Dynamic effects of ventral hippocampal NRG3/ERBB4 signaling on nicotine withdrawal-induced responses.

Tobacco smoking remains a leading cause of preventable death in the United States, with approximately a 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH) of male and female mice. We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

Dynamic Effects of Ventral Hippocampal NRG3/ERBB4 Signaling on Nicotine Withdrawal-Induced Responses.

Tobacco smoking remains a leading cause of preventable death in the United States, with a less than 5% success rate for smokers attempting to quit. High relapse rates have been linked to several genetic factors, indicating that the mechanistic relationship between genes and drugs of abuse is a valuable avenue for the development of novel smoking cessation therapies. For example, various single nucleotide polymorphisms (SNPs) in the gene for neuregulin 3 (NRG3) and its cognate receptor, the receptor tyrosine-protein kinase erbB-4 (ERBB4), have been linked to nicotine addiction. Our lab has previously shown that ERBB4 plays a role in anxiety-like behavior during nicotine withdrawal (WD); however, the neuronal mechanisms and circuit-specific effects of NRG3-ERBB4 signaling during nicotine and WD are unknown. The present study utilizes genetic, biochemical, and functional approaches to examine the anxiety-related behavioral and functional role of NRG3-ERBB4 signaling, specifically in the ventral hippocampus (VH). We report that 24hWD from nicotine is associated with altered synaptic expression of VH NRG3 and ERBB4, and genetic disruption of VH ErbB4 leads to an elimination of anxiety-like behaviors induced during 24hWD. Moreover, we observed attenuation of GABAergic transmission as well as alterations in Ca2+-dependent network activity in the ventral CA1 area of VH ErbB4 knock-down mice during 24hWD. Our findings further highlight contributions of the NRG3-ERBB4 signaling pathway to anxiety-related behaviors seen during nicotine WD.

ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes.

Ca2+ is a major second messenger involved in cellular and subcellular signaling in a wide range of cells, including astrocytes, which use calcium ions to communicate with other cells in the brain. Even though a variety of genetically encoded Ca2+ indicators have been developed to study astrocyte calcium signaling, understanding the dynamics of endoplasmic reticulum calcium signaling is greatly limited by the currently available tools. To address this, we developed an endoplasmic reticulum-targeted calcium indicator, ER-GCaMP6f, which is anchored to the cytosolic side of the organelle and measures signaling that occurs in close proximity to the endoplasmic reticulum of astrocytes. Using a combination of confocal and super-resolution microscopy techniques, we demonstrate the localization of the indicator in the endoplasmic reticulum in both cell soma and processes of astrocytes. Combining ER-GCaMP6f with total internal reflection fluorescence microscopy, we show that Ca2+ fluctuations in small astrocytic processes can be detected, which are otherwise not observable with existing indicators and standard wide-field and confocal techniques. We also compared the ER-GCaMP6f indicator against currently used plasma membrane-tethered and cytosolic GCaMP6f indicators. ER-GCaMP6f identifies dynamics in calcium signaling of endoplasmic reticulum resident receptors that are missed by plasma membrane-anchored indicators. We also generated an adeno-associated virus (AAV5) and demonstrate that ER-GCaMP6f can be expressed in vivo and by measured calcium activity in brain slices. ER-GCaMP6f provides a powerful tool to study calcium signaling in close proximity to the endoplasmic reticulum in astrocyte cell soma and processes both in culture and in brain slices.

Microglia morphology and proinflammatory signaling in the nucleus accumbens during nicotine withdrawal.

Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum-a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.

Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

Neuregulin 3 Signaling Mediates Nicotine-Dependent Synaptic Plasticity in the Orbitofrontal Cortex and Cognition.

Neuregulin 3 (NRG3) and ErbB4 have been linked to nicotine addiction; however, the neuronal mechanisms and behavioral consequences of NRG3-ErbB4 sensitivity to nicotine remain elusive. Recent literature suggests that relapse to smoking is due to a lack of impulsive control, which is thought to be due to altered functioning within the orbitofrontal cortex (OFC). Therefore, we examined circuitry changes within this structure following nicotine application. We report that nicotine controls synaptic plasticity in the OFC through NRG3/ErbB4-dependent regulation of GABAergic inhibition. We observed that both nicotine and NRG3 facilitated the conversion of long-term potentiation into long-term depression at cortical layer 3/5 synapses. Induction of long-term depression by nicotine relied on nicotinic receptor activation and key regulators of NRG3 signaling: (1) release of intracellular calcium, (2) activation of the BACE1 beta-secretase, and (3) ErbB4 receptor activation. Nicotine-induced synaptic plasticity was also associated with accumulation of intracellular GABA and was completely blocked by GABAA/GABAB antagonists. To test whether these mechanisms underlie OFC-dependent behavior, we evaluated the effects of nicotine in the go/no-go task. Nicotine-impaired stimulus discrimination in this task was rescued by pharmacologic disruption of the NRG3 receptor, ErbB4. Altogether, our data indicate that nicotine-induced synaptic plasticity in the OFC and cognitive changes depend on NRG3-ErbB4 signaling. We propose that nicotine activation of this pathway may contribute to nicotine addiction, particularly in individuals with genetic variation in NRG3.

Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing.

The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs) are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages. Integrase-deficient lentiviral vectors (IDLVs) present an attractive means for delivery of CRISPR/Cas9 components because: (1) they are capable of transducing a broad range of cells and tissues, (2) have superior packaging capacity compared to other vectors (e.g., adeno-associated viral vectors), and (3) they are expressed transiently and demonstrate very weak integration capability. In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in human embryonic kidney (HEK293T) cells and post-mitotic brain neurons in vivo, via transient expression and with higher gene-targeting specificity than the corresponding integrase-competent vectors.

Glucagon-like peptide-1 receptor activation in the nucleus accumbens core suppresses feeding by increasing glutamatergic AMPA/kainate signaling.

Glucagon-like peptide-1 receptor (GLP-1R) activation in the nucleus accumbens (NAc) core is pharmacologically and physiologically relevant for regulating palatable food intake. Here, we assess whether GLP-1R signaling in the NAc core of rats modulates GABAergic medium spiny neurons (MSNs) through presynaptic-glutamatergic and/or presynaptic-dopaminergic signaling to control feeding. First, ex vivo fast-scan cyclic voltammetry showed that the GLP-1R agonist exendin-4 (Ex-4) does not alter dopamine release in the NAc core. Instead, support for a glutamatergic mechanism was provided by ex vivo electrophysiological analyses showing that Ex-4 activates presynaptic GLP-1Rs in the NAc core to increase the activity of MSNs via a glutamatergic, AMPA/kainate receptor-mediated mechanism, indicated by increased mEPSC frequency and decreased paired pulse ratio in core MSNs. Only a small, direct excitatory effect on MSNs by Ex-4 was observed, suggesting that the contribution of postsynaptic GLP-1R to MSN activity is minimal. The behavioral relevance of the electrophysiological data was confirmed by the finding that intracore injection of the AMPA/kainate receptor antagonist CNQX attenuated the ability of NAc core GLP-1R activation by Ex-4 microinjection to suppress food intake and body weight gain; in contrast, intracore NMDA receptor blockade by AP-5 did not inhibit the energy balance effects of NAc core Ex-4. Together, these data provide evidence for a novel glutamatergic, but not dopaminergic, mechanism by which NAc core GLP-1Rs promote negative energy balance.

Selective induction of astrocytic gliosis generates deficits in neuronal inhibition.

Reactive astrocytosis develops in many neurologic diseases, including epilepsy. Astrocytotic contributions to pathophysiology are poorly understood. Studies examining this are confounded by comorbidities accompanying reactive astrocytosis. We found that high-titer transduction of astrocytes with enhanced green fluorescent protein (eGFP) via adeno-associated virus induced reactive astrocytosis without altering the intrinsic properties or anatomy of neighboring neurons. We examined the consequences of selective astrocytosis induction on synaptic transmission in mouse CA1 pyramidal neurons. Neurons near eGFP-labeled reactive astrocytes had reduced inhibitory, but not excitatory, synaptic currents. This inhibitory postsynaptic current (IPSC) erosion resulted from a failure of the astrocytic glutamate-glutamine cycle. Reactive astrocytes downregulated expression of glutamine synthetase. Blockade of this enzyme normally induces rapid synaptic GABA depletion. In astrocytotic regions, residual inhibition lost sensitivity to glutamine synthetase blockade, whereas exogenous glutamine administration enhanced IPSCs. Astrocytosis-mediated deficits in inhibition triggered glutamine-reversible hyperexcitability in hippocampal circuits. Thus, reactive astrocytosis could generate local synaptic perturbations, leading to broader functional deficits associated with neurologic disease.