Zona incerta neurons projecting to the ventral tegmental area promote action initiation towards feeding.

KEY POINTS: The zona incerta (ZI) and ventral tegmental area (VTA) are brain areas that are both implicated in feeding behaviour. The ZI projects to the VTA, although it has not yet been investigated whether this projection regulates feeding. We experimentally (in)activated the ZI to VTA projection by using dual viral vector technology, and studied the effects on feeding microstructure, the willingness to work for food, general activity and body temperature. Activity of the ZI to VTA projection promotes feeding by facilitating action initiation towards food, as reflected in meal frequency and the willingness to work for food reward, without affecting general activity or directly modulating body temperature. We show for the first time that activity of the ZI to VTA projection promotes feeding, which improves the understanding of the neurobiology of feeding behaviour and body weight regulation. ABSTRACT: Both the zona incerta (ZI) and the ventral tegmental area (VTA) have been implicated in feeding behaviour. The ZI provides prominent input to the VTA, although it has not yet been investigated whether this projection regulates feeding. Therefore, we investigated the role of ZI to VTA projection neurons in the regulation of several aspects of feeding behaviour. We determined the effects of (in)activation of ZI to VTA projection neurons on feeding microstructure, food-motivated behaviour under a progressive ratio schedule of reinforcement, locomotor activity and core body temperature. To activate or inactivate ZI neurons projecting to the VTA, we used a combination of canine adenovirus-2 in the VTA, as well as Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) or tetanus toxin (TetTox) light chain in the ZI. TetTox-mediated inactivation of ZI to VTA projection neurons reduced food-motivated behaviour and feeding by reducing meal frequency. Conversely, DREADD-mediated chemogenetic activation of ZI to VTA projection neurons promoted food-motivated behaviour and feeding. (In)activation of ZI to VTA projection neurons did not affect locomotor activity or directly regulate core body temperature. Taken together, ZI neurons projecting to the VTA exert bidirectional control overfeeding behaviour. More specifically, activity of ZI to VTA projection neurons facilitate action initiation towards feeding, as reflected in both food-motivated behaviour and meal initiation, without affecting general activity.

Is leptin resistance the cause or the consequence of diet-induced obesity?

BACKGROUND/OBJECTIVES: Obesity is strongly associated with leptin resistance. It is unclear whether leptin resistance results from the (over)consumption of energy-dense diets or if reduced leptin sensitivity is also a pre-existing factor in rodent models of diet-induced obesity (DIO). We here tested whether leptin sensitivity on a chow diet predicts subsequent weight gain and leptin sensitivity on a free choice high-fat high-sucrose (fcHFHS) diet. METHODS: Based upon individual leptin sensitivity on chow diet, rats were grouped in leptin sensitive (LS, n = 22) and leptin resistant (LR, n = 19) rats (P = 0.000), and the development of DIO on a fcHFHS diet was compared. The time-course of leptin sensitivity was measured over weeks in individual rats. RESULTS: Both on a chow and a fcHFHS diet, high variability in leptin sensitivity was observed between rats, but not over time per individual rat. Exposure to the fcHFHS diet revealed that LR rats were more prone to develop DIO (P = 0.013), which was independent of caloric intake (p ≥ 0.320) and the development of diet-induced leptin resistance (P = 0.769). Reduced leptin sensitivity in LR compared with LS rats before fcHFHS diet exposure, was associated with reduced leptin-induced phosphorylated signal transducer and activator of transcription 3 (pSTAT3) levels in the dorsomedial and ventromedial hypothalamus (P ≤ 0.049), but not the arcuate nucleus (P = 0.558). CONCLUSIONS: A pre-existing reduction in leptin sensitivity determines the susceptibility to develop excessive DIO after fcHFHS diet exposure. Rats with a pre-existing reduction in leptin sensitivity develop excessive DIO without eating more calories or altering their leptin sensitivity.

Rats that are predisposed to excessive obesity show reduced (leptin-induced) thermoregulation even in the preobese state.

Both feeding behavior and thermogenesis are regulated by leptin. The sensitivity to leptin's anorexigenic effects on chow diet was previously shown to predict the development of diet-induced obesity. In this study, we determined whether the sensitivity to leptin's anorexigenic effects correlates with leptin's thermogenic response, and if this response is exerted at the level of the dorsomedial hypothalamus (DMH), a brain area that plays an important role in thermoregulation. Based on the feeding response to injected leptin on a chow diet, rats were divided into leptin-sensitive (LS) and leptin-resistant (LR) groups. The effects of leptin on core body, brown adipose tissue (BAT) and tail temperature were compared after intravenous versus intra-DMH leptin administration. After intravenous leptin injection, LS rats increased their BAT thermogenesis and reduced heat loss via the tail, resulting in a modest increase in core body temperature. The induction of these thermoregulatory mechanisms with intra-DMH leptin was smaller, but in the same direction as with intravenous leptin administration. In contrast, LR rats did not show any thermogenic response to either intravenous or intra-DMH leptin. These differences in the thermogenic response to leptin were associated with a 1°C lower BAT temperature and reduced UCP1 expression in LR rats under ad libitum feeding. The preexisting sensitivity to the anorexigenic effects of leptin, a predictor for obesity, correlates with the sensitivity to the thermoregulatory effects of leptin, which appears to be exerted, at least in part, at the level of the DMH.

Anatomical projections of the dorsomedial hypothalamus to the periaqueductal grey and their role in thermoregulation: a cautionary note.

The DMH is known to regulate brown adipose tissue (BAT) thermogenesis via projections to sympathetic premotor neurons in the raphe pallidus, but there is evidence that the periaqueductal gray (PAG) is also an important relay in the descending pathways regulating thermogenesis. The anatomical projections from the DMH to the PAG subdivisions and their function are largely elusive, and may differ per anterior-posterior level from bregma. We here aimed to investigate the anatomical projections from the DMH to the PAG along the entire anterior-posterior axis of the PAG, and to study the role of these projections in thermogenesis in Wistar rats. Anterograde channel rhodopsin viral tracing showed that the DMH projects especially to the dorsal and lateral PAG. Retrograde rabies viral tracing confirmed this, but also indicated that the PAG receives a diffuse input from the DMH and adjacent hypothalamic subregions. We aimed to study the role of the identified DMH to PAG projections in thermogenesis in conscious rats by specifically activating them using a combination of canine adenovirus-2 (CAV2Cre) and Cre-dependent designer receptor exclusively activated by designer drugs (DREADD) technology. Chemogenetic activation of DMH to PAG projections increased BAT temperature and core body temperature, but we cannot exclude the possibility that at least some thermogenic effects were mediated by adjacent hypothalamic subregions due to difficulties in specifically targeting the DMH and distinct subdivisions of the PAG because of diffuse virus expression. To conclude, our study shows the complexity of the anatomical and functional connection between the hypothalamus and the PAG, and some technical challenges in studying their connection.

Individual differences in voluntary alcohol intake in rats: relationship with impulsivity, decision making and Pavlovian conditioned approach.

RATIONALE: Alcohol use disorder (AUD) has been associated with suboptimal decision making, exaggerated impulsivity, and aberrant responses to reward-paired cues, but the relationship between AUD and these behaviors is incompletely understood. OBJECTIVES: This study aims to assess decision making, impulsivity, and Pavlovian-conditioned approach in rats that voluntarily consume low (LD) or high (HD) amounts of alcohol. METHODS: LD and HD were tested in the rat gambling task (rGT) or the delayed reward task (DRT). Next, the effect of alcohol (0-1.0 g/kg) was tested in these tasks. Pavlovian-conditioned approach (PCA) was assessed both prior to and after intermittent alcohol access (IAA). Principal component analyses were performed to identify relationships between the most important behavioral parameters. RESULTS: HD showed more optimal decision making in the rGT. In the DRT, HD transiently showed reduced impulsive choice. In both LD and HD, alcohol treatment increased optimal decision making in the rGT and increased impulsive choice in the DRT. PCA prior to and after IAA was comparable for LD and HD. When PCA was tested after IAA only, HD showed a more sign-tracking behavior. The principal component analyses indicated dimensional relationships between alcohol intake, impulsivity, and sign-tracking behavior in the PCA task after IAA. CONCLUSIONS: HD showed a more efficient performance in the rGT and DRT. Moreover, alcohol consumption enhanced approach behavior to reward-predictive cues, but sign-tracking did not predict the level of alcohol consumption. Taken together, these findings suggest that high levels of voluntary alcohol intake are associated with enhanced cue- and reward-driven behavior.

Cardiac ion channel trafficking defects and drugs.

Fine control over the functional expression of cardiac ion channels is required to maintain normal action potential (AP) duration and QTc times. A growing number of drugs interfere with normal trafficking of ion channels to and from the plasma membrane, thereby altering the number of channels on the cell surface. Most drugs do this at clinically relevant concentrations, which may lead to potentially life-threatening cardiac arrhythmias. Recently, major progress has been made in the understanding of the subcellular mechanisms by which drugs affect the trafficking of ion channels, which is of great benefit for the development of ways to counteract these adverse drug effects. Pharmacological correction seems to be a promising approach to address the trafficking defects induced by several drugs. However, as pharmacological correction is hampered by concomitant direct channel block or unspecific effects, further studies are needed to improve its potential as a clinical therapy.

Efficient and specific cardiac IK1 inhibition by a new pentamidine analogue.

AIMS: In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK1) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK1 by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK1 inhibitors based on the P structure. METHODS AND RESULTS: We analysed seven pentamidine analogues (PA-1 to PA-7) for IK1 blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC50 values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK1 in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 µM. However, PA-6 at 10 µM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 µM. CONCLUSION: PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK1 block.