Stress-driven potentiation of lateral hypothalamic synapses onto ventral tegmental area dopamine neurons causes increased consumption of palatable food.

Stress can cause overconsumption of palatable high caloric food. Despite the important role of stress eating in obesity and (binge) eating disorders, its underlying neural mechanisms remain unclear. Here we demonstrate in mice that stress alters lateral hypothalamic area (LHA) control over the ventral tegmental area (VTA), thereby promoting overconsumption of palatable food. Specifically, we show that glutamatergic LHA neurons projecting to the VTA are activated by social stress, after which their synapses onto dopamine neurons are potentiated via AMPA receptor subunit alterations. We find that stress-driven strengthening of these specific synapses increases LHA control over dopamine output in key target areas like the prefrontal cortex. Finally, we demonstrate that while inducing LHA-VTA glutamatergic potentiation increases palatable fat intake, reducing stress-driven potentiation of this connection prevents such stress eating. Overall, this study provides insights in the neural circuit adaptations caused by stress that drive overconsumption of palatable food.

Characterizing and TRAPing a Social Stress-Activated Neuronal Ensemble in the Ventral Tegmental Area.

Social stress is a major contributor to neuropsychiatric issues such as depression, substance abuse and eating disorders. The ventral tegmental area (VTA) is involved in the effects of stress on cognitive and emotional processes perturbed in these disorders. However, the VTA is a cellularly heterogeneous brain area and it remains unclear which of its neuronal populations make up the social stress-sensitive ensemble. The current study characterizes the molecular, topographical and functional properties of VTA social stress-activated cells. First, we used immunohistochemical analysis of Fos protein, a marker of recent increased neuronal activity, to show that acute social stress activates a mainly neuronal ensemble in the VTA (VTASocial stress neurons). Topographical analysis showed that this ensemble, which comprises ∼11% of all VTA neurons, occurs across VTA subregions. Further analysis showed that approximately half of the VTASocial stress neurons express the dopamine synthesis rate-limiting enzyme tyrosine hydroxylase (TH). In a minority of cases this occurred with coexpression of vesicular glutamate transporter 2 (Vglut2). Also part of the ensemble were VTA cells expressing just Vglut2 without TH, and cells expressing the vesicular GABA transporter (VGAT) without TH. Next, using targeted recombination in active populations (TRAP2), we showed that VTASocial stress neurons can be permanently tagged and made tractable for future functional investigations. Using a combination of TRAP2 and patch-clamp electrophysiology we demonstrate that VTASocial stress neurons exhibit higher excitability than their non-TRAPed neighbor cells. Overall, this study shows that acute social stress activates an ensemble of neurons throughout the VTA, comprising distinct molecular identities, and with specific electrophysiological features. It also identifies TRAP2 as a tool to make this ensemble tractable for future functional studies.

Identification of Novel Neurocircuitry Through Which Leptin Targets Multiple Inputs to the Dopamine System to Reduce Food Reward Seeking.

BACKGROUND: Leptin reduces the motivation to obtain food by modulating activity of the mesolimbic dopamine (DA) system upon presentation of cues that predict a food reward. Although leptin directly reduces the activity of ventral tegmental area (VTA) DA neurons, the majority of leptin receptor (LepR)-expressing DA neurons do not project to the nucleus accumbens, the projection implicated in driving food reward seeking. Therefore, the precise locus of leptin action to modulate motivation for a food reward is unresolved. METHODS: We used transgenic mice expressing Cre recombinase under the control of the LepR promoter, anatomical tracing, optogenetics-assisted patch-clamp electrophysiology, in vivo optogenetics with fiber photometric calcium measurements, and chemogenetics to unravel how leptin-targeted neurocircuitry inhibits food reward seeking. RESULTS: A large number of DA neurons projecting to the nucleus accumbens are innervated by local VTA LepR-expressing GABA (gamma-aminobutyric acid) neurons. Leptin enhances the activity of these GABA neurons and thereby inhibits nucleus accumbens-projecting DA neurons. In addition, we find that lateral hypothalamic LepR-expressing neurons projecting to the VTA are inhibited by leptin and that these neurons modulate DA neurons indirectly via inhibition of VTA GABA neurons. In accordance with such a disinhibitory function, optogenetically stimulating lateral hypothalamic LepR projections to the VTA potently activates DA neurons in vivo. Moreover, we found that chemogenetic activation of lateral hypothalamic LepR neurons increases the motivation to obtain a food reward only when mice are in a positive energy balance. CONCLUSIONS: We identify neurocircuitry through which leptin targets multiple inputs to the DA system to reduce food reward seeking.

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.

Effects of GABA and Leptin Receptor-Expressing Neurons in the Lateral Hypothalamus on Feeding, Locomotion, and Thermogenesis.

OBJECTIVE: The lateral hypothalamus (LH) is known for its role in feeding, and it also regulates other aspects of energy homeostasis. How genetically defined LH neuronal subpopulations mediate LH effects on energy homeostasis remains poorly understood. The behavioral effects of chemogenetically activating LH gamma-aminobutyric acid (GABA) and the more selective population of LH GABA neurons that coexpress the leptin receptor (LepR) were compared. METHODS: LepR-cre and VGAT-cre mice were injected with AAV5-hSyn-DIO-hM3DGq-mCherry in the LH. The behavioral effects of LH GABA or LH LepR neuronal activation on feeding, locomotion, thermogenesis, and body weight were assessed. RESULTS: The activation of LH GABA neurons increased body temperature (P ≤ 0.008) and decreased body weight (P ≤ 0.01) despite decreased locomotor activity (P = 0.03) and transiently increased chow intake (P ≤ 0.009). Also, similar to other studies, this study found that activation of LH GABA neurons induced gnawing on both food and nonfood (P = 0.001) items. Activation of LH LepR neurons decreased body weight (P ≤ 0.01) and chow intake when presented on the cage floor (P ≤ 0.04) but not when presented in the cage top and increased locomotor activity (P = 0.002) and body temperature (P = 0.03). CONCLUSIONS: LH LepR neurons are a subset of LH GABA neurons, and LH LepR activation more specifically regulates energy homeostasis to promote a negative energy balance.

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.

Chemogenetic activation of dopamine neurons in the ventral tegmental area, but not substantia nigra, induces hyperactivity in rats.

Hyperactivity is a core symptom in various psychiatric disorders, including attention-deficit/hyperactivity disorder, schizophrenia, bipolar disorders, and anorexia nervosa. Although hyperactivity has been linked to dopaminergic signalling, the causal relationship between midbrain dopamine neuronal activity and locomotor hyperactivity remains unknown. In this study, we test whether increased dopamine neuronal activity is sufficient to induce locomotor hyperactivity. To do so, we used designer receptors exclusively activated by designer drugs (DREADD) to chemogenetically enhance neuronal activity in two main midbrain dopamine neuron populations, i.e. the ventral tegmental area (VTA) and substantia nigra pars compacta (SN), in TH:Cre rats. We found that activation of VTA dopamine neurons induced a pronounced and long-lasting hyperactive phenotype, whilst SN dopamine neuron activation only modestly increased home cage locomotion. Furthermore, this hyperactive phenotype was replicated by selective activation of the neuronal pathway from VTA to the nucleus accumbens (NAC). These results show a clear functional difference between neuronal subpopulations in the VTA and SN with regards to inducing locomotor hyperactivity, and suggest that the dopaminergic pathway from VTA to NAC may be a promising target for the treatment of hyperactivity disorders.

Mapping of a FEB3 homologous febrile seizure locus on mouse chromosome 2 containing candidate genes Scn1a and Scn3a.

Febrile seizures (FS) are the most common seizure type in children. Recurrent FS are a risk factor for developing temporal lobe epilepsy later in life and are known to have a strong genetic component. Experimental FS (eFS) can be elicited in mice by warm-air induced hyperthermia. We used this model to screen the chromosome substitution strain (CSS) panel derived from C57BL/6J and A/J for FS susceptibility and identified C57BL/6J-Chr2A /NaJ (CSS2), as the strain with the strongest FS susceptibility phenotype. The aim of this study was to map FS susceptibility loci and select candidate genes on mouse chromosome 2. We generated an F2 population by intercrossing the hybrids (F1 ) that were derived from CSS2 and C57BL/6J mice. All CSS2-F2 individuals were genotyped and phenotyped for eFS susceptibility, and QTL analysis was performed. Candidate gene selection was based on bioinformatics analyses and differential brain expression between CSS2 and C57BL/6J strains determined by microarray analysis. Genetic mapping of the eFS susceptibility trait identified two significant loci: FS-QTL2a (LOD-score 3.6) and FS-QTL2b (LOD-score 6.2). FS-QTL2a contained 44 genes expressed in the brain at post natal day 14. Four of these (Arl6ip6, Cytip, Fmnl2 Ifih1) contained a non-synonymous SNP comparing CSS2 and C57BL/6J, six genes (March7, Nr4a2, Gpd2, Grb14, Scn1a, Scn3a) were differentially expressed between these strains. A region within FS-QTL2a is homologous to the human FEB3 locus. The fact that we identify mouse FS-QTL2a with high FEB3 homology is strong support for the validity of the eFS mouse model to study genetics of human FS.

Expression Profiling after Prolonged Experimental Febrile Seizures in Mice Suggests Structural Remodeling in the Hippocampus.

Febrile seizures are the most prevalent type of seizures among children up to 5 years of age (2-4% of Western-European children). Complex febrile seizures are associated with an increased risk to develop temporal lobe epilepsy. To investigate short- and long-term effects of experimental febrile seizures (eFS), we induced eFS in highly febrile convulsion-susceptible C57BL/6J mice at post-natal day 10 by exposure to hyperthermia (HT) and compared them to normotherm-exposed (NT) mice. We detected structural re-organization in the hippocampus 14 days after eFS. To identify molecular candidates, which entrain this structural re-organization, we investigated temporal changes in mRNA expression profiles eFS 1 hour to 56 days after eFS. We identified 931 regulated genes and profiled several candidates using in situ hybridization and histology at 3 and 14 days after eFS. This is the first study to report genome-wide transcriptome analysis after eFS in mice. We identify temporal regulation of multiple processes, such as stress-, immune- and inflammatory responses, glia activation, glutamate-glutamine cycle and myelination. Identification of the short- and long-term changes after eFS is important to elucidate the mechanisms contributing to epileptogenesis.

Identification of Srp9 as a febrile seizure susceptibility gene.

OBJECTIVE: Febrile seizures (FS) are the most common seizure type in young children. Complex FS are a risk factor for mesial temporal lobe epilepsy (mTLE). To identify new FS susceptibility genes we used a forward genetic strategy in mice and subsequently analyzed candidate genes in humans. METHODS: We mapped a quantitative trait locus (QTL1) for hyperthermia-induced FS on mouse chromosome 1, containing the signal recognition particle 9 (Srp9) gene. Effects of differential Srp9 expression were assessed in vivo and in vitro. Hippocampal SRP9 expression and genetic association were analyzed in FS and mTLE patients. RESULTS: Srp9 was differentially expressed between parental strains C57BL/6J and A/J. Chromosome substitution strain 1 (CSS1) mice exhibited lower FS susceptibility and Srp9 expression than C57BL/6J mice. In vivo knockdown of brain Srp9 reduced FS susceptibility. Mice with reduced Srp9 expression and FS susceptibility, exhibited reduced hippocampal AMPA and NMDA currents. Downregulation of neuronal Srp9 reduced surface expression of AMPA receptor subunit GluA1. mTLE patients with antecedent FS had higher SRP9 expression than patients without. SRP9 promoter SNP rs12403575(G/A) was genetically associated with FS and mTLE. INTERPRETATION: Our findings identify SRP9 as a novel FS susceptibility gene and indicate that SRP9 conveys its effects through endoplasmic reticulum (ER)-dependent synthesis and trafficking of membrane proteins, such as glutamate receptors. Discovery of this new FS gene and mechanism may provide new leads for early diagnosis and treatment of children with complex FS at risk for mTLE.

Mapping an X-linked locus that influences heat-induced febrile seizures in mice.

PURPOSE: Febrile seizures (FS) are the most common seizure type in children between the age of 6 months and 5 years. Although FS are largely benign, recurrent FS are a major risk factor for developing temporal lobe epilepsy (TLE) later in life. The mechanisms underlying FS are largely unknown; however, family and twin studies indicate that FS susceptibility is under complex genetic control. We have recently developed a phenotypic screen to study the genetics of FS susceptibility in mice. Using this screen in a phenotype-driven genetic strategy we analyzed the C57BL/6J-Chr #(A)/NaJ chromosome substitution strain (CSS) panel. In each CSS line one chromosome of the A/J strain is substituted in a genetically homogeneous C57BL/6J background. The analysis of the CSS panel revealed that A/J chromosomes 1, 2, 6, 10, 13, and X carry at least one quantitative trait locus (QTL) for heat-induced FS susceptibility. The fact that many X-linked genes are highly expressed in the brain and have been implicated in human developmental disorders often presenting with seizures (like fragile X mental retardation) prompted us to map the chromosome X QTL. METHODS: C57BL/6J mice were mated with C57BL/6J-Chr X(A) /NaJ (CSSX) to generate F(2)-generations-CXBL6 and BL6CX-originating from CSSX or C57BL/6J mothers, respectively. Heat-induced FS were elicited on postnatal day 14 by exposure to a controlled warm airstream of 50°C. The latency to heat-induced FS is our phenotype. This phenotype has previously been validated by video-electroencephalography (EEG) monitoring. After phenotyping and genotyping the F(2)-population, QTL analysis was performed using R/QTL software. KEY FINDINGS: QTL analysis revealed a significant peak with an LOD-score of 3.25. The 1-LOD confidence interval (149,886,866-158,836,462 bp) comprises 52 protein coding genes, of which 34 are known to be brain expressed. Two of these brain-expressed genes have previously been linked to X-linked epilepsies, namely Cdkl5 and Pdha1. SIGNIFICANCE: Our results show that the mouse genetics of X-linked FS susceptibility is complex, and that our heat-induced FS-driven genetic approach is a powerful tool for use in unraveling the complexities of this trait in mice. Fine-mapping and functional studies will be required to further identify the X-linked FS susceptibility genes.

Home seeking behavior in rat pups: Attachment vs. kin selection, oxytocin vs. vasopressin.

We are interested in the rat as an animal model for infant-mother attachment. In the first experiment we tried to distinguish between a preference for familiar animals (attachment theory) and a preference for genetically related animals (kin selection theory) with the use of an early cross-fostering procedure. Genetic relationships did not influence preferences in cross-fostered pups on postnatal day 17, only familiarity did. Subsequently we investigated if peptides known to influence affiliative behaviors were also effective in the present paradigm. Injection of oxytocin into the cisterna magna did not yield significant effects on preference, while vasopressin and desglycinamide-[Arg8]vasopressin reduced the preference in a dose dependent manner. The effect of vasopressin was completely blocked by pretreatment with the vasopressin V(1A) receptor antagonist d(CH2)5Tyr(Me)(2),Arg(8)-vasopressin. We discuss the explanatory power of attachment theory and kin selection theory with regard to preference experiments in rats and the usefulness of the rat as an animal model for infant-mother attachment.

A method for gender determination in newborn dark pigmented mice.

In many studies using mice, investigators must determine pups' gender at a very early postnatal stage. The gender of mouse pups is typically assessed by measuring the anogenital distance, which is greater in males than in females. This method, however, has proven to be difficult and not completely reliable. The authors describe a quick, easy and reliable method to establish the gender of pigmented mice. In male mice, a pigment spot on the scrotum is visible to the naked eye from the first day of life onwards, whereas female pups lack visible pigmentation in the anogenital region. In lightly pigmented or albino mice, the pigmentation is not obvious or not at all visible. The authors show that identifying this pigment spot is a more accurate and efficient method of determining pup gender compared with measurement of the anogenital distance. This 'spot on' method would therefore be a useful adjunct to conventional methods for determining the gender of pigmented neonatal mice.

Neonatal amygdala lesions affect appetitive motivational and consummatory aspects of social behavior in the rat.

In the present study, rats received amygdala lesions (AMX) on either Postnatal Day 7 (PD 7; immature brain) or PD 21 (almost mature brain), and adult social activity was studied after short-term isolation housing. Sham-operated rats demonstrated increased following and approaching behavior after 7 days of isolation compared with after 4 days of isolation, an effect that was absent in AMX-PD 7 and AMX-PD 21 rats. Furthermore, AMX-PD 7 rats, but not AMX-PD 21 rats, displayed a reduction in investigatory behavior after prolonged isolation. This indicates that in AMX-PD 21 rats, mainly appetitive motivational aspects of social behavior were affected, whereas in AMX-PD 7 rats both motivational and consummatory aspects were disturbed. Finally, the reported deficits in AMX-PD 7 rats may reflect neurodevelopmental deficits of structures connected with the amygdala.