Behavioral encoding across timescales by region-specific dopamine dynamics.

The dorsal (DS) and ventral striatum (VS) receive dopaminergic projections that control motor functions and reward-related behavior. It remains poorly understood how dopamine release dynamics across different temporal scales in these regions are coupled to behavioral outcomes. Here, we employ the dopamine sensor dLight1.3b together with multiregion fiber photometry and machine learning-based analysis to decode dopamine dynamics across the striatum during self-paced exploratory behavior in mice. Our data show a striking coordination of rapidly fluctuating signal in the DS, carrying information across dopamine levels, with a slower signal in the VS, consisting mainly of slow-paced transients. Importantly, these release dynamics correlated with discrete behavioral motifs, such as turns, running, and grooming on a subsecond-to-minute time scale. Disruption of dopamine dynamics with cocaine caused randomization of action selection sequencing and disturbance of DS-VS coordination. The data suggest that distinct dopamine dynamics of DS and VS jointly encode behavioral sequences during unconstrained activity with DS modulating the stringing together of actions and VS the signal to initiate and sustain the selected action.

Disruption of the PDZ domain-binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis, and reward motivation.

The dopamine (DA) transporter (DAT) is part of a presynaptic multiprotein network involving interactions with scaffold proteins via its C-terminal PDZ domain-binding sequence. Using a mouse model expressing DAT with mutated PDZ-binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here, we show by application of direct stochastic reconstruction microscopy not only that the striatal level of transporter is reduced in DAT-AAA mice but also that the nanoscale distribution of this transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal DA adaptations and changes in DA-related behaviors distinct from those seen in other genetic DAT mouse models. DA levels in the striatum are reduced to ∼45% of that of WT, accompanied by elevated DA turnover. Nonetheless, fast-scan cyclic voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared with WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels, whereas immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed ratio of one and progressive ratio schedule of reinforcement but a reduction compared with WT when using cocaine as reinforcer. In summary, our data demonstrate how disruption of PDZ domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics and related behaviors.

Disruption of hippocampal synaptic transmission and long-term potentiation by psychoactive synthetic cannabinoid 'Spice' compounds: comparison with Δ9 -tetrahydrocannabinol.

There has been a marked increase in the availability of synthetic drugs designed to mimic the effects of marijuana. These cannabimimetic drugs, sold illicitly as 'Spice' and related products, are associated with serious medical complications in some users. In vitro studies suggest that synthetic cannabinoids in these preparations are potent agonists at central cannabinoid CB1 receptors (CB1Rs), but few investigations have delineated their cellular effects, particularly in comparison with the psychoactive component of marijuana, Δ9 -tetrahydrocannabinol (Δ9 -THC). We compared the ability of three widely abused synthetic cannabinoids and Δ9 -THC to alter glutamate release and long-term potentiation in the mouse hippocampus. JWH-018 was the most potent inhibitor of hippocampal synaptic transmission (EC50 ~15 nM), whereas its fluoropentyl derivative, AM2201, inhibited synaptic transmission with slightly lower potency (EC50 ~60 nM). The newer synthetic cannabinoid, XLR-11, displayed much lower potency (EC50 ~900 nM) that was similar to Δ9 -THC (EC50 ~700 nM). The effects of all compounds occurred via activation of CB1Rs, as demonstrated by reversal with the selective antagonist/inverse agonist AM251 or the neutral CB1R antagonist PIMSR1. Moreover, AM2201 was without effect in the hippocampus of transgenic mice lacking the CB1R. Hippocampal slices exposed to either synthetic cannabinoids or Δ9 -THC exhibited significantly impaired long-term potentiation (LTP). We find that, compared with Δ9 -THC, the first-generation cannabinoids found in Spice preparations display higher potency, whereas a recent synthetic cannabinoid is roughly equipotent with Δ9 -THC. The disruption of synaptic function by these synthetic cannabinoids is likely to lead to profound impairments in cognitive and behavioral function.

DNA-PAINT adaptors make for efficient multiplexing.

Multiplexed super-resolution imaging offers a route to spatial proteomics; however, time-efficient mapping of many protein species has been challenging. Two recent works in Cell highlight SUM-PAINT and FLASH-PAINT, methods that leverage adaptor DNA strand design to combine advances in multiplexing with increases in speed of label exchange. These advances permit unbiased omics-style analyses to advance biological insights from super-resolution images.

Within-Mice Comparison of Microdialysis and Fiber Photometry-Recorded Dopamine Biosensor during Amphetamine Response.

A fundamental concept in neuroscience is the transmission of information between neurons via neurotransmitters, -modulators, and -peptides. For the past decades, the gold standard for measuring neurochemicals in awake animals has been microdialysis (MD). The emergence of genetically encoded fluorescence-based biosensors, as well as in vivo optical techniques such as fiber photometry (FP), has introduced technologically distinct means of measuring neurotransmission. To directly compare MD and FP, we performed concurrent within-animal recordings of extracellular dopamine (DA) in the dorsal striatum (DS) before and after administration of amphetamine in awake, freely behaving mice expressing the dopamine sensor dLight1.3b. We show that despite temporal differences, MD- and FP-based readouts of DA correlate well within mice. Down-sampling of FP data showed temporal correlation to MD data, with less variance observed using FP. We also present evidence that DA fluctuations periodically reach low levels, and naïve animals have rapid, predrug DA dynamics measured with FP that correlate to the subsequent pharmacodynamics of amphetamine as measured with MD and FP.

Insulin-like growth factor 1 supplementation supports motor coordination and affects myelination in preterm pigs.

Introduction: Preterm infants have increased risk of impaired neurodevelopment to which reduced systemic levels of insulin-like growth factor 1 (IGF-1) in the weeks after birth may play a role. Hence, we hypothesized that postnatal IGF-1 supplementation would improve brain development in preterm pigs, used as a model for preterm infants. Methods: Preterm pigs delivered by cesarean section received recombinant human IGF-1/IGF binding protein-3 complex (rhIGF-1/rhIGFBP-3, 2.25 mg/kg/day) or vehicle from birth to postnatal day 19. Motor function and cognition were assessed by monitoring of in-cage and open field activities, balance beam test, gait parameters, novel object recognition and operant conditioning tests. Collected brains were subject to magnetic resonance imaging (MRI), immunohistochemistry, gene expression analyses and protein synthesis measurements. Results: The IGF-1 treatment increased cerebellar protein synthesis rates (both in vivo and ex vivo). Performance in the balance beam test was improved by IGF-1 but not in other neurofunctional tests. The treatment decreased total and relative caudate nucleus weights, without any effects to total brain weight or grey/white matter volumes. Supplementation with IGF-1 reduced myelination in caudate nucleus, cerebellum, and white matter regions and decreased hilar synapse formation, without effects to oligodendrocyte maturation or neuron differentiation. Gene expression analyses indicated enhanced maturation of the GABAergic system in the caudate nucleus (decreased NKCC1:KCC2 ratio) with limited effects in cerebellum or hippocampus. Conclusion: Supplemental IGF-1 during the first three weeks after preterm birth may support motor function by enhancing GABAergic maturation in the caudate nucleus, despite reduced myelination. Supplemental IGF-1 may support postnatal brain development in preterm infants, but more studies are required to identify optimal treatment regimens for subgroups of very or extremely preterm infants.

A density-based enrichment measure for assessing colocalization in single-molecule localization microscopy data.

Dual-color single-molecule localization microscopy (SMLM) provides unprecedented possibilities for detailed studies of colocalization of different molecular species in a cell. However, the informational richness of the data is not fully exploited by current analysis tools that often reduce colocalization to a single value. Here, we describe a tool specifically designed for determination of co-localization in both 2D and 3D from SMLM data. The approach uses a function that describes the relative enrichment of one molecular species on the density distribution of a reference species. The function reframes the question of colocalization by providing a density-context relevant to multiple biological questions. Moreover, the function visualize enrichment (i.e. colocalization) directly in the images for easy interpretation. We demonstrate the approach's functionality on both simulated data and cultured neurons, and compare it to current alternative measures. The method is available in a Python function for easy and parameter-free implementation.

Modeling the early stages of Alzheimer's disease by administering intracerebroventricular injections of human native Aß oligomers to rats.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease characterized by the accumulation of aggregated amyloid beta (Aß) and hyperphosphorylated tau along with a slow decline in cognitive functions. Unlike advanced AD, the initial steps of AD pathophysiology have been poorly investigated, partially due to limited availability of animal models focused on the early, plaque-free stages of the disease. The aim of this study was to evaluate the early behavioral, anatomical and molecular alterations in wild-type rats following intracerebroventricular injections of human Aß oligomers (AßOs). Bioactive human AD and nondemented control brain tissue extracts were characterized using ELISA and proteomics approaches. Following a bilateral infusion, rats underwent behavioral testing, including the elevated plus maze, social recognition test, Morris water maze and Y-maze within 6 weeks postinjection. An analysis of brain structure was performed with manganese-enhanced MRI. Collected brain tissues were analyzed using stereology, immunohistochemistry, ELISA and qPCR. No sensorimotor deficits affecting motor performance on different maze tasks were observed, nor was spatial memory disturbed in AD rats. In contrast, a significant impairment of social memory became evident at 21 days postinjection. This deficit was associated with a significantly decreased volume of the lateral entorhinal cortex and a tendency toward a decrease in the total brain volume. Significant increase of cleaved caspase-3-positive cells, microglial activation and proinflammatory responses accompanied by altered expression of synaptic markers were observed in the hippocampus of AD rats with immunohistochemical and qPCR approaches at 6 weeks postinjection. Our data suggest that the social memory impairment observed in AßO-injected rats might be determined by neuroinflammatory responses and synaptopathy. An infusion of native oligomeric Aß in the rat brain represents a feasible tool to model early plaque-free events associated with AD.

Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity.

The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show, by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the DA transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and DA D2 autoreceptor activation in a manner dependent on Ca2+ influx via N-type voltage-gated Ca2+ channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2 autoreceptor, syntaxin-1, and clathrin nanodomains. The data reveal a mechanism for rapid alterations of nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.

Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis.

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

Identifying dominant-negative actions of a dopamine transporter variant in patients with parkinsonism and neuropsychiatric disease.

Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis.

Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains.

Dopamine regulates reward, cognition, and locomotor functions. By mediating rapid reuptake of extracellular dopamine, the dopamine transporter is critical for spatiotemporal control of dopaminergic neurotransmission. Here, we use super-resolution imaging to show that the dopamine transporter is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to, but not overlapping with, the dopamine transporter nanodomains. The molecular organization of the dopamine transporter in nanodomains is reversibly reduced by short-term activation of NMDA-type ionotropic glutamate receptors, implicating dopamine transporter nanodomain distribution as a potential mechanism to modulate dopaminergic neurotransmission in response to excitatory input.The dopamine transporter (DAT) has a crucial role in the regulation of neurotransmission. Here, the authors use super-resolution imaging to show that DAT clusters into cholesterol-dependent membrane regions that are reversibly regulated by ionotropic glutamate receptors activation.

Wheel running exercise attenuates vulnerability to self-administer nicotine in rats.

BACKGROUND: Preventing or postponing tobacco use initiation could greatly reduce the number of tobacco-related deaths. While evidence suggests that exercise is a promising treatment for tobacco addiction, it is not clear whether exercise could prevent initial vulnerability to tobacco use. Thus, using an animal model, we examined whether exercise attenuates vulnerability to the use and reinforcing effects of nicotine, the primary addictive chemical in tobacco. METHODS: Initial vulnerability was assessed using an acquisition procedure wherein exercising (unlocked running wheel, n=10) and sedentary (locked or no wheel, n=12) male adolescent rats had access to nicotine infusions (0.01-mg/kg) during daily 21.5-h sessions beginning on postnatal day 30. Exercise/sedentary sessions (2-h/day) were conducted prior to each of the acquisition sessions. The effects of exercise on nicotine's reinforcing effects were further assessed in separate groups of exercising (unlocked wheel, n=7) and sedentary (no wheel, n=5) rats responding for nicotine under a progressive-ratio schedule with exercise/sedentary sessions (2-h/day) conducted before the daily progressive-ratio sessions. RESULTS: While high rates of acquisition of nicotine self-administration were observed among both groups of sedentary controls, acquisition was robustly attenuated in the exercise group with only 20% of exercising rats meeting the acquisition criterion within the 16-day testing period as compared to 67% of the sedentary controls. Exercise also decreased progressive-ratio responding for nicotine as compared to baseline and to sedentary controls. CONCLUSIONS: Exercise may effectively prevent the initiation of nicotine use in adolescents by reducing the reinforcing effects of nicotine.

Diminished role of dopamine D1-receptor signaling with the development of an addicted phenotype in rats.

BACKGROUND: Although considerable evidence implicates dopamine D1-receptor signaling in the nucleus accumbens in motivation for cocaine during early stages of addiction, less is known with regard to its role after the development of addiction. Here, we examined its role in the development of an addicted phenotype in intact male and female rats, and in female rats that were either resistant or vulnerable to developing this phenotype. METHODS: Intact males, females, and ovariectomized (OVX) females with and without estradiol (vulnerable, OVX+E; resistant, OVX+Veh) were given either short access (ShA) (three fixed-ratio 1 sessions, maximum of 20 infusions) or 24-hour extended access (ExA) to cocaine for 10 days (4 trials/hour). Motivation for cocaine was assessed after a 14-day abstinence period with a progressive-ratio schedule. Once responding stabilized, the effects of intra-accumbens infusion of the D1-receptor antagonist, SCH-23390 (0, .3, 1.0, 3.0 µg), were examined. RESULTS: Motivation for cocaine was markedly higher after abstinence from ExA versus ShA self-administration in intact males and females, indicating the development of an addicted phenotype in these groups. Motivation for cocaine was also higher than ShA control subjects in OVX+E but not OVX+Veh females after ExA self-administration, confirming the categorization of these groups as vulnerable versus resistant. After ExA self-administration, intact males and females and OVX+E but not OVX+Veh females were less sensitive to the effects of D1-receptor antagonism as compared with their ShA counterparts. CONCLUSIONS: These results suggest that the role of D1-receptor signaling, although critical in "nonaddicted" stages, becomes diminished once addiction has developed.

Acute and chronic administration of a low-dose combination of topiramate and ondansetron reduces ethanol's reinforcing effects in male alcohol preferring (P) rats.

Topiramate (a GABA/glutamate modulator) and ondansetron (a serotonin-3 antagonist) have shown promise as treatments for alcohol use disorders (AUDs), although efficacy is modest/variable for both medications. We recently showed in animal models of consumption and relapse that acute treatment with a combination of these medications was more efficacious than either alone. To determine whether the mechanism for its beneficial effects is through modulation of ethanol's reinforcing effects, we measured the effect of this combination in male alcohol preferring (P) rats (N = 22) responding for ethanol under a progressive-ratio (PR) schedule. Low doses, which either do not affect (ondansetron; 0.001 mg/kg) or only modestly affect (topiramate; 10 mg/kg) alcohol-related behaviors on their own, were selected in an attempt to maximize their combined efficacy while minimizing potential side effects. In addition to acute treatment (1 day), the effects of chronic administration (10 days) were examined in an attempt to model human treatment approaches. The effects of the combination were compared with the low dose of topiramate alone hypothesizing that the combination would be more efficacious than topiramate alone. Although both topiramate and the combination similarly reduced PR responding for ethanol following acute treatment and during the initial phase of chronic treatment (Days 1-5), after repeated administration (Days 6-10), only the combination produced a sustained reduction in ethanol-maintained responding. These results suggest an advantage of the combination over topiramate alone at producing a sustained reduction in ethanol's reinforcing effects following prolonged treatment, and lend further support for its use as a potential treatment for AUDs.