Johannes W. de Jong

Combined use of the canine adenovirus-2 and DREADD-technology to activate specific neural pathways in vivo.

We here describe a technique to transiently activate specific neural pathways in vivo. It comprises the combined use of a CRE-recombinase expressing canine adenovirus-2 (CAV-2) and an adeno-associated virus (AAV-hSyn-DIO-hM3D(Gq)-mCherry) that contains the floxed inverted sequence of the designer receptor exclusively activated by designer drugs (DREADD) hM3D(Gq)-mCherry. CAV-2 retrogradely infects projection neurons, which allowed us to specifically express hM3D(Gq)-mCherry in neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (Acb), the majority of which were dopaminergic. Activation of hM3D(Gq)-mCherry by intraperitoneal (i.p.) injections of clozapine-N-oxide (CNO) leads to increases in neuronal activity, which enabled us to specifically activate VTA to Acb projection neurons. The VTA to Acb pathway is part of the mesolimbic dopamine system and has been implicated in behavioral activation and the exertion of effort. Injections of all doses of CNO led to increases in progressive ratio (PR) performance. The effect of the lowest dose of CNO was suppressed by administration of a DRD1-antagonist, suggesting that CNO-induced increases in PR-performance are at least in part mediated by DRD1-signaling. We hereby validate the combined use of CAV-2 and DREADD-technology to activate specific neural pathways and determine consequent changes in behaviorally relevant paradigms.

Ghrelin mediates anticipation to a palatable meal in rats.

Food anticipatory activity (FAA) is displayed in rats when access to food is restricted to a specific time frame of their circadian phase, a behavior thought to reflect both hunger and the motivation to eat. Rats also display FAA in a feeding schedule with ad libitum access to normal chow, but limited availability of a palatable meal, which is thought to involve mainly motivational aspects. The orexigenic hormone ghrelin has been implicated in FAA in rodents with restricted access to chow. Because ghrelin plays an important role not only in the control of food intake, but also in reward, we sought to determine the role of ghrelin in anticipation to a palatable meal. Plasma ghrelin levels of non‐restricted rats that anticipated chocolate correlated positively with FAA and were increased compared with chow‐fed control rats. Furthermore, centrally injected ghrelin increased, whereas an antagonist of the ghrelin receptor decreased, the anticipation to chocolate. Therefore, we hypothesize that central ghrelin signaling is able to mediate the motivational drive to eat.

Reducing Ventral Tegmental Dopamine D2 Receptor Expression Selectively Boosts Incentive Motivation

Altered mesolimbic dopamine signaling has been widely implicated in addictive behavior. For the most part, this work has focused on dopamine within the striatum, but there is emerging evidence for a role of the auto-inhibitory, somatodendritic dopamine D2 receptor (D2R) in the ventral tegmental area (VTA) in addiction. Thus, decreased midbrain D2R expression has been implicated in addiction in humans. Moreover, knockout of the gene encoding the D2R receptor (Drd2) in dopamine neurons has been shown to enhance the locomotor response to cocaine in mice. Therefore, we here tested the hypothesis that decreasing D2R expression in the VTA of adult rats, using shRNA knockdown, promotes addiction-like behavior in rats responding for cocaine or palatable food. Rats with decreased VTA D2R expression showed markedly increased motivation for both sucrose and cocaine under a progressive ratio schedule of reinforcement, but the acquisition or maintenance of cocaine self-administration were not affected. They also displayed enhanced cocaine-induced locomotor activity, but no change in basal locomotion. This robust increase in incentive motivation was behaviorally specific, as we did not observe any differences in fixed ratio responding, extinction responding, reinstatement or conditioned suppression of cocaine, and sucrose seeking. We conclude that VTA D2R knockdown results in increased incentive motivation, but does not directly promote other aspects of addiction-like behavior.

Low Control over Palatable Food Intake in Rats Is Associated with Habitual Behavior and Relapse Vulnerability: Individual Differences

The worldwide obesity epidemic poses an enormous and growing threat to public health. However, the neurobehavioral mechanisms of overeating and obesity are incompletely understood. It has been proposed that addiction-like processes may underlie certain forms of obesity, in particular those associated with binge eating disorder. To investigate the role of addiction-like processes in obesity, we adapted a model of cocaine addiction-like behavior in rats responding for highly palatable food. Here, we tested whether rats responding for highly palatable chocolate Ensure would come to show three criteria of addiction-like behavior, i.e., high motivation, continued seeking despite signaled non-availability and persistence of seeking despite aversive consequences. We also investigated whether exposure to a binge model (a diet consisting of alternating periods of limited food access and access to highly palatable food), promotes the appearance of food addiction-like behavior. Our data show substantial individual differences in control over palatable food seeking and taking, but no distinct subgroup of animals showing addiction-like behavior could be identified. Instead, we observed a wide range extending from low to very high control over palatable food intake. Exposure to the binge model did not affect control over palatable food seeking and taking, however. Animals that showed low control over palatable food intake (i.e., scored high on the three criteria for addiction-like behavior) were less sensitive to devaluation of the food reward and more prone to food-induced reinstatement of extinguished responding, indicating that control over palatable food intake is associated with habitual food intake and vulnerability to relapse. In conclusion, we present an animal model to assess control over food seeking and taking. Since diminished control over food intake is a major factor in the development of obesity, understanding its behavioral and neural underpinnings may facilitate improved management of the obesity epidemic.

The mesolimbic system and eating addiction: what sugar does and does not do

Obesity and obesity-related disorders are a major threat to public health. It has been suggested that food addiction is a valid clinical concept and that food addiction is a contributing factor to the obesity epidemic. Research involving restricted access ‘binge’ diets has shown that rodents will display sucrose-related behavior that is reminiscent of substance addiction, under certain conditions. A question that remains, however, is if food or certain components of food possess addictive qualities akin to drugs of abuse. The alternative is that ‘food addiction’ (or rather ‘eating addiction’) is not a substance use disorder in the sense that people are addicted to any specific substance or component of food, but rather an addictive disorder involving disinhibition of food intake in general that shares similarities with behavioral addictions such as problem gambling. Here we describe how sugar (a candidate addictive component of frequently consumed foods) has short and long term effects on the brain and compare this to how addictive substances functionally alter the mesolimbic dopamine system. We focus on this system since plasticity changes in the mesolimbic system have been implicated in the development of drug addiction. We conclude that sugar has a strong direct influence on the dopamine system, which underlies its profound reinforcing qualities. However, at present there is limited evidence to suggest that sugar intake induces plasticity changes comparable to those induced by drugs of abuse. Thus, based on current literature we propose that it is probably that the long term effects of sugar on the brain are both qualitatively as well as quantitatively different from those of addictive substances.

Enhancing excitability of dopamine neurons promotes motivational behaviour through increased action initiation

Motivational deficits are a key symptom in multiple psychiatric disorders, including major depressive disorder, schizophrenia and addiction. A likely neural substrate for these motivational deficits is the brain dopamine (DA) system. In particular, DA signalling in the nucleus accumbens, which originates from DA neurons in the ventral tegmental area (VTA), has been identified as a crucial substrate for effort-related and activational aspects of motivation. Unravelling how VTA DA neuronal activity relates to motivational behaviours is required to understand how motivational deficits in psychiatry can be specifically targeted. In this study, we therefore used designer receptors exclusively activated by designer drugs (DREADD) in TH:Cre rats, in order to determine the effects of chemogenetic DA neuron activation on different aspects of motivational behaviour. We found that chemogenetic activation of DA neurons in the VTA, but not substantia nigra, significantly increased responding for sucrose under a progressive ratio schedule of reinforcement. More specifically, high effort exertion was characterized by increased initiations of reward-seeking actions. This effect was dependent on effort requirements and instrumental contingencies, but was not affected by sucrose pre-feeding. Together, these findings indicate that VTA DA neuronal activation drives motivational behaviour by facilitating action initiation. With this study, we show that enhancing excitability of VTA DA neurons is a viable strategy to improve motivational behaviour.

A novel approach to map induced activation of neuronal networks using chemogenetics and functional neuroimaging in rats: A proof-of-concept study on the mesocorticolimbic system

&NA; Linking neural circuit activation at whole‐brain level to neuronal activity at cellular level remains one of the major challenges in neuroscience research. We set up a novel functional neuroimaging approach to map global effects of locally induced activation of specific midbrain projection neurons using chemogenetics (Designer Receptors Exclusively Activated by Designer Drugs (DREADD)‐technology) combined with pharmacological magnetic resonance imaging (phMRI) in the rat mesocorticolimbic system. Chemogenetic activation of DREADD‐targeted mesolimbic or mesocortical pathways, i.e. projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) or medial prefrontal cortex (mPFC), respectively, induced significant blood oxygenation level‐dependent (BOLD) responses in areas with DREADD expression, but also in remote defined neural circuitry without DREADD expression. The time‐course of brain activation corresponded with the behavioral output measure, i.e. locomotor (hyper)activity, in the mesolimbic pathway‐targeted group. Chemogenetic activation specifically increased neuronal activity, whereas functional connectivity assessed with resting state functional MRI (rs‐fMRI) remained stable. Positive and negative BOLD responses distinctively reflected simultaneous ventral pallidum activation and substantia nigra pars reticulata deactivation, respectively, demonstrating the concept of mesocorticolimbic network activity with concurrent activation of the direct and indirect pathways following stimulation of specific midbrain projection neurons. The presented methodology provides straightforward and widely applicable opportunities to elucidate relationships between local neuronal activity and global network activity in a controllable manner, which will increase our understanding of the functioning and dysfunctioning of large‐scale neuronal networks in health and disease. HighlightsChemogenetics and fMRI bridge functional brain studies from cell to systems level.DREADD‐phMRI maps large‐scale network activity after projection‐specific activation.DREADD‐phMRI identifies activated and deactivated neural circuits.Activated VTA projection neurons distinctively trigger direct and indirect pathways.

FTO knockdown in rat ventromedial hypothalamus does not affect energy balance

Single nucleotide polymorphisms (SNPs) clustered in the first intron of the fat mass and obesity‐associated (FTO) gene has been associated with obesity. FTO expression is ubiquitous, with particularly high levels in the hypothalamic area of the brain. To investigate the region‐specific role of FTO, AAV technology was applied to knockdown FTO in the ventromedial hypothalamus (VMH). No effect of FTO knockdown was observed on bodyweight or parameters of energy balance. Animals were exposed twice to an overnight fast, followed by a high‐fat high‐sucrose (HFHS) diet for 1 week. FTO knockdown did not result in a different response to the diets. A region‐specific role for FTO in the VMH in the regulation of energy balance could not be found.

Abstract 3005: Vascular endothelial growth factor A - a systematic review and meta-analysis of expression patterns in breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background Vascular Endothelial Growth Factor-A (VEGF-A), pivotal for neo-angiogenesis, is an interesting target for breast cancer molecular imaging. To assess its potential clinical value, we performed a systematic review and meta-analysis of VEGF-A expression rates in normal breast tissue, benign and (pre-)invasive breast disease, and investigated associations with clinicopathological characteristics. Methods We systematically searched EMBASE and MEDLINE for articles describing VEGF-A expression in breast disease assessed by immunohistochemistry (IHC) or Enzyme-Linked Immunosorbent Assay (ELISA). We pooled IHC expression rates with random-effects models and applied meta-regression to identify associations with clinicopathological characteristics. We summarized findings for ELISA results. Results Of 2,711 unique articles retrieved, 157 studies described IHC results (16,046 tissue samples). Pooled expression rates were lower in studies applying antibody clone VG1 (-21%, p=0.018) or C-1 (-24%, p=0.016), compared to A-20. Expression compared to invasive carcinoma was lower in normal tissue (-51%, p<0.001) and benign breast diseases (-34%, p<0.001), but comparable for carcinoma in situ (+5%, p=0.289). Expression rates increased with tumour grade (II: +14%, p<0.001; III: +25%, p<0.001), stage (II: +11%, p<0.001; III: +22%, p<0.001), size (T3/4: +11%, p=0.004), HER2-positivity (+14%, p<0.001), lymph node positivity(+11%, p<0.001), and decreased in hormone receptor positive (PR-positive: -7%, p<0.001; ER-positive:-8%, p<0.001) and lobular cancers (-14%, p<0.001). Substantial heterogeneity was observed in study results. Furthermore, expression rates strongly depended on VEGF-A positivity threshold. VEGF-A levels assessed by ELISA (42 studies, 8,498 samples) were highly variable. Conclusion Although reported VEGF-A expression rates in breast cancer were variable, we found significant correlations with clinicopathological characteristics indicating aggressive disease. Also, expression rates were low in normal breast tissue and benign breast disease. As expression rates strongly depended on VEGF-A positivity - arguing against an on/off phenomenon - applicability of VEGF-A for molecular imaging purposes will depend on sensitivity of the molecular imaging modality, target population, and envisioned applications. Acknowledgment This research was supported by the Center for Translational Molecular Medicine (MAMMOTH). Citation Format: Arthur Adams, Jeroen Vermeulen, Peter Zuithoff, Elsken Van der Wall, Laetitia Lamberts, Elisabeth de Vries, Johannes de Jong, Gooitzen Van Dam, Paul J. van Diest, Willem P.Th.M. Mali, Sjoerd G. Elias. Vascular endothelial growth factor A - a systematic review and meta-analysis of expression patterns in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3005. doi:10.1158/1538-7445.AM2014-3005