Molecular determinants of endocannabinoid-mediated brain development

The enchanting process of brain development entails a fragile and finite time window, throughout which biochemical imbalances and external insults can potentially affect developing neuronal circuits. Within the numerous exquisitely orchestrated events taking place in the foetal brain, axonal pathfinding is of paramount importance. If aberrant, axonal outgrowth can lead to errors in synaptogenesis and impaired connectivity, with potential long-lasting behavioural phenotypes. Despite the prominent role of endocannabinoids (eCBs) in axonal guidance, the molecular determinants of eCB-mediated axonal regulation remain largely unexplored. Aiming to interrogate the eCB machinery in less explored subcortical territories, study I investigates the role of the eCB system and its underlying components in foetal cholinergic projection neurons. Prenatal manipulation of cannabinoid receptor 1 (CB1R) permanently reshaped septo-hippocampal cholinergic projections. Nerve growth factor (NGF) was identified as an upstream molecule capable of regulating 2-arachydonoylglycerol (2-AG) via monoacylglycerol lipase (MAGL) degradation. The compartmentalization of each eCB machinery component along extending neurites is therefore crucial. However, axonal pathfinding takes place within the extracellular matrix, migrating glia and developing oligodendrocytes, generating a convoluted scenario where each growth cone interacts with multiple guidance proteins. Cannabinoid receptor 2 (CB2R)-expressing oligodendrocytes are introduced in study II as an additional player able to drive aberrant callosal axons spread upon supra-physiological 2-AG levels. Excess 2-AG engaged CB2R-mediated premature proliferation of oligodendrocyte end-feet. The interaction between the oligodendrocyte-derived chemorepellant molecule Slit2 and its receptor roundabout 1 (Robo1) on corticofugal axonal ends induced errant CB1R-expressing corticofugal axon pathfinding. In addition to being an endogenous receptor of eCBs, CB1R is also a target for the principal psychoactive compound of cannabis, ∆9-tetrahydrocannabinol (THC). In study III we undertook an unbiased proteomics screening of the embryonic cortical plate following maternal THC exposure. Superior Cervical Ganglion 10 (SCG10), a microtubule-binding protein present in extending growth cones, was identified as a direct molecular target of THC. Thus, microtubule dynamics represent a novel target of prenatal THC exposure, whose impairment promotes axonal defects and long-lasting synaptic connectivity impairment. Cholecystokinin (Cck)-containing interneurons, due to their high expression of CB1Rs and early appearance in the developing cortex, are likely targets of in utero eCB imbalances. Taking advantage of the recent CckBAC/DsRed mouse line in study IV we achieved a systematic anatomical and physiological characterization of Cck-CB1R-expressing cells from embryonic day (E)10.5 to adulthood. In sum, multiple upstream and downstream molecular components of the eCBs system were assessed over the course of this thesis investigation, within both physiological brain development and upon maternal cannabis exposure

A selective projection from the subthalamic nucleus to parvalbumin-expressing interneurons of the striatum

The striatum and subthalamic nucleus (STN) are considered to be the primary input nuclei of the basal ganglia. Projection neurons of both striatum and STN can extensively interact with other basal ganglia nuclei, and there is growing anatomical evidence of direct axonal connections from the STN to striatum. There remains, however, a pressing need to elucidate the organization and impact of these subthalamostriatal projections in the context of the diverse cell types constituting the striatum. To address this, we carried out monosynaptic retrograde tracing from genetically-defined populations of dorsal striatal neurons in adult male and female mice, quantifying the connectivity from STN neurons to spiny projection neurons, GABAergic interneurons, and cholinergic interneurons. In parallel, we used a combination of ex vivo electrophysiology and optogenetics to characterize the responses of a complementary range of dorsal striatal neuron types to activation of STN axons. Our tracing studies showed that the connectivity from STN neurons to striatal parvalbumin-expressing interneurons is significantly higher (∼ four- to eight-fold) than that from STN to any of the four other striatal cell types examined. In agreement, our recording experiments showed that parvalbumin-expressing interneurons, but not the other cell types tested, commonly exhibited robust monosynaptic excitatory responses to subthalamostriatal inputs. Taken together, our data collectively demonstrate that the subthalamostriatal projection is highly selective for target cell type. We conclude that glutamatergic STN neurons are positioned to directly and powerfully influence striatal activity dynamics by virtue of their enriched innervation of GABAergic parvalbumin-expressing interneurons.Significance StatementPlacing the subthalamostriatal projection within schemes of basal ganglia circuit organization is challenging because of the diversity of cell types within striatum. Here, we shed new light on the structural and electrophysiological substrates by which STN neurons can exert direct and biased influences on the striatal microcircuit. We discovered that STN innervation of parvalbumin-expressing interneurons is relatively enriched and impactful as compared to innervation of other types of striatal neuron. Accordingly, the STN joins a growing list of subcortical structures that, although not considered 'canonical' sources of inputs to striatum, selectively target striatal interneurons. Our results are important in supporting the concept that the glutamatergic subthalamostriatal projection is positioned to fulfil diverse and likely unique roles within basal ganglia circuits

Life-long epigenetic programming of cortical architecture by maternal ‘Western’ diet during pregnancy

Funding: European Research Council (SECRET-CELLS, ERC-2015-AdG-695136; T.H.); Wellcome Trust grant number 094476/Z/10/Z, which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St. Andrews.The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with ‘Western’ diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched ‘Western’ diets can reprogram maternal bodily metabolism with maternal nutrient supply precipitating the body-wide imprinting of molecular and cellular adaptations at the level of long-range intercellular signaling networks in the unborn fetus. Even though unfavorable neurological outcomes are amongst the most common complications of intrauterine ω-6 PUFA excess, cellular underpinnings of life-long modifications to brain architecture remain unknown. Here, we show that nutritional ω-6 PUFA-derived endocannabinoids desensitize CB1 cannabinoid receptors, thus inducing epigenetic repression of transcriptional regulatory networks controlling neuronal differentiation. We found that cortical neurons lose their positional identity and axonal selectivity when mouse fetuses are exposed to excess ω-6 PUFAs in utero. Conversion of ω-6 PUFAs into endocannabinoids disrupted the temporal precision of signaling at neuronal CB1 cannabinoid receptors, chiefly deregulating Stat3-dependent transcriptional cascades otherwise required to execute neuronal differentiation programs. Global proteomics identified the immunoglobulin family of cell adhesion molecules (IgCAMs) as direct substrates, with DNA methylation and chromatin accessibility profiling uncovering epigenetic reprogramming at >1400 sites in neurons after prolonged cannabinoid exposure. We found anxiety and depression-like behavioral traits to manifest in adult offspring, which is consistent with genetic models of reduced IgCAM expression, to suggest causality for cortical wiring defects. Overall, our data uncover a regulatory mechanism whose disruption by maternal food choices could limit an offspring’s brain function for life.PostprintPeer reviewe

Complete representation of action space and value in all dorsal striatal pathways

The dorsal striatum plays a central role in the selection, execution, and evaluation of actions. An emerging model attributes action selection to the matrix and evaluation to the striosome compartment. Here, we use large-scale cell-type-specific calcium imaging to determine the activity of striatal projection neurons (SPNs) during motor and decision behaviors in the three major outputs of the dorsomedial striatum: Oprm1+ striosome versus D1+ direct and A2A+ indirect pathway SPNs. We find that Oprm1+ SPNs show complex tunings to simple movements and value-guided actions, which are conserved across many sessions in a single task but remap between contexts. During decision making, the SPN tuning profiles form a complete representation in which sequential SPN activity jointly encodes task progress and value. We propose that the three major output pathways in the dorsomedial striatum share a similarly complete representation of the entire action space, including task- and phase-specific signals of action value and choice.QC 20231101</p

Life-long epigenetic programming of cortical architecture by maternal 'Western' diet during pregnancy

The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with 'Western' diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched 'Western' diets can reprogram maternal bodily metabolism with maternal nutrient supply precipitating the body-wide imprinting of molecular and cellular adaptations at the level of long-range intercellular signaling networks in the unborn fetus. Even though unfavorable neurological outcomes are amongst the most common complications of intrauterine ω-6 PUFA excess, cellular underpinnings of life-long modifications to brain architecture remain unknown. Here, we show that nutritional ω-6 PUFA-derived endocannabinoids desensitize CB1 cannabinoid receptors, thus inducing epigenetic repression of transcriptional regulatory networks controlling neuronal differentiation. We found that cortical neurons lose their positional identity and axonal selectivity when mouse fetuses are exposed to excess ω-6 PUFAs in utero. Conversion of ω-6 PUFAs into endocannabinoids disrupted the temporal precision of signaling at neuronal CB1 cannabinoid receptors, chiefly deregulating Stat3-dependent transcriptional cascades otherwise required to execute neuronal differentiation programs. Global proteomics identified the immunoglobulin family of cell adhesion molecules (IgCAMs) as direct substrates, with DNA methylation and chromatin accessibility profiling uncovering epigenetic reprogramming at >1400 sites in neurons after prolonged cannabinoid exposure. We found anxiety and depression-like behavioral traits to manifest in adult offspring, which is consistent with genetic models of reduced IgCAM expression, to suggest causality for cortical wiring defects. Overall, our data uncover a regulatory mechanism whose disruption by maternal food choices could limit an offspring's brain function for life

Adverse effects of Δ9-tetrahydrocannabinol on neuronal bioenergetics during postnatal development

This work was supported by a grant from FWF (P 34121-B; EK), GW Pharmaceuticals, as well as funding from the Swedish Research Council (2018-02838; TH), the European Research Council (SECRET-CELLS, ERC-2015-AdG-695136; TH), and the Wellcome Trust (grant no. 094476/Z/10/Z, which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St. Andrews).Ongoing societal changes in views on the medical and recreational roles of cannabis increased the use of concentrated plant extracts with a Δ9-tetrahydrocannabinol (THC) content of more than 90%. Even though prenatal THC exposure is widely considered adverse for neuronal development, equivalent experimental data for young age cohorts are largely lacking. Here, we administered plant-derived THC (1 or 5 mg/kg) to mice daily during P5–P16 and P5–P35 and monitored its effects on hippocampal neuronal survival and specification by high-resolution imaging and iTRAQ proteomics, respectively. We found that THC indiscriminately affects pyramidal cells and both cannabinoid receptor 1+ (CB1R)+ and CB1R– interneurons by P16. THC particularly disrupted the expression of mitochondrial proteins (complexes I–IV), a change that had persisted even 4 months after the end of drug exposure. This was reflected by a THC-induced loss of membrane integrity occluding mitochondrial respiration and could be partially or completely rescued by pH stabilization, antioxidants, bypassed glycolysis, and targeting either mitochondrial soluble adenylyl cyclase or the mitochondrial voltage-dependent anion channel. Overall, THC exposure during infancy induces significant and long-lasting reorganization of neuronal circuits through mechanisms that, in large part, render cellular bioenergetics insufficient to sustain key developmental processes in otherwise healthy neurons.Publisher PDFPeer reviewe