Yury M. Morozov

Hardwiring the Brain: Endocannabinoids Shape Neuronal Connectivity

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB1 cannabinoid receptors (CB1Rs) are enriched in the axonal growth cones of γ-aminobutyric acid–containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB1R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB1Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

Molecular and morphological heterogeneity of neural precursors in the mouse neocortical proliferative zones.

The proliferative ventricular zone (VZ) is the main source of projection neurons for the overlying cerebral neocortex. The number and diversity of neocortical neurons is determined, in part, by factors controlling the proliferation and specification of VZ cells during embryonic development. We used a variety of methods, including in utero electroporation with specific cellular markers, computer-assisted serial EM cell reconstruction, and time-lapse multiphoton imaging to characterize the molecular and morphological characteristics of the VZ constituents and to capture their behavior during cell division. Our analyses reveal at least two types of dividing cells in the VZ: (1) radial glial cells (RGCs) that span the entire neocortical wall and maintain contact both at the ventricular and pial surfaces throughout mitotic division, and (2) short neural precursors (SNPs) that possess a ventricular endfoot and a basal process of variable length that is retracted during mitotic division. These two precursor cell classes are present concomitantly in the VZ, but their relative number changes over the course of cortical neurogenesis. Moreover, the SNPs are morphologically, ultrastructurally and molecularly distinct from dividing RGCs. For example, SNPs are marked by their preferential expression of the tubulin α-1 promoter whereas RGCs instead express the glutamate–aspartate transporter and brain lipid binding protein promoters. In contrast to recent studies that suggest that RGCs are the sole type of VZ precursor, the present study indicates that the VZ in murine dorsal telencephalon is similar to that in human and nonhuman primates, because it contains multiple types of neuronal precursors.

Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling

Primary cilia are present on mammalian neurons and glia, but their function is largely unknown. We generated conditional homozygous mutant mice for a gene we termed Stumpy. Mutants lack cilia and have conspicuous abnormalities in postnatally developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in neural stem cells known as astrocyte-like neural precursors (ALNPs). Previous studies suggested that primary cilia mediate sonic hedgehog (Shh) signaling. Here, we find that loss of ALNP cilia leads to abrogated Shh activity, increased cell cycle exit, and morphological abnormalities in ALNPs. Processing of Gli3, a mediator of Shh signaling, is also altered in the absence of cilia. Further, key mediators of the Shh pathway localize to ALNP cilia. Thus, selective targeting of Shh machinery to primary cilia confers to ALNPs the ability to differentially respond to Shh mitogenic signals compared to neighboring cells. Our data suggest these organelles are cellular “antennae” critically required to modulate ALNP behavior.

Hypothalamic POMC neurons promote cannabinoid-induced feeding

Hypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide β-endorphin. CB1R activation selectively increases β-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.

Differential Subcellular Recruitment of Monoacylglycerol Lipase Generates Spatial Specificity of 2-Arachidonoyl Glycerol Signaling during Axonal Pathfinding

Endocannabinoids, particularly 2-arachidonoyl glycerol (2-AG), impact the directional turning and motility of a developing axon by activating CB1 cannabinoid receptors (CB1Rs) in its growth cone. Recent findings posit that sn-1-diacylglycerol lipases (DAGLα/β) synthesize 2-AG in the motile axon segment of developing pyramidal cells. Coincident axonal targeting of CB1Rs and DAGLs prompts the hypothesis that autocrine 2-AG signaling facilitates axonal outgrowth. However, DAGLs alone are insufficient to account for the spatial specificity and dynamics of 2-AG signaling. Therefore, we hypothesized that local 2-AG degradation by monoacylglycerol lipase (MGL) must play a role. We determined how subcellular recruitment of MGL is temporally and spatially restricted to establish the signaling competence of 2-AG during axonal growth. MGL is expressed in central and peripheral axons of the fetal nervous system by embryonic day 12.5. MGL coexists with DAGLα and CB1Rs in corticofugal axons of pyramidal cells. Here, MGL and DAGLα undergo differential axonal targeting with MGL being excluded from the motile neurite tip. Thus, spatially confined MGL activity generates a 2-AG-sensing microdomain and configures 2-AG signaling to promote axonal growth. Once synaptogenesis commences, MGL disperses in stationary growth cones. The axonal polarity of MGL is maintained by differential proteasomal degradation because inhibiting the ubiquitin proteasome system also induces axonal MGL redistribution. Because MGL inactivation drives a CB1R-dependent axonal growth response, we conclude that 2-AG may act as a focal protrusive signal for developing neurons and whose regulated metabolism is critical for attaining correct axonal complexity.

Origin, Early Commitment, Migratory Routes, and Destination of Cannabinoid Type 1 Receptor-Containing Interneurons

It is now well established that inhibitory interneurons of the cerebral cortex display large diversity, but where each subclass originates and how they acquire final position and physiological characteristics is only begin to be elucidated. Recent studies indicate that the phenotypes of many forebrain interneurons are specified in the ganglionic eminence (GE) at the time of their origin. However, developmental history of cannabinoid type 1 receptor (CB(1)) positive (+) interneurons is not known. Here, we focus on the origin and migratory routs of prospective CB(1)/cholecystokinin (CCK)+ and CB(1)/reelin/calretinin+ gamma-aminobutyric acid (GABA)-ergic hippocampal interneurons. We have used variety of markers and a combination of methods, including immunocytochemistry at light and electron microscopic level, and in utero electroporation, to identify a subpopulation of CB(1)+ cells at the time of their origin in the caudal GE and pallial-subpallial boundary at the 11th-12th embryonic days. We have followed their migration, first radially to the marginal zone, then tangentially in the lateral-to-medial direction within the dorsal telencephalon, before they reach their final destination in the hippocampus proper and the dentate gyrus where they differentiate into CB(1)/CCK+ or CB(1)/reelin/calretinin+ GABAergic interneurons. Thus, the specific subclasses of CB(1)+ inhibitory interneurons, similar to the projection neurons, are determined at the time and place of last cell division and follow their own complex migratory pattern to the final positions.

Post-natal development of type 1 cannabinoid receptor immunoreactivity in the rat hippocampus.

Type 1 cannabinoid receptors, selectively located on axon terminals of GABAergic interneurons in the hippocampus, are known to be involved in endocannabinoid‐mediated retrograde synaptic signalling. The question arises whether type 1 cannabinoid receptors appear on these axons during early post‐natal life, when GABAergic transmission is still depolarizing, and whether there are any developmental changes in the cellular or subcellular expression pattern. Here we demonstrate, using single and double immunocytochemical methods at the light and electron microscopic levels, that type 1 cannabinoid receptors are expressed only on the membrane of axon terminals and pre‐terminal axons but not on the soma‐dendritic membrane at all examined timepoints between post‐natal days 0 and 20, similar to the adult distribution. All type 1 cannabinoid receptor‐positive boutons formed symmetric synapses. Granular labelling in the somata was already strong at post‐natal day 0 and corresponded to multivesicular bodies, lysosomes, Golgi apparatus and rough endoplasmic reticulum. The type 1 cannabinoid receptor‐positive axons were shown to originate largely from cholecystokinin‐immunoreactive basket and bistratified neurons throughout the hippocampus (90% of all type 1 cannabinoid receptor‐containing cells) and dentate gyrus (70% of all type 1 cannabinoid receptor‐containing cells). The remaining cells have not been identified but probably belong to the somatostatin‐ and/or neuropeptide Y‐containing subsets, as cholecystokinin‐negative, type 1 cannabinoid receptor‐positive axons have been observed in strata moleculare and lacunosum‐moleculare of the dentate gyrus and CA1–3, respectively, where these neurons are known to arborize. No cell types were found that expressed type 1 cannabinoid receptors transiently at some developmental stage. We conclude that the cellular and subcellular pattern of type 1 cannabinoid receptor expression during early post‐natal life is similar to the adult pattern and type 1 cannabinoid receptors are expressed on the cholecystokinin‐containing axons as soon as synapse formation begins. This suggests that retrograde synaptic signalling by endocannabinoids is required for the normal operation of GABAergic neurotransmission even before it becomes hyperpolarizing.

Translocation of Synaptically Connected Interneurons across the Dentate Gyrus of the Early Postnatal Rat Hippocampus

Most neurons in the developing mammalian brain migrate to their final destinations by translocation of the cell nucleus within their leading process and immature bipolar body that is devoid of synaptic connections. Here, we used a combination of immunohistochemistry at light- and electron-microscopic (EM) levels and time-lapse imaging in slice cultures to analyze migration of synaptically interconnected, cholecystokinin-immunopositive [CCK(+)] interneurons in the dentate gyrus in the rat hippocampus during early postnatal ages. We observed dynamic morphogenetic transformation of the CCK(+) interneurons, from a horizontal bipolar shape situated in the molecular layer, through a transitional triangular and then vertical bipolar form that they acquire while traversing the granular layer to finally assume an adult-like pyramidal-shaped morphology on entering the hilus. Immunostaining with anti-glial fibrillary acidic protein and three-dimensional reconstructions from serial EM images indicate that, unlike granule cells, which migrate from the hilus to the granular layer, interneurons traverse this layer in the opposite direction without apparent surface-mediated guidance of the radial glial cells. Importantly, the somas, dendrites, and axons of the CCK(+) transitional forms maintain old and acquire new synaptic contacts while migrating across the dentate plate. The migration of synaptically interconnected neurons that may occur in response to local functional demand represents a novel mode of cell movement and form of neuroplasticity.

Antibodies to cannabinoid type 1 receptor co-react with stomatin-like protein 2 in mouse brain mitochondria

Anti‐cannabinoid type 1 receptor (CB1) polyclonal antibodies are widely used to detect the presence of CB1 in a variety of brain cells and their organelles, including neuronal mitochondria. Surprisingly, we found that anti‐CB1 sera, in parallel with CB1, also recognize the mitochondrial protein stomatin‐like protein 2. In addition, we show that the previously reported effect of synthetic cannabinoid WIN 55,212‐2 on mitochondrial complex III respiration is not detectable in purified mitochondrial preparations. Thus, our study indicates that a direct relationship between endocannabinoid signaling and mitochondrial functions in the cerebral cortex seems unlikely, and that caution should be taken interpreting findings obtained using anti‐CB1 antibodies.

The spatial and temporal pattern of fatty acid amide hydrolase expression in rat hippocampus during postnatal development

GABAergic synaptic transmission is efficiently controlled by endogenous cannabinoids in cortical structures. Fatty acid amide hydrolase (FAAH) is one of the metabolizing enzymes of endocannabinoids in the brain. In this study we investigated the cellular and subcellular distribution of FAAH at various timepoints during the first postnatal weeks, when GABA is still depolarizing, and plays a crucial role in network events. FAAH immunoreactivity is strong in the CA3 region already at postnatal day 0 (P0), but in CA1 only after P8. During this period, FAAH levels in hilar mossy cells decrease and in granule cells slowly increase. Pyramidal cells express FAAH first in the soma and proximal dendrites, and gradually in more distal segments, reaching adult levels in the most distal dendrites only at P22. Transient expression of FAAH was found in a small number of stratum radiatum cells that may be interneurons and in ependymal cells at the border of the alveus and corpus callosum between P2 and P8. At the ultrastructural level, FAAH distribution at all ages was very similar to the adult pattern, i.e. it was largely associated with the membrane of cytoplasmic vesicles, mitochondria and endoplasmic reticulum. During postnatal development of the hippocampus, the spatio‐temporal expression of FAAH correlates well with the general pattern of neuronal maturation, but not with the arrival of afferent pathways, which suggests that FAAH – and its major endocannabinoid substrate, anandamide – is unlikely to be involved in the presynaptic control of neurotransmission. Instead, FAAH may subserve general roles as the inactivating enzyme for many fatty acid amides, in addition to anandamide.