Premature Death in Bodybuilders: What Do We Know?

Premature deaths in bodybuilders regularly make headlines and are cited as evidence that bodybuilding is a dangerous activity. A wealth of research has revealed elite athletes typically enjoy lower mortality rates than non-athletes, but research on bodybuilder lifespan is surprisingly limited. Anabolic androgenic steroid (AAS) use is commonly cited as a key contributor to morbidity and premature mortality in bodybuilders, but this area of research is highly nuanced and influenced by numerous confounders unique to bodybuilding. It is quite possible that bodybuilders are at elevated risk and that AAS use is the primary reason for this, but there remains much unknown in this realm. As global participation in bodybuilding increases, and healthcare providers play a more active role in monitoring bodybuilder health, there is a need to identify how numerous factors associated with bodybuilding ultimately influence short- and long-term health and mortality rate. In this Current Opinion, we discuss what is currently known about the bodybuilder lifespan, identify the nuances of the literature regarding bodybuilder health and AAS use, and provide recommendations for future research on this topic.

Subcellular localization of D2 receptors in the murine substantia nigra.

G-protein-coupled D2 autoreceptors expressed on dopamine neurons (D2Rs) inhibit transmitter release and cell firing at axonal endings and somatodendritic compartments. Mechanistic details of somatodendritic dopamine release remain unresolved, partly due to insufficient information on the subcellular distribution of D2Rs. Previous studies localizing D2Rs have been hindered by a dearth of antibodies validated for specificity in D2R knockout animals and have been limited by the small sampling areas imaged by electron microscopy. This study utilized sub-diffraction fluorescence microscopy and electron microscopy to examine D2 receptors in a superecliptic pHlourin GFP (SEP) epitope-tagged D2 receptor knockin mouse. Incubating live slices with an anti-SEP antibody achieved the selective labeling of plasma membrane-associated receptors for immunofluorescent imaging over a large area of the substantia nigra pars compacta (SNc). SEP-D2Rs appeared as puncta-like structures along the surface of dendrites and soma of dopamine neurons visualized by antibodies to tyrosine hydroxylase (TH). TH-associated SEP-D2Rs displayed a cell surface density of 0.66 puncta/µm2, which corresponds to an average frequency of 1 punctum every 1.50 µm. Separate ultrastructural experiments using silver-enhanced immunogold revealed that membrane-bound particles represented 28% of total D2Rs in putative dopamine cells within the SNc. Structures immediately adjacent to dendritic membrane gold particles were unmyelinated axons or axon varicosities (40%), astrocytes (19%), other dendrites (7%), or profiles unidentified (34%) in single sections. Some apposed profiles also expressed D2Rs. Fluorescent and ultrastructural analyses also provided the first visualization of membrane D2Rs at the axon initial segment, a compartment critical for action potential generation. The punctate appearance of anti-SEP staining indicates there is a population of D2Rs organized in discrete signaling sites along the plasma membrane, and for the first time, a quantitative estimate of spatial frequency is provided.

The residence of synaptically released dopamine on D2 autoreceptors.

Neuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition.

A Gain-of-Function Variant in Dopamine D2 Receptor and Progressive Chorea and Dystonia Phenotype.

BACKGROUND: We describe a 4-generation Dutch pedigree with a unique dominantly inherited clinical phenotype of a combined progressive chorea and cervical dystonia carrying a novel heterozygous dopamine D2 receptor (DRD2) variant. OBJECTIVES: The objective of this study was to identify the genetic cause of the disease and to further investigate the functional consequences of the genetic variant. METHODS: After detailed clinical and neurological examination, whole-exome sequencing was performed. Because a novel variant in the DRD2 gene was found as the likely causative gene defect in our pedigree, we sequenced the DRD2 gene in a cohort of 121 Huntington-like cases with unknown genetic cause (Germany). Moreover, functional characterization of the DRD2 variant included arrestin recruitment, G protein activation, and G protein-mediated inhibition of adenylyl cyclase determined in a cell model, and G protein-regulated inward-rectifying potassium channels measured in midbrain slices of mice. RESULT: We identified a novel heterozygous variant c.634A > T, p.Ile212Phe in exon 5 of DRD2 that cosegregated with the clinical phenotype. Screening of the German cohort did not reveal additional putative disease-causing variants. We demonstrated that the D2S/L -I212 F receptor exhibited increased agonist potency and constitutive activation of G proteins in human embryonic kidney 239 cells as well as significantly reduced arrestin3 recruitment. We further showed that the D2S -I212 F receptor exhibited aberrant receptor function in mouse midbrain slices. CONCLUSIONS: Our results support an association between the novel p.Ile212Phe variant in DRD2, its modified D2 receptor activity, and the hyperkinetic movement disorder reported in the 4-generation pedigree. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

RIM is essential for stimulated but not spontaneous somatodendritic dopamine release in the midbrain.

Action potentials trigger neurotransmitter release at active zones, specialized release sites in axons. Many neurons also secrete neurotransmitters or neuromodulators from their somata and dendrites. However, it is unclear whether somatodendritic release employs specialized sites for release, and the molecular machinery for somatodendritic release is not understood. Here, we identify an essential role for the active zone protein RIM in stimulated somatodendritic dopamine release in the midbrain. In mice in which RIMs are selectively removed from dopamine neurons, action potentials failed to evoke significant somatodendritic release detected via D2 receptor-mediated currents. Compellingly, spontaneous dopamine release was normal upon RIM knockout. Dopamine neuron morphology, excitability, and dopamine release evoked by amphetamine, which reverses dopamine transporters, were also unaffected. We conclude that somatodendritic release employs molecular scaffolds to establish secretory sites for rapid dopamine signaling during firing. In contrast, basal release that is independent of action potential firing does not require RIM.

Cocaine-induced adaptation of dopamine D2S, but not D2L autoreceptors.

The dopamine D2 receptor has two splice variants, D2S (Short) and D2L (Long). In dopamine neurons, both variants can act as autoreceptors to regulate neuronal excitability and dopamine release, but the roles of each variant are incompletely characterized. In a previous study we used viral receptor expression in D2 receptor knockout mice to show distinct effects of calcium signaling on D2S and D2L autoreceptor function (Gantz et al., 2015). However, the cocaine-induced plasticity of D2 receptor desensitization observed in wild type mice was not recapitulated with this method of receptor expression. Here we use mice with genetic knockouts of either the D2S or D2L variant to investigate cocaine-induced plasticity in D2 receptor signaling. Following a single in vivo cocaine exposure, the desensitization of D2 receptors from neurons expressing only the D2S variant was reduced. This did not occur in D2L-expressing neurons, indicating differential drug-induced plasticity between the variants.

Desensitized D2 autoreceptors are resistant to trafficking.

Dendritic release of dopamine activates dopamine D2 autoreceptors, which are inhibitory G protein-coupled receptors (GPCRs), to decrease the excitability of dopamine neurons. This study used tagged D2 receptors to identify the localization and distribution of these receptors in living midbrain dopamine neurons. GFP-tagged D2 receptors were found to be unevenly clustered on the soma and dendrites of dopamine neurons within the substantia nigra pars compacta (SNc). Physiological signaling and desensitization of the tagged receptors were not different from wild type receptors. Unexpectedly, upon desensitization the tagged D2 receptors were not internalized. When tagged D2 receptors were expressed in locus coeruleus neurons, a desensitizing protocol induced significant internalization. Likewise, when tagged µ-opioid receptors were expressed in dopamine neurons they too were internalized. The distribution and lack of agonist-induced internalization of D2 receptors on dopamine neurons indicate a purposefully regulated localization of these receptors.

Distinct regulation of dopamine D2S and D2L autoreceptor signaling by calcium.

D2 autoreceptors regulate dopamine release throughout the brain. Two isoforms of the D2 receptor, D2S and D2L, are expressed in midbrain dopamine neurons. Differential roles of these isoforms as autoreceptors are poorly understood. By virally expressing the isoforms in dopamine neurons of D2 receptor knockout mice, this study assessed the calcium-dependence and drug-induced plasticity of D2S and D2L receptor-dependent G protein-coupled inwardly rectifying potassium (GIRK) currents. The results reveal that D2S, but not D2L receptors, exhibited calcium-dependent desensitization similar to that exhibited by endogenous autoreceptors. Two pathways of calcium signaling that regulated D2 autoreceptor-dependent GIRK signaling were identified, which distinctly affected desensitization and the magnitude of D2S and D2L receptor-dependent GIRK currents. Previous in vivo cocaine exposure removed calcium-dependent D2 autoreceptor desensitization in wild type, but not D2S-only mice. Thus, expression of D2S as the exclusive autoreceptor was insufficient for cocaine-induced plasticity, implying a functional role for the co-expression of D2S and D2L autoreceptors.

Separate GABA afferents to dopamine neurons mediate acute action of opioids, development of tolerance, and expression of withdrawal.

GABA release from interneurons in VTA, projections from the nucleus accumbens (NAc), and rostromedial tegmental nucleus (RMTg) was selectively activated in rat brain slices. The inhibition induced by µ-opioid agonists was pathway dependent. Morphine induced a 46% inhibition of IPSCs evoked from the RMTg, 18% from NAc, and IPSCs evoked from VTA interneurons were almost insensitive (11% inhibition). In vivo morphine treatment resulted in tolerance to the inhibition of RMTg, but not local interneurons or NAc, inputs. One common sign of opioid withdrawal is an increase in adenosine-dependent inhibition. IPSCs evoked from the NAc were potently inhibited by activation of presynaptic adenosine receptors, whereas IPSCs evoked from RMTg were not changed. Blockade of adenosine receptors selectively increased IPSCs evoked from the NAc during morphine withdrawal. Thus, the acute action of opioids, the development of tolerance, and the expression of withdrawal are mediated by separate GABA afferents to dopamine neurons.

Drosophila larvae as a model to study physiological alcohol dependence.

Alcohol addiction is a disease that includes a diverse set of phenotypes. Functional alcohol tolerance is an adaptation to the effects of alcohol that restores neuronal homeostatic balance while the drug is present. When the drug is suddenly withheld, these adaptations unbalance the nervous system and are thought to be the origin of some withdrawal symptoms. Withdrawal symptoms, which can be a motivating factor for alcoholics to relapse, are taken as evidence of physiological ethanol dependence. Both tolerance and withdrawal symptoms are diagnostic criteria for alcoholism. Recent studies have demonstrated that the larvae of Drosophila show conserved alcohol tolerance and withdrawal phenotypes indicating that Drosophila genetics can now be used in studying this endophenotype of alcohol addiction.

Is alcoholism learned? Insights from the fruit fly.

Alcohol addiction is a complex, unique human disease. Breaking addiction down into contributing endophenotypes enables its study in a variety of model systems. The Drosophila model system has been most often used to study alcohol sensitivity, tolerance, and physiological dependence. However, none of these endophenotypes can account for the near-permanent quality of the addicted state. It has been recently discussed that addictive drugs may hijack the learning-and-memory machinery to produce persistent behavioral changes. Learning and memory is amenable to experimental study, and provides us with a window into how alcohol affects higher-order mental functions that are likely to contribute compulsive drug use. Here, we review the Drosophila literature that links alcohol-related behaviors to learning and memory.

Neural adaptation leads to cognitive ethanol dependence.

Physiological alcohol dependence is a key adaptation to chronic ethanol consumption that underlies withdrawal symptoms, is thought to directly contribute to alcohol addiction behaviors, and is associated with cognitive problems such as deficits in learning and memory. Based on the idea that an ethanol-adapted (dependent) animal will perform better in a learning assay than an animal experiencing ethanol withdrawal will, we have used a learning paradigm to detect physiological ethanol dependence in Drosophila. Moderate ethanol consumption initially degrades the capacity of larvae to learn, but they eventually adapt and are able to learn as well as ethanol-naive animals. However, withholding ethanol from ethanol-adapted larvae impairs learning. Ethanol reinstatement restores the capacity to learn, thus demonstrating cognitive dependence on ethanol. The larval nervous system also shows ethanol-withdrawal hyperexcitability. Larvae reach ethanol concentrations equivalent to 0.05 to 0.08 blood-alcohol concentration-levels that would be mildly intoxicating in humans. These ethanol-induced changes in learning are not the product of sensory deficits or state-dependent learning. This is the first demonstration of cognitive ethanol dependence in an invertebrate genetic model system.

A low concentration of ethanol impairs learning but not motor and sensory behavior in Drosophila larvae.

Drosophila melanogaster has proven to be a useful model system for the genetic analysis of ethanol-associated behaviors. However, past studies have focused on the response of the adult fly to large, and often sedating, doses of ethanol. The pharmacological effects of low and moderate quantities of ethanol have remained understudied. In this study, we tested the acute effects of low doses of ethanol (∼7 mM internal concentration) on Drosophila larvae. While ethanol did not affect locomotion or the response to an odorant, we observed that ethanol impaired associative olfactory learning when the heat shock unconditioned stimulus (US) intensity was low but not when the heat shock US intensity was high. We determined that the reduction in learning at low US intensity was not a result of ethanol anesthesia since ethanol-treated larvae responded to the heat shock in the same manner as untreated animals. Instead, low doses of ethanol likely impair the neuronal plasticity that underlies olfactory associative learning. This impairment in learning was reversible indicating that exposure to low doses of ethanol does not leave any long lasting behavioral or physiological effects.

Olfactory conditioning in the third instar larvae of Drosophila melanogaster using heat shock reinforcement.

Adult Drosophila melanogaster has long been a popular model for learning and memory studies. Now the larval stage of the fruit fly is also being used in an increasing number of classical conditioning studies. In this study, we employed heat shock as a novel negative reinforcement for larvae and obtained high learning scores following just one training trial. We demonstrated heat-shock conditioning in both reciprocal and non-reciprocal paradigms and observed that the time window of association for the odor and heat shock reinforcement is on the order of a few minutes. This is slightly wider than the time window for electroshock conditioning reported in previous studies, possibly due to lingering effects of the high temperature. To test the utility of this simplified assay for the identification of new mutations that disrupt learning, we examined flies carrying mutations in the dnc gene. While the sensitivity to heat shock, as tested by writhing, was similar for wild type and dnc homozygotes, dnc mutations strongly diminished learning. We confirmed that the learning defect in dnc flies was indeed due to mutation in the dnc gene using non-complementation analysis. Given that heat shock has not been employed as a reinforcement for larvae in the past, we explored learning as a function of heat shock intensity and found that optimal learning occurred around 41 °C, with higher and lower temperatures both resulting in lower learning scores. In summary, we have developed a very simple, robust paradigm of learning in fruit fly larvae using heat shock reinforcement.

The morphology of supragranular pyramidal neurons in the human insular cortex: a quantitative Golgi study.

Although the primate insular cortex has been studied extensively, a comprehensive investigation of its neuronal morphology has yet to be completed. To that end, neurons from 20 human subjects (10 males and 10 females; N = 600) were selected from the secondary gyrus brevis, precentral gyrus, and postcentral gyrus of the left insula. The secondary gyrus brevis was generally more complex in terms of dendritic/spine extent than either the precentral or postcentral insular gyri, which is consistent with the posterior-anterior gradient of dendritic complexity observed in other cortical regions. The male insula had longer, spinier dendrites than the female insula, potentially reflecting sex differences in interoception. In comparing the current insular data with regional dendritic data quantified from other Brodmann's areas (BAs), insular total dendritic length (TDL) was less than the TDL of high integration cortices (BA6beta, 10, 11, 39), but greater than the TDL of low integration cortices (BA3-1-2, 4, 22, 44). Insular dendritic spine number was significantly greater than both low and high integration regions. Overall, the insula had spinier, but shorter neurons than did high integration cortices, and thus may represent a specialized type of heteromodal cortex, one that integrates crude multisensory information crucial to interoceptive processes.