Phosphorylation of pyruvate dehydrogenase inversely associates with neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Prenatal nicotine alters development of the laterodorsal tegmentum: Possible role for attention-deficit/hyperactivity disorder and drug dependence.

As we cycle between the states of wakefulness and sleep, a bilateral cholinergic nucleus in the pontine brain stem, the laterodorsal tegmentum (LDT), plays a critical role in controlling salience processing, attention, behavioral arousal, and electrophysiological signatures of the sub- and microstates of sleep. Disorders involving abnormal alterations in behavioral and motivated states, such as drug dependence, likely involve dysfunctions in LDT signaling. In addition, as the LDT exhibits connectivity with the thalamus and mesocortical circuits, as well as receives direct, excitatory input from the prefrontal cortex, a role for the LDT in cognitive symptoms characterizing attention-deficit/hyperactivity disorder (ADHD) including impulsivity, inflexibility, and dysfunctions of attention is suggested. Prenatal nicotine exposure (PNE) is associated with a higher risk for later life development of drug dependence and ADHD, suggesting alteration in development of brain regions involved in these behaviors. PNE has been shown to alter glutamate and cholinergic signaling within the LDT. As glutamate and acetylcholine are major excitatory mediators, these alterations would likely alter excitatory output to target regions in limbic motivational circuits and to thalamic and cortical networks mediating executive control. Further, PNE alters neuronal development and transmission within prefrontal cortex and limbic areas that send input to the LDT, which would compound effects of differential processing within the PNE LDT. When taken together, alterations in signaling in the LDT are likely to play a role in negative behavioral outcomes seen in PNE individuals, including a heightened risk of drug dependence and ADHD behaviors.

Hippocampal disruptions of synaptic and astrocyte metabolism are primary events of early amyloid pathology in the 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-ß (Aß) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, this crucial metabolic interplay during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aß accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in the 5xFAD hippocampus. This hyperactive neuronal phenotype coincided with decreased hippocampal tricarboxylic acid (TCA) cycle metabolism mapped by stable 13C isotope tracing. Particularly, reduced astrocyte TCA cycle activity and decreased glutamine synthesis led to hampered neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, the cerebral cortex of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism, which may suggest a metabolic compensation in this brain region. We found limited changes when we explored the brain proteome and metabolome of the 5xFAD mice, supporting that the functional metabolic disturbances between neurons and astrocytes are early primary events in AD pathology. In addition, synaptic mitochondrial and glycolytic function was selectively impaired in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex regional and cell-specific metabolic adaptations in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunctions in AD.

Prenatal exposure to nicotine in mice is associated with alterations in development and cellular and synaptic effects of alcohol in a brainstem arousal nucleus.

Using drugs of abuse while pregnant has tremendous negative consequences for the offspring, including an enhanced risk for substance use disorder (SUD). This vulnerability suggests that gestational exposure to drugs alters the developmental trajectory of neurons important in SUD processes, which could lead to later life changes in responsiveness to motivationally salient stimuli. The laterodorsal tegmentum (LDT) gates the behaviorally relevant firing pattern signaling stimuli saliency in mesoaccumbal circuits. Accordingly, any alterations in LDT functionality could alter output, and play a role in negative outcomes on motivated behavior associated with early-life nicotine exposure. Therefore, we investigated whether prenatal exposure to nicotine (PNE), which is a known teratogen, altered responsiveness of LDT neurons to alcohol by conducting electrophysiology in brain slices. Alcohol induced an outward current in control LDT cells, which was not seen in PNE LDT neurons. The frequency of mEPSCs was significantly decreased by alcohol in LDT PNE cells and accompanied by a decrease in action potential frequency, which were actions not seen in controls. Changes in baseline activity of PNE LDT cells were also observed. In summary, PNE LDT neurons showed alterations in baseline activity and membrane and synaptic responses to postnatal exposures to alcohol. The differences in PNE baseline activity and alcohol responses likely lead to differential output from the LDT to mesoaccumbal targets that could play a role in biasing coding of relevant stimuli, which could participate in the enhanced proclivity for development of SUD in those exposed during gestation to nicotine.

Prenatal nicotine exposure in mice induces sex-dependent anxiety-like behavior, cognitive deficits, hyperactivity, and changes in the expression of glutamate receptor associated-genes in the prefrontal cortex.

In rodents, prenatal nicotine exposure (PNE) has been associated with increased risk for development of cognitive and emotional disturbances, but the findings are somewhat conflicting. Lack of behavioral alterations following PNE could be due to the variety of methods available for nicotine delivery, exposure time and species used, with inbred strains being mostly employed. Such differences suggest the need to investigate the behavioral phenotype in each PNE model available if we are to find models with enhanced translational value. In this study, we assessed sex-dependent effects of PNE on ADHD-related behaviors and on the levels of mRNA coding for glutamate receptor subunits within the prefrontal cortex in the outbred NMRI mice exposed to nicotine via maternal drinking water during gestation. Cotinine levels were assessed in newborn pups. Behaviors related to anxiety, compulsivity, working memory, and locomotion were evaluated in both sexes of young adult offspring using the elevated zero maze, marble burying, spontaneous alternation behavior, and locomotor activity tests. Expression of mRNA coding for different glutamate receptors subunits within the prefrontal cortex (PFC) was measured using RT-qPCR. Cotinine levels in the serum of newborns confirmed fetal nicotine exposure. Both male and female offspring showed ADHD-like behaviors, such as deficit in the SAB test and hyperactivity. In addition, PNE male mice displayed anxiety- and compulsive-like behaviors, effects that were absent in female offspring. Finally, PNE reduced the mRNA expression of GluN1-, GluN2B-, and mGluR2-related genes within the PFC of male offspring, whereas it reduced the expression of mRNA coding for GluA2 subunit in female mice. PNE in NMRI mice induced sex-dependent behavioral changes, which parallels clinical findings following maternal cigarette smoke exposure. Alterations detected in PFC mRNA glutamate receptor proteins could contribute to the abnormal behavioral responses observed, but other signaling pathways or brain regions are likely involved in the behavioral susceptibility of PNE individuals.

Plasticity in the Brainstem: Prenatal and Postnatal Experience Can Alter Laterodorsal Tegmental (LDT) Structure and Function.

The brainstem has traditionally been considered an area of the brain with autonomous control of mostly homeostatic functions such as heart rate, respiration, and the sleep and wakefulness state, which would preclude the necessity to exhibit the high degree of synaptic or cellular mechanisms of plasticity typical of regions of the brain responsible for flexible, executive control, such as the medial prefrontal cortex or the hippocampus. The perception that the brainstem does not share the same degree of flexibility to alter synaptic strength and/or wiring within local circuits makes intuitive sense, as it is not easy to understand how "soft wiring" would be an advantage when considering the importance of faithful and consistent performance of the homeostatic, autonomic functions that are controlled by the brainstem. However, many of the molecular and cellular requirements which underlie strengthening of synapses seen in brain regions involved in higher-level processing are present in brainstem nuclei, and recent research suggest that the view of the brainstem as "hard wired," with rigid and static connectivity and with unchanging synaptic strength, is outdated. In fact, information from studies within the last decades, including work conducted in our group, leads us to propose that the brainstem can dynamically alter synaptic proteins, and change synaptic connections in response to prenatal or postnatal stimuli, and this would likely alter functionality and output. This article reviews recent research that has provided information resulting in our revision of the view of the brainstem as static and non-changing by using as example recent information gleaned from a brainstem pontine nucleus, the laterodorsal tegmentum (LDT). The LDT has demonstrated mechanisms underlying synaptic plasticity, and plasticity has been exhibited in the postnatal LDT following exposure to drugs of abuse. Further, exposure of the brain during gestation to drugs of abuse results in alterations in development of signaling pathways in the LDT. As the LDT provides a high degree of innervation of mesoaccumbal and mesocortical circuits involved in salience, as well as thalamocortical circuits involved in control of arousal and orientation, changes in synaptic strength would be expected to alter output, which would significantly impact behavioral state, motivated behavior and directed attention. Further, alterations in developmental trajectory within the LDT following prenatal exposure to drugs of abuse would be expected to impact on later life expression of motivation and arousal.

Cellular and Molecular Changes in Hippocampal Glutamate Signaling and Alterations in Learning, Attention, and Impulsivity Following Prenatal Nicotine Exposure.

Over 70 million European pregnant women are smokers during their child-bearing years. Consumption of tobacco-containing products during pregnancy is associated with several negative behavioral outcomes for the offspring, including a higher susceptibility for the development of attention-deficit/hyperactive disorder (ADHD). In efforts to minimize fetal exposure to tobacco smoke, many women around the world switch to nicotine replacement therapies (NRTs) during the gestational period; however, prenatal nicotine exposure (PNE) in any form has been associated with alterations in cognitive processes, including learning, memory, and attention. These processes are controlled by glutamatergic signaling of hippocampal pyramidal neurons within the CA1 region, suggesting actions of nicotine on glutamatergic transmission in this region if present prenatally. Accordingly, we aimed to investigate hippocampal glutamatergic function following PNE treatment in NMRI mice employing molecular, cellular electrophysiology, and pharmacological approaches, as well as to evaluate cognition in the rodent continuous performance task (rCPT), a recently developed mouse task allowing assessment of learning, attention, and impulsivity. PNE induced increases in the expression levels of mRNA coding for different glutamate receptors and subunits within the hippocampus. Functional alterations in AMPA and NMDA receptors on CA1 pyramidal neurons of PNE mice were suggestive of higher GluA2-lacking and lower GluN2A-containing receptors, respectively. Finally, PNE was associated with reduced learning, attention, and enhanced impulsivity in the rCPT. Alterations in glutamatergic functioning in CA1 neurons parallel changes seen in the spontaneously hypertensive rat ADHD model and likely contribute to the lower cognitive performance in the rCPT.

Alterations in NMDAR-mediated signaling within the laterodorsal tegmental nucleus are associated with prenatal nicotine exposure.

Cigarette smoking during pregnancy has been clinically associated with a variety of poorbehavioral outcomes for the exposed individuals, including higher risks for drug abuse and development of attention/deficit-hyperactive disorder (ADHD). Experimental studies support the hypothesis that nicotine might contribute to these risks, since prenatal nicotine exposure (PNE) in rodents was associated with greater addiction liability, hyperactivity, social impairments and a wide range of emotional and cognitive deficits. Alterations of glutamate signaling within brain regions involved in behavioral circuits could contribute to these outcomes. The pontine laterodorsal tegmental nucleus (LDT) exerts cholinergic modulation within the ventral tegmental area, nucleus accumbens, and cortical-projecting thalamic centers and PNE-associated alterations in LDT glutamate signaling could impact cholinergic output to these LDT targets. We have previously demonstrated that PNE alters AMPA-mediated signaling within LDT neurons, and in the present investigation, we focused on changes of NMDA receptors (NMDARs) and presence of silent synapses as an indicator of metaplastic processes in LDT cells associated with PNE treatment. PNE was associated with a decreased functional presence of GluN2B NMDAR subunits in synapses of large, putatively cholinergic neurons, whereas an increased function of this subunit was detected in small, likely GABAergic cells. In addition, PNE was associated with functional alterations of extrasynaptic NMDARs in putative cholinergic neurons, suggestive of an increased presence of GluN3A-containing NMDARs. An increased number of silent synapses was exclusively seen in the small cells. When taken together, we hypothesize that NMDA-mediated signaling changes within LDT neurons following PNE treatment would result in reductions of excitatory cholinergic modulatory tone in target brain regions, which would be expected to contribute to the behavioral deficits found among these individuals.

Calcium rises induced by AMPA and nicotine receptors in the ventral tegmental area show differences in mouse brain slices prenatally exposed to nicotine.

Nicotine exposure during gestation is associated with a higher risk of adverse behavioral outcomes including a heightened liability for dependency to drugs of abuse, which can exhibit drug-specificity influenced by gender. This enhanced liability suggests that nicotine use during pregnancy alters neural development in circuits involved in motivation and reward-based learning. The ventral tegmental area (VTA) is critical in motivated behaviors and we hypothesized that gestational exposure to nicotine alters the development of excitatory circuits in this nucleus. Accordingly, in VTA brain slices from male and female mice exposed to nicotine during the prenatal period (PNE) and controls, we compared cellular rises in calcium induced by AMPA receptor and nicotinic acetylcholine receptor (nAChR) stimulation by use of the ratiometric calcium binding dye, Fura-2AM. We found that AMPA induced smaller amplitude calcium rises in the PNE VTA, which was an effect only detected in males. Further, while the amplitude did not vary between treatment and control in females, a greater number of cells responded with rises in calcium in the PNE. Conversely, the proportions of cells responding with calcium rises induced by nAChR stimulation did not change in either gender according to treatment. However, larger rises in calcium in PNE females were detected. When taken together our data show that excitatory signaling in the VTA is altered in a gender-specific manner by PNE and suggest that alterations in signaling could play a role in drug-specific differences in maladaptive, motivated behaviors exhibited by males and females born to mothers exposed to nicotine during pregnancy. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 2018.

Prenatal nicotine exposure alters postsynaptic AMPA receptors and glutamate neurotransmission within the laterodorsal tegmentum (LDT) of juvenile mice.

Despite dissemination of information regarding the harm on fetal development of smoking while pregnant, the number of pregnancies associated with nicotine exposure appears to have stagnated. Presence of nicotine during neural formulation is associated with a higher susceptibility of drug dependence, suggesting an altered development of neurons in circuits involved in saliency and motivation. The laterodorsal tegmental nucleus (LDT) plays a role in coding stimuli valence via afferents to mesolimbic nuclei. Accordingly, alterations in development of neural mechanisms in the LDT could be involved in vulnerability to drug dependency. Therefore, we examined the effect of prenatal nicotine exposure (PNE) on glutamatergic functioning of LDT neurons in mouse brain slices using whole-cell, patch clamp concurrent with fluorescence-based calcium imaging. PNE was associated with larger amplitudes of AMPA-induced currents, and greater AMPA-mediated rises in intracellular calcium. AMPA/NMDA ratios and the AMPA-current rectification index were lower and higher, respectively, consistent with changes in the functionality of AMPA receptors in the PNE, which was substantiated by a greater inhibition of evoked and spontaneous glutamatergic synaptic events by a selective inhibitor of GluA2-lacking AMPA receptors. Paired pulse ratios showed a decreased probability of glutamate release from presynaptic inputs, and fluorescent imaging indicated a decreased action potential-dependent calcium increase associated with PNE. When taken together, our data suggest that PNE alters LDT glutamatergic functioning, which could alter output to mesolimbic targets. Such an alteration could play a role in altered coding of relevancy of drug stimuli that could enhance risk for development of drug dependency.

Loss of Lypd6 leads to reduced anxiety-like behaviour and enhanced responses to nicotine.

Nicotine consumption through smoking affects anxious states in humans. However, the precise role of nicotinic acetylcholine receptor (nAChR) circuitry in the regulation of anxiety remains elusive. The Lynx protein Lypd6 is highly enriched in synaptic loci and has been previously identified as an endogenous inhibitor of neuronal nAChR function in vitro. Here, we investigate the effect of Lypd6 in anxiety-related behaviour and examine the molecular underpinnings of its function in the brain. We employ the marble burying (MB) and elevated zero maze (EZM) tests in Lypd6 knock-out (KO) and wild-type (WT) mice and find that loss of Lypd6 leads to decreased digging behaviour in the MB test and increased time spent in the open area in the EZM test. Moreover, we demonstrate that acute nicotine administration reduces digging in the MB test in both KO and WT mice and further accentuates the inherent genotype difference. Using in vitro electrophysiology in dorsal raphe nuclei (DRN) neurons from Lypd6 KO mice, we show that nicotine-evoked whole-cell currents are enhanced in the absence of Lypd6. Collectively, these data are the first to indicate the involvement of Lypd6 in circuits associated with anxiety and suggest that a possible underlying neurobiological mechanism is the modulation of cholinergic responses in the DRN.