Multiomic foundations of human prefrontal cortex tissue function.

The prefrontal cortex (PFC) is a region of the brain that in humans is involved in the production of higher-order functions such as cognition, emotion, perception, and behavior. Neurotransmission in the PFC produces higher-order functions by integrating information from other areas of the brain. At the foundation of neurotransmission, and by extension at the foundation of higher-order brain functions, are an untold number of coordinated molecular processes involving the DNA sequence variants in the genome, RNA transcripts in the transcriptome, and proteins in the proteome. These "multiomic" foundations are poorly understood in humans, perhaps in part because most modern studies that characterize the molecular state of the human PFC use tissue obtained when neurotransmission and higher-order brain functions have ceased (i.e., the postmortem state). Here, analyses are presented on data generated for the Living Brain Project (LBP) to investigate whether PFC tissue from individuals with intact higher-order brain function has characteristic multiomic foundations. Two complementary strategies were employed towards this end. The first strategy was to identify in PFC samples obtained from living study participants a signature of RNA transcript expression associated with neurotransmission measured intracranially at the time of PFC sampling, in some cases while participants performed a task engaging higher-order brain functions. The second strategy was to perform multiomic comparisons between PFC samples obtained from individuals with intact higher-order brain function at the time of sampling (i.e., living study participants) and PFC samples obtained in the postmortem state. RNA transcript expression within multiple PFC cell types was associated with fluctuations of dopaminergic, serotonergic, and/or noradrenergic neurotransmission in the substantia nigra measured while participants played a computer game that engaged higher-order brain functions. A subset of these associations - termed the "transcriptional program associated with neurotransmission" (TPAWN) - were reproduced in analyses of brain RNA transcript expression and intracranial neurotransmission data obtained from a second LBP cohort and from a cohort in an independent study. RNA transcripts involved in TPAWN were found to be (1) enriched for RNA transcripts associated with measures of neurotransmission in rodent and cell models, (2) enriched for RNA transcripts encoded by evolutionarily constrained genes, (3) depleted of RNA transcripts regulated by common DNA sequence variants, and (4) enriched for RNA transcripts implicated in higher-order brain functions by human population genetic studies. In PFC excitatory neurons of living study participants, higher expression of the genes in TPAWN tracked with higher expression of RNA transcripts that in rodent PFC samples are markers of a class of excitatory neurons that connect the PFC to deep brain structures. TPAWN was further reproduced by RNA transcript expression patterns differentiating living PFC samples from postmortem PFC samples, and significant differences between living and postmortem PFC samples were additionally observed with respect to (1) the expression of most primary RNA transcripts, mature RNA transcripts, and proteins, (2) the splicing of most primary RNA transcripts into mature RNA transcripts, (3) the patterns of co-expression between RNA transcripts and proteins, and (4) the effects of some DNA sequence variants on RNA transcript and protein expression. Taken together, this report highlights that studies of brain tissue obtained in a safe and ethical manner from large cohorts of living individuals can help advance understanding of the multiomic foundations of brain function.

Dopamine and serotonin in human substantia nigra track social context and value signals during economic exchange.

Dopamine and serotonin are hypothesized to guide social behaviours. In humans, however, we have not yet been able to study neuromodulator dynamics as social interaction unfolds. Here, we obtained subsecond estimates of dopamine and serotonin from human substantia nigra pars reticulata during the ultimatum game. Participants, who were patients with Parkinson's disease undergoing awake brain surgery, had to accept or reject monetary offers of varying fairness from human and computer players. They rejected more offers in the human than the computer condition, an effect of social context associated with higher overall levels of dopamine but not serotonin. Regardless of the social context, relative changes in dopamine tracked trial-by-trial changes in offer value-akin to reward prediction errors-whereas serotonin tracked the current offer value. These results show that dopamine and serotonin fluctuations in one of the basal ganglia's main output structures reflect distinct social context and value signals.

Noradrenaline tracks emotional modulation of attention in human amygdala.

The noradrenaline (NA) system is one of the brain's major neuromodulatory systems; it originates in a small midbrain nucleus, the locus coeruleus (LC), and projects widely throughout the brain.1,2 The LC-NA system is believed to regulate arousal and attention3,4 and is a pharmacological target in multiple clinical conditions.5,6,7 Yet our understanding of its role in health and disease has been impeded by a lack of direct recordings in humans. Here, we address this problem by showing that electrochemical estimates of sub-second NA dynamics can be obtained using clinical depth electrodes implanted for epilepsy monitoring. We made these recordings in the amygdala, an evolutionarily ancient structure that supports emotional processing8,9 and receives dense LC-NA projections,10 while patients (n = 3) performed a visual affective oddball task. The task was designed to induce different cognitive states, with the oddball stimuli involving emotionally evocative images,11 which varied in terms of arousal (low versus high) and valence (negative versus positive). Consistent with theory, the NA estimates tracked the emotional modulation of attention, with a stronger oddball response in a high-arousal state. Parallel estimates of pupil dilation, a common behavioral proxy for LC-NA activity,12 supported a hypothesis that pupil-NA coupling changes with cognitive state,13,14 with the pupil and NA estimates being positively correlated for oddball stimuli in a high-arousal but not a low-arousal state. Our study provides proof of concept that neuromodulator monitoring is now possible using depth electrodes in standard clinical use.

Toward isolating reward changes in diet-induced obesity: A demand analysis.

Although hormonal and metabolic factors are well known to influence obesity, recent evidence suggests that obesity may be influenced also by changes in reward sensitivity akin to that seen in other 'reward pathologies', like substance use disorders. The current study sought to isolate changes in reward that may occur after the onset of diet-induced obesity by characterizing the economic demand for caloric (sucrose) and non-caloric (saccharin) reinforcers in a preclinical model of diet-induced obesity (DIO). We utilized economic demand analysis to measure baseline demand intensity (Q0) and demand elasticity (α) for sucrose and saccharin reinforcers in rats. After baseline measures were collected, rats were assigned randomly to a high-fat (HF) diet or low-fat (LF) control diet. After 8-weeks of diet exposure, HF rats were divided into obesity-resistant (OR) or obesity-prone (OP) groups based on weight after the 8-week HF diet exposure. Post-DIO demand data for each reinforcer were reassessed. At baseline, rats had higher demand intensity and lower elasticity for sucrose compared to saccharin. After 8-weeks of the high-fat diet, OP rats had significantly greater weight gain and lower demand elasticity for sucrose and saccharin and higher demand intensity for saccharin. The changes in sucrose and saccharin elasticity suggest that DIO-induced changes in food-related behavior are associated with changes in reward processes. The changes in demand intensity for saccharin suggest that demand intensity, as a measure of 'set point', is not directly linked to metabolic processes. The current study shows that microeconomic theory and demand analysis is able to isolate independent aspects of diet-induced reward changes related to caloric and non-caloric reinforcers.

The role of glutamate signaling in incentive salience: second-by-second glutamate recordings in awake Sprague-Dawley rats.

The attribution of incentive salience to reward-predictive stimuli has been shown to be associated with substance abuse-like behavior such as increased drug taking. Evidence suggests that glutamate neurotransmission and sequential N-methyl-D-aspartate (NMDA) activation are involved in the attribution of incentive salience. Here, we further explore the role of second-by-second glutamate neurotransmission in the attribution of incentive salience to reward-predictive stimuli by measuring sign-tracking behavior during a Pavlovian conditioned approach procedure using ceramic-based microelectrode arrays configured for sensitive measures of extracellular glutamate in awake behaving Sprague-Dawley rats. Specifically, we show that there is an increase in extracellular glutamate levels in the prelimbic cortex (PrL) and the nucleus accumbens core (NAcC) during sign-tracking behavior to a food-predictive conditioned stimulus (CS+) compared to the presentation of a non-predictive conditioned stimulus (CS-). Furthermore, the results indicate greater increases in extracellular glutamate levels in the PrL compared to NAcC in response to the CS+, including differences in glutamate release and signal decay. Taken together, the present research suggests that there is differential glutamate signaling in the NAcC and PrL during sign-tracking behavior to a food-predictive CS+.

Differential stimulus control of drug-seeking: multimodal reinstatement.

In animal models of substance-use disorder, individuals that repeatedly self-administer drugs of abuse have long-lasting neuronal adaptations that do not occur ostensibly in control animals only exposed to natural reinforcers (e.g. food). Because any treatment for substance-use disorder will be given to individuals with drug-taking histories, adequate dissociation of the specific neurobehavioral mechanisms underlying drug reinforcement, natural reinforcement and their associated cue effects requires an experimental model that exposes individuals to both reinforcer conditions, along with their associated stimuli. Furthermore, contingent stimuli that reinforce drug seeking through second-order relationships may produce reinstatement of drug seeking through different neurobehavioral means than non-contingent exposure to stimuli that signal the availability of a drug reinforcer, effectively producing different modes of stimulus-induced reinstatement. Toward experimental isolation of the relationships mentioned, herein, we used a within-session multiple schedule of reinforcement containing both discriminative (SD ) and conditioned (CS) stimuli to study stimulus control of drug-taking and food-taking behavior, along with how these functionally distinct cues may differentially reinstate drug-seeking and food-seeking behavior within a single animal. We demonstrate specific stimulus control over drug and food taking; furthermore, we demonstrate that the same stimulus (i.e. cue light) operating as an SD or CS produced differential reinstatement of drug-taking and food-taking behavior. The results suggest that contingent CSs and non-contingent SD s produce reinstatement through different neurobehavioral processes and, within-session multiple schedules, can be used to study different modes of specific stimulus control over drug and food seeking in a single animal with both drug-taking and food-taking history.

Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn-1 gene.

INTRODUCTION: In kindling, repeated electrical stimulation of certain brain areas causes progressive and permanent intensification of epileptiform activity resulting in generalized seizures. We focused on the role(s) of glutamate and a negative regulator of glutamate release, STXBP5/tomosyn-1, in kindling. METHODS: Stimulating electrodes were implanted in the amygdala and progression to two successive Racine stage 5 seizures was measured in wild-type and STXBP5/tomosyn-1-/- (Tom-/-) animals. Glutamate release measurements were performed in distinct brain regions using a glutamate-selective microelectrode array (MEA). RESULTS: Naïve Tom-/- mice had significant increases in KCl-evoked glutamate release compared to naïve wild type as measured by MEA of presynaptic release in the hippocampal dentate gyrus (DG). Kindling progression was considerably accelerated in Tom-/- mice, requiring fewer stimuli to reach a fully kindled state. Following full kindling, MEA measurements of both kindled Tom+/+ and Tom-/- mice showed significant increases in KCl-evoked and spontaneous glutamate release in the DG, indicating a correlation with the fully kindled state independent of genotype. Resting glutamate levels in all hippocampal subregions were significantly lower in the kindled Tom-/- mice, suggesting possible changes in basal control of glutamate circuitry in the kindled Tom-/- mice. CONCLUSIONS: Our studies demonstrate that increased glutamate release in the hippocampal DG correlates with acceleration of the kindling process. Although STXBP5/tomosyn-1 loss increased evoked glutamate release in naïve animals contributing to their prokindling phenotype, the kindling process can override any attenuating effect of STXBP5/tomosyn-1. Loss of this "braking" effect of STXBP5/tomosyn-1 on kindling progression may set in motion an alternative but ultimately equally ineffective compensatory response, detected here as reduced basal glutamate release.

Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aß-Bearing Mice.

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.

Role of ionotropic glutamate receptors in delay and probability discounting in the rat.

RATIONALE: Discounting of delayed and probabilistic reinforcement is linked to increased drug use and pathological gambling. Understanding the neurobiology of discounting is important for designing treatments for these disorders. Glutamate is considered to be involved in addiction-like behaviors; however, the role of ionotropic glutamate receptors (iGluRs) in discounting remains unclear. OBJECTIVES: The current study examined the effects of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor blockade on performance in delay and probability discounting tasks. METHODS: Following training in either delay or probability discounting, rats (n = 12, each task) received pretreatments of the NMDA receptor antagonists MK-801 (0, 0.01, 0.03, 0.1, or 0.3 mg/kg, s.c.) or ketamine (0, 1.0, 5.0, or 10.0 mg/kg, i.p.), as well as the AMPA receptor antagonist CNQX (0, 1.0, 3.0, or 5.6 mg/kg, i.p.). Hyperbolic discounting functions were used to estimate sensitivity to delayed/probabilistic reinforcement and sensitivity to reinforcer amount. RESULTS: An intermediate dose of MK-801 (0.03 mg/kg) decreased sensitivity to both delayed and probabilistic reinforcement. In contrast, ketamine did not affect the rate of discounting in either task but decreased sensitivity to reinforcer amount. CNQX did not alter sensitivity to reinforcer amount or delayed/probabilistic reinforcement. CONCLUSIONS: These results show that blockade of NMDA receptors, but not AMPA receptors, decreases sensitivity to delayed/probabilistic reinforcement (MK-801) and sensitivity to reinforcer amount (ketamine). The differential effects of MK-801 and ketamine demonstrate that sensitivities to delayed/probabilistic reinforcement and reinforcer amount are pharmacologically dissociable.

P301L tau expression affects glutamate release and clearance in the hippocampal trisynaptic pathway.

Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium-evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4- and 7-fold increase in potassium-evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal-dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability. Pre-synaptic vesicular glutamate transporters (vGLUTs) package glutamate into vesicles before exocytosis into the synaptic cleft. Once in the extracellular space, glutamate acts on glutamate receptors. Glutamate is removed from the extracellular space by excitatory amino acid transporters, including GLT-1, predominantly localized to glia. P301L tau expression increases vGLUT expression and glutamate release, while also decreasing GLT-1 expression and glutamate clearance.