Characterization of orexin input to dopamine neurons of the ventral tegmental area projecting to the medial prefrontal cortex and shell of nucleus accumbens

Orexin neurons are involved in homeostatic regulatory processes, including arousal and feeding, and provide a major input from the hypothalamus to the ventral tegmental area (VTA) of the midbrain. VTA neurons are a central hub processing reward and motivation and target the medial prefrontal cortex (mPFC) and the shell part of nucleus accumbens (NAcs). We investigated whether subpopulations of dopamine (DA) neurons in the VTA projecting either to the mPFC or the medial division of shell part of nucleus accumbens (mNAcs) receive differential input from orexin neurons and whether orexin exerts differential electrophysiological effects upon these cells. VTA neurons projecting to the mPFC or the mNAcs were traced retrogradely by Cav2-Cre virus and identified by expression of yellow fluorescent protein (YFP). Immunocytochemical analysis showed that a higher proportion of all orexin-innervated DA neurons projected to the mNAcs (34.5%) than to the mPFC (5.2%). Of all sampled VTA neurons projecting either to the mPFC or mNAcs, the dopaminergic (68.3 vs. 79.6%) and orexin-innervated DA neurons (68.9 vs. 64.4%) represented the major phenotype. Whole-cell current clamp recordings were obtained from fluorescently labeled neurons in slices during baseline periods and bath application of orexin A. Orexin similarly increased the firing rate of VTA dopamine neurons projecting to mNAcs (1.99 ± 0.61 Hz to 2.53 ± 0.72 Hz) and mPFC (0.40 ± 0.22 Hz to 1.45 ± 0.56 Hz). Thus, the hypothalamic orexin system targets mNAcs and to a lesser extent mPFC-projecting dopaminergic neurons of the VTA and exerts facilitatory effects on both clusters of dopamine neurons

GelMap: intrinsic calibration and deformation mapping for expansion microscopy

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground-truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows

GelMap: intrinsic calibration and deformation mapping for expansion microscopy

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground-truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows

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 (VTA Social 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 VTA Social 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 VTA Social 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 VTA Social 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

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

ALS-associated C21ORF2 variant disrupts DNA damage repair, mitochondrial metabolism, neuronal excitability and NEK1 levels in human motor neurons

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS, but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore, we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2, KIF5A, NEK1, TBK1, and TUBA4A) which highlighted many novel binding partners, and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system, and of ALS-associated C21ORF2 variants is largely unknown. Therefore, we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First, our data show C21ORF2 expression in ALS-relevant mouse and human neurons, such as spinal and cortical motor neurons. Further, the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients, but not isogenic controls, show increased apoptosis, and changes in DNA damage response, mitochondria and neuronal excitability. In addition, C21ORF2-V58L induced post-transcriptional downregulation of NEK1, an ALS-associated protein implicated in apoptosis and DDR. In all, our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS

Correction:How the COVID-19 pandemic highlights the necessity of animal research (vol 30, pg R1014, 2020)

(Current Biology 30, R1014–R1018; September 21, 2020) As a result of an author oversight in the originally published version of this article, a number of errors were introduced in the author list and affiliations. First, the middle initials were omitted from the names of several authors. Second, the surname of Dr. van Dam was mistakenly written as “Dam.” Third, the first name of author Bernhard Englitz was misspelled as “Bernard” and the surname of author B.J.A. Pollux was misspelled as “Pullox.” Finally, Dr. Keijer's first name was abbreviated rather than written in full. These errors, as well as various errors in the author affiliations, have now been corrected online

Cocaine withdrawal reduces GABAB R transmission at entopeduncular nucleus - lateral habenula synapses

Lateral habenula (LHb) hyperactivity plays a pivotal role in the emergence of negative emotional states, including those occurring during withdrawal from addictive drugs. We have previously implicated cocaine-driven adaptations at synapses from the entopeduncular nucleus (EPN) to the LHb in this process. Specifically, ionotropic GABAA receptor (R)-mediated neurotransmission at EPN-to-LHb synapses is reduced during cocaine withdrawal, due to impaired vesicle filling. Recent studies have shown that metabotropic GABAB R signaling also controls LHb activity, although its role at EPN-to-LHb synapses during drug withdrawal is unknown. Here, we predicted that cocaine treatment would reduce GABAB R-mediated neurotransmission at EPN-to-LHb synapses. We chronically treated mice with saline or cocaine, prepared brain slices after two days of withdrawal and performed voltage-clamp recordings from LHb neurons whilst optogenetically stimulating EPN terminals. Compared with controls, mice in cocaine withdrawal exhibited reduced GABAA R-mediated input to LHb neurons, and a reduced occurrence of GABAB R-signaling at EPN-to-LHb synapses. We then assessed the underlying mechanism of this decrease. Application of GABAB R agonist baclofen evoked similar postsynaptic responses in EPN-innervated LHb neurons in saline- and cocaine-treated mice. Release probability at EPN-to-LHb GABAergic synapses was also comparable between groups. However, incubating brain slices in glutamine to facilitate GABA vesicle filling, normalized GABAB R-currents at EPN-to-LHb synapses in cocaine-treated mice. Overall, we show that during cocaine withdrawal, together with reduced GABAA R transmission, also GABAB R-mediated inhibitory signaling is diminished at EPN-to-LHb synapses, likely via the same presynaptic deficit. In concert, these alterations are predicted to contribute to the emergence of drug withdrawal symptoms, facilitating drug relapse