of energy surfeit are associated with positive affect. Harnessing the positive valence of signals such as leptin may attenuate the negative affect associated with hunger, providing a compelling new approach for weight loss maintenance.

Females are less sensitive than males to the motivational- and dopamine-suppressing effects of kappa opioid receptor activation.

The neuropeptide dynorphin (DYN) activates kappa opioid receptors (KORs) in the brain to produce depressive-like states and decrease motivation. KOR-mediated suppression of dopamine release in the nucleus accumbens (NAc) is considered one underlying mechanism. We previously showed that, regardless of estrous cycle stage, female rats are less sensitive than males to KOR agonist-mediated decreases in motivation to respond for brain stimulation reward, measured with intracranial self-stimulation (ICSS). However, the explicit roles of KORs, circulating gonadal hormones, and their interaction with dopamine signaling in motivated behavior are not known. As such, we measured the effects of the KOR agonist U50,488 on ICSS stimulation thresholds before and after gonadectomy (or sham surgery). We found that ovariectomized females remained less sensitive than sham or castrated males to KOR-mediated decreases in brain stimulation reward, indicating that circulating gonadal hormones do not play a role. We used qRT-PCR to examine whether sex differences in gene expression in limbic brain regions are associated with behavioral sex differences. We found no sex differences in Pdyn or Oprk1 mRNA in the NAc and ventral tegmental area (VTA), but tyrosine hydroxylase (Th) mRNA was significantly higher in female compared to male VTA. To further explore sex-differences in KOR-mediated suppression of dopamine, we used fast scan cyclic voltammetry (FSCV) and demonstrated that U50,488 was less effective in suppressing evoked NAc dopamine release in females compared to males. These data raise the possibility that females are protected from KOR-mediated decreases in motivation by an increased capacity to produce and release dopamine.

Brain Stimulation Reward Supports More Consistent and Accurate Rodent Decision-Making than Food Reward.

Animal models of decision-making rely on an animal's motivation to decide and its ability to detect differences among various alternatives. Food reinforcement, although commonly used, is associated with problematic confounds, especially satiety. Here, we examined the use of brain stimulation reward (BSR) as an alternative reinforcer in rodent models of decision-making and compared it with the effectiveness of sugar pellets. The discriminability of various BSR frequencies was compared to differing numbers of sugar pellets in separate free-choice tasks. We found that BSR was more discriminable and motivated greater task engagement and more consistent preference for the larger reward. We then investigated whether rats prefer BSR of varying frequencies over sugar pellets. We found that animals showed either a clear preference for sugar reward or no preference between reward modalities, depending on the frequency of the BSR alternative and the size of the sugar reward. Overall, these results suggest that BSR is an effective reinforcer in rodent decision-making tasks, removing food-related confounds and resulting in more accurate, consistent, and reliable metrics of choice.