The ANC-1 (Nesprin-1/2) organelle-anchoring protein functions through mitochondria to polarize axon growth in response to SLT-1.

A family of giant KASH proteins, including C. elegans ANC-1 and mammalian Nesprin-1 and -2, are involved in organelle anchoring and are associated with multiple neurodevelopmental disorders including autism, bipolar disorder, and schizophrenia. However, little is known about how these proteins function in neurons. Moreover, the role of organelle anchoring in axon development is poorly understood. Here, we report that ANC-1 functions with the SLT-1 extracellular guidance cue to polarize ALM axon growth. This role for ANC-1 is specific to its longer ANC-1A and ANC-1C isoforms, suggesting that it is mechanistically distinct from previously described roles for ANC-1. We find that ANC-1 is required for the localization of a cluster of mitochondria to the base of the proximal axon. Furthermore, genetic and pharmacological studies indicate that ANC-1 functions with mitochondria to promote polarization of ALM axon growth. These observations reveal a mechanism whereby ANC-1 functions through mitochondria to polarize axon growth in response to SLT-1.

cAMP-mediated upregulation of HCN channels in VTA dopamine neurons promotes cocaine reinforcement.

Chronic cocaine exposure induces enduring neuroadaptations that facilitate motivated drug taking. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to modulate neuronal firing and pacemaker activity in ventral tegmental area (VTA) dopamine neurons. However, it remained unknown whether cocaine self-administration affects HCN channel function and whether HCN channel activity modulates motivated drug taking. We report that rat VTA dopamine neurons predominantly express Hcn3-4 mRNA, while VTA GABA neurons express Hcn1-4 mRNA. Both neuronal types display similar hyperpolarization-activated currents (Ih), which are facilitated by acute increases in cAMP. Acute cocaine application decreases voltage-dependent activation of Ih in VTA dopamine neurons, but not in GABA neurons. Unexpectedly, chronic cocaine self-administration results in enhanced Ih selectively in VTA dopamine neurons. This differential modulation of Ih currents is likely mediated by a D2 autoreceptor-induced decrease in cAMP as D2 (Drd2) mRNA is predominantly expressed in dopamine neurons, whereas D1 (Drd1) mRNA is barely detectable in the VTA. Moreover, chronically decreased cAMP via Gi-DREADD stimulation leads to an increase in Ih in VTA dopamine neurons and enhanced binding of HCN3/HCN4 with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), an auxiliary subunit that is known to facilitate HCN channel surface trafficking. Finally, we show that systemic injection and intra-VTA infusion of the HCN blocker ivabradine reduces cocaine self-administration under a progressive ratio schedule and produces a downward shift of the cocaine dose-response curve. Our results suggest that cocaine self-administration induces an upregulation of Ih in VTA dopamine neurons, while HCN inhibition reduces the motivation for cocaine intake.

Application of optogenetics and in vivo imaging approaches for elucidating the neurobiology of addiction.

The neurobiology of addiction has been an intense topic of investigation for more than 50 years. Over this time, technological innovation in methods for studying brain function rapidly progressed, leading to increasingly sophisticated experimental approaches. To understand how specific brain regions, cell types, and circuits are affected by drugs of abuse and drive behaviors characteristic of addiction, it is necessary both to observe and manipulate neural activity in addiction-related behavioral paradigms. In pursuit of this goal, there have been several key technological advancements in in vivo imaging and neural circuit modulation in recent years, which have shed light on the cellular and circuit mechanisms of addiction. Here we discuss some of these key technologies, including circuit modulation with optogenetics, in vivo imaging with miniaturized single-photon microscopy (miniscope) and fiber photometry, and how the application of these technologies has garnered novel insights into the neurobiology of addiction.

An autism-causing calcium channel variant functions with selective autophagy to alter axon targeting and behavior.

Common and rare variants of the CACNA1C voltage-gated calcium channel gene have been associated with autism and other neurodevelopmental disorders including schizophrenia, bipolar disorder and ADHD. However, little is known about how CACNA1C variants affect cellular processes to alter neurodevelopment. The Timothy syndrome mutation is a rare de novo gain-of-function variant in CACNA1C that causes autism with high penetrance, providing a powerful avenue into investigating the role of CACNA1C variants in neurodevelopmental disorders. Here, we use egl-19, the C. elegans homolog of CACNA1C, to investigate the role of voltage-gated calcium channels in autism. We show that an egl-19(gof) mutation that is equivalent to the Timothy syndrome mutation can alter axon targeting and affect behavior in C. elegans. We find that wildtype egl-19 negatively regulates axon termination. The egl-19(gof) mutation represses axon termination to cause axon targeting defects that lead to the misplacement of electrical synapses and alterations in habituation to light touch. Moreover, genetic interactions indicate that the egl-19(gof) mutation functions with genes that promote selective autophagy to cause defects in axon termination and behavior. These results reveal a novel genetic mechanism whereby a de novo mutation in CACNA1C can drive alterations in circuit formation and behavior.

Treadmill Exercise Reduces Neuroinflammation, Glial Cell Activation and Improves Synaptic Transmission in the Prefrontal Cortex in 3 × Tg-AD Mice.

Physical exercise improves memory and cognition in physiological aging and Alzheimer's disease (AD), but the mechanisms remain poorly understood. Here, we test the hypothesis that Aß oligomer accumulation, neuroinflammation, and glial cell activation may lead to disruption of synaptic transmission in the prefrontal cortex of 3 × Tg-AD Mice, resulting in impairment of learning and memory. On the other hand, treadmill exercise could prevent the pathogenesis and exert neuroprotective effects. Here, we used immunohistochemistry, western blotting, enzyme-linked immunosorbent assay, and slice electrophysiology to analyze the levels of GSK3ß, Aß oligomers (Aß dimers and trimers), pro-inflammatory cytokines (IL-1ß, IL-6, and TNFα), the phosphorylation of CRMP2 at Thr514, and synaptic currents in pyramidal neurons in the prefrontal cortex. We show that 12-week treadmill exercise beginning in three-month-old mice led to the inhibition of GSK3ß kinase activity, decreases in the levels of Aß oligomers, pro-inflammatory cytokines (IL-1ß, IL-6, and TNFα), and the phosphorylation of CRMP2 at Thr514, reduction of microglial and astrocyte activation, and improvement of excitatory and inhibitory synaptic transmission of pyramidal neurons in the prefrontal cortex of 3 × Tg-AD Mice. Thus, treadmill exercise reduces neuroinflammation, glial cell activation and improves synaptic transmission in the prefrontal cortex in 3 × Tg-AD mice, possibly related to the inhibition of GSK3ß kinase activity.

Psilocybin analog 4-OH-DiPT enhances fear extinction and GABAergic inhibition of principal neurons in the basolateral amygdala.

Psychedelics such as psilocybin show great promise for the treatment of depression and PTSD, but their long duration of action poses practical limitations for patient access. 4-OH-DiPT is a fast-acting and shorter-lasting derivative of psilocybin. Here we characterized the pharmacological profile of 4-OH-DiPT and examined its impact on fear extinction learning as well as a potential mechanism of action. First, we profiled 4-OH-DiPT at all 12 human 5-HT GPCRs. 4-OH-DiPT showed strongest agonist activity at all three 5-HT2A/2B/2C receptors with near full agonist activity at 5-HT2A. Notably, 4-OH-DiPT had comparable activity at mouse and human 5-HT2A/2B/2C receptors. In a fear extinction paradigm, 4-OH-DiPT significantly reduced freezing responses to conditioned cues in a dose-dependent manner with a greater potency in female mice than male mice. Female mice that received 4-OH-DiPT before extinction training had reduced avoidance behaviors several days later in the light dark box, elevated plus maze and novelty-suppressed feeding test compared to controls, while male mice did not show significant differences. 4-OH-DiPT produced robust increases in spontaneous inhibitory postsynaptic currents (sIPSCs) in basolateral amygdala (BLA) principal neurons and action potential firing in BLA interneurons in a 5-HT2A-dependent manner. RNAscope demonstrates that Htr2a mRNA is expressed predominantly in BLA GABA interneurons, Htr2c mRNA is expressed in both GABA interneurons and principal neurons, while Htr2b mRNA is absent in the BLA. Our findings suggest that 4-OH-DiPT activates BLA interneurons via the 5-HT2A receptor to enhance GABAergic inhibition of BLA principal neurons, which provides a potential mechanism for suppressing learned fear.

Epac2 in midbrain dopamine neurons contributes to cocaine reinforcement via enhancement of dopamine release.

Repeated exposure to drugs of abuse results in an upregulation of cAMP signaling in the mesolimbic dopamine system, a molecular adaptation thought to be critically involved in the development of drug dependence. Exchange protein directly activated by cAMP (Epac2) is a major cAMP effector abundantly expressed in the brain. However, it remains unknown whether Epac2 contributes to cocaine reinforcement. Here, we report that Epac2 in the mesolimbic dopamine system promotes cocaine reinforcement via enhancement of dopamine release. Conditional knockout of Epac2 from midbrain dopamine neurons (Epac2-cKO) and the selective Epac2 inhibitor ESI-05 decreased cocaine self-administration in mice under both fixed-ratio and progressive-ratio reinforcement schedules and across a broad range of cocaine doses. In addition, Epac2-cKO led to reduced evoked dopamine release, whereas Epac2 agonism robustly enhanced dopamine release in the nucleus accumbens in vitro. This mechanism is central to the behavioral effects of Epac2 disruption, as chemogenetic stimulation of ventral tegmental area (VTA) dopamine neurons via deschloroclozapine (DCZ)-induced activation of Gs-DREADD increased dopamine release and reversed the impairment of cocaine self-administration in Epac2-cKO mice. Conversely, chemogenetic inhibition of VTA dopamine neurons with Gi-DREADD reduced dopamine release and cocaine self-administration in wild-type mice. Epac2-mediated enhancement of dopamine release may therefore represent a novel and powerful mechanism that contributes to cocaine reinforcement.

CB2 Agonist GW842166x Protected against 6-OHDA-Induced Anxiogenic- and Depressive-Related Behaviors in Mice.

In addition to motor dysfunction, patients with Parkinson's disease (PD) are often affected by neuropsychiatric disorders, such as anxiety and depression. In animal models, activation of the endocannabinoid (eCB) system produces anxiolytic and antidepressant-like behavioral effects. CB2 agonists have demonstrated neuroprotective effects against neurotoxin-induced dopamine neuron loss and deficits in motor function. However, it remains unknown whether CB2 agonism ameliorates anxiogenic- and depressive-like behaviors in PD models. Here, we report that the selective CB2 agonist GW842166x exerted neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced loss of dopaminergic terminals and dopamine release in the striatum, which were blocked by the CB2 antagonist AM630. We found that 6-OHDA-treated mice exhibited anxiogenic- and depressive-like behaviors in the open-field, sucrose preference, novelty-suppressed feeding, marble burying, and forced swim tests but did not show significant changes in the elevated plus-maze and light-dark box test. GW842166x treatments ameliorated 6-OHDA-induced anxiogenic- and depressive-like behaviors, but the effects were blocked by CB2 antagonism, suggesting a CB2-dependent mechanism. These results suggest that the CB2 agonist GW842166x not only reduces 6-OHDA-induced motor function deficits but also anxiogenic- and depressive-like behaviors in 6-OHDA mouse models of PD.

Ibudilast attenuates cocaine self-administration and prime- and cue-induced reinstatement of cocaine seeking in rats.

Ibudilast is a non-selective phosphodiesterase (PDE) inhibitor and glial cell modulator which has shown great promise for the treatment of drug and alcohol use disorders in recent clinical studies. However, it is unknown whether and how ibudilast affects cocaine seeking behavior. Here we show that systemic administration of ibudilast dose-dependently reduced cocaine self-administration under fixed- and progressive-ratio reinforcement schedules in rats and shifted cocaine dose-response curves downward. In addition, ibudilast decreased cocaine prime- and cue-induced reinstatement of cocaine seeking. These results indicate that ibudilast was effective in reducing the reinforcing effects of cocaine and relapse to cocaine seeking. Chronic cocaine exposure induces cAMP-related neuroadaptations in the reward circuitry of the brain. To investigate potential mechanisms for ibudilast-induced attenuation of cocaine self-administration, we recorded from ventral tegmental area (VTA) dopamine neurons in ex vivo midbrain slices prepared from rats that had undergone saline and cocaine self-administration. We found cocaine self-administration led to a decrease in inhibitory postsynaptic currents (IPSCs), an increase in the AMPAR/NMDAR ratio, and an increase in the excitation to inhibition (E/I) ratio. Ibudilast pretreatments enhanced GABAergic inhibition and did not further change cocaine-induced potentiation of excitation, leading to normalization of the E/I ratio. Restoration of the balance between excitation and inhibition in VTA dopamine neurons may contribute to the attenuation of cocaine self-administration by ibudilast.

The Neuroprotective Effects of the CB2 Agonist GW842166x in the 6-OHDA Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder associated with dopamine neuron loss and motor dysfunction. Neuroprotective agents that prevent dopamine neuron death hold great promise for slowing the disease's progression. The activation of cannabinoid (CB) receptors has shown neuroprotective effects in preclinical models of neurodegenerative disease, traumatic brain injury, and stroke, and may provide neuroprotection against PD. Here, we report that the selective CB2 agonist GW842166x exerted protective effects against the 6-hydroxydopamine (6-OHDA)-induced loss of dopamine neurons and its associated motor function deficits in mice, as shown by an improvement in balance beam walking, pole, grip strength, rotarod, and amphetamine-induced rotation tests. The neuroprotective effects of GW842166x were prevented by the CB2 receptor antagonist AM630, suggesting a CB2-dependent mechanism. To investigate potential mechanisms for the neuroprotective effects of GW842166x, we performed electrophysiological recordings from substantia nigra pars compacta (SNc) dopamine neurons in ex vivo midbrain slices prepared from drug-naïve mice. We found that the bath application of GW842166x led to a decrease in action potential firing, likely due to a decrease in hyperpolarization-activated currents (Ih) and a shift of the half-activation potential (V1/2) of Ih to a more hyperpolarized level. Taken together, the CB2 agonist GW842166x may reduce the vulnerability of dopamine neurons to 6-OHDA by decreasing the action potential firing of these neurons and the associated calcium load.