The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB 1 cannabinoid receptors on neurons and CB 2 cannabinoid Correspondence should be addressed to N.S. (nstella@uw.edu). 11 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Neuroscience website. Competing Financial Interests:The authors declare no competing financial interests.Reprints and permissions information is available online at http://www.nature.com/reprintsandpermissions/. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2011 February 1. Published in final edited form as:Nat Neurosci. 2010 August ; 13(8): 951-957. doi:10.1038/nn.2601. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase α-β-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB 2 -mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activitydependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB 1 -dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.In the nervous system, the endocannabinoids (eCBs) arachidonoylethanolamide (anandamide) and 2-AG are produced and inactivated by neurons and glia 1,2 . The production of eCBs increases in response to specific stimuli, including membrane receptor activation, ion channel opening and calcium influx 2 . eCBs are inactivated by cellular uptake followed by intracellular enzymatic hydrolysis 3,4 . The balance between this production and inactivation dictates the levels of extracellular eCB accumulation and the ensuing activation of CB 1 receptors expressed by neurons (regulating neurotransmitter release) and CB 2 receptors expressed by microglia (regulating their motility and ability to produce immunomodulators) [4][5][6][7] . Thus, the enzymatic steps that control the production and inactivation of eCBs constitute promising molecular targets for indirectly modulating CB 1 and CB 2 receptor activity, and thereby controlling neurotransmission and neuroinflammation.Of all the steps that control the accumulation of eCBs, the hydrolytic enzymes that inactivate anandamide and 2-AG represent the most promising pharmacological and genetic targets for fine-tuning the local accumulation of these lipid transmitters. Inhibition of fatty acid amide hydrolase (FAAH) increases...

Endocannabinoid-mediated retrograde signaling exerts powerful control over synaptic transmission in many brain areas. However, in the neocortex, the precise laminar, cellular, and subcellular localization of the type 1 cannabinoid receptor (CB 1 ) as well as its function has been elusive. Here we combined multiple immunolabeling with whole-cell recordings to investigate the morpho-functional characteristics of cannabinoid signaling in rat somatosensory cortex.Immunostaining for CB 1 revealed axonal and somatic labeling with striking layer specificity: a high density of CB 1 -positive fibers was seen in layers II-III, in layer VI, and in upper layer V, whereas other layers had sparse (layer IV) or hardly any (layer I) staining. Membrane staining for CB 1 was only found in axon terminals, all of which contained GABA and formed symmetric synapses. Double immunostaining also revealed that CB 1 -positive cells formed two neurochemically distinct subpopulations: two-thirds were cholecystokinin positive and one-third expressed calbindin, each subserving specific inhibitory functions in cortical networks.In addition, cannabinoid sensitivity of GABAergic input showed striking layer specificity, as revealed by both electrophysiological and anatomical experiments. We found a unique population of large pyramidal neurons in layer VB that received much less perisomatic innervation from CB 1 -expressing GABAergic axon terminals and, accordingly, showed no depolarization-induced suppression of inhibition, unlike pyramidal cells in layer II, and a population of small pyramidal cells in layer V. This suggests that inhibitory control of pyramidal cells involved in intracortical or corticostriatal processing is fine-tuned by activity-dependent endocannabinoid signaling, whereas inhibition of pyramidal cells relaying cortical information to lower subcortical effector centers often lacks this plasticity.

Learning from experience depends at least in part on changes in neuronal connections. We present the largest map of connectivity to date between cortical neurons of a defined type (L2/3 pyramidal cells), which was enabled by automated analysis of serial section electron microscopy images with improved handling of image defects. We used the map to identify constraints on the learning algorithms employed by the cortex. Previous cortical studies modeled a continuum of synapse sizes (Arellano et al. , 2007) by a log-normal distribution (Loewenstein, Kuras and Rumpel, 2011;de Vivo et al. , 2017;Santuy et al. , 2018) . A continuum is consistent with most neural network models of learning, in which synaptic strength is a continuously graded analog variable. Here we show that synapse size, when restricted to synapses between L2/3 pyramidal cells, is well-modeled by the sum of a binary variable and an analog variable drawn from a log-normal distribution. Two synapses sharing the same presynaptic and postsynaptic cells are known to be correlated in size (Sorra and Harris, 1993;Koester and Johnston, 2005;Bartol et al. , 2015;Kasthuri et al. , 2015;Dvorkin and Ziv, 2016;Bloss et al. , 2018;Motta et al. , 2019) . We show that the binary variables of the two synapses are highly correlated, while the analog variables are not. Binary variation could be the outcome of a Hebbian or other synaptic plasticity rule depending on activity signals that are relatively uniform across neuronal arbors, while analog variation may be dominated by other influences. We discuss the implications for the stability-plasticity dilemma. †Correspondence to svenmd@princeton.edu ,

No abstract

Giant inhibitory terminals with multiple synapses, the counterparts of excitatory "detonator" or "driver" terminals, have not been described in the forebrain. Using three-dimensional reconstructions of electron microscopic images, we quantitatively characterize a GABAergic pathway that establishes synaptic contacts exclusively via multiple synapses. Axon terminals of the nigrothalamic pathway formed, on average, 8.5 synapses on large-diameter dendrites and somata of relay cells in the ventromedial nucleus of the rat thalamus. All synapses of a given terminal converged on a single postsynaptic element. The vast majority of the synapses established by a single terminal were not separated by astrocytic processes. Nigrothalamic terminals in the macaque monkey showed the same ultrastructural features both in qualitative and quantitative terms (the median number of synapse per target was also 8.5). The individual synapses were closely spaced in both species. The nearest-neighbor synaptic distances were 169 nm in the rat and 178 nm in the monkey. The average number of synapses within 0.75 m from any given synapse was 3.8 in the rat and 3.5 in the monkey. The arrangement of synapses described in this study creates favorable conditions for intersynaptic spillover of GABA among the multiple synapses of a single bouton, which can result in larger charge transfer. This could explain faithful and efficient GABAergic signal transmission in the nigrothalamic pathway in the healthy condition and during Parkinson's disease. In addition, our structural data suggest that the rodent nigrothalamic pathway can be a valid model of the primate condition, when the mechanism of GABAergic transmission is studied.

Inhibitory neurons in mammalian cortex exhibit diverse physiological, morphological, molecular and connectivity signatures. While considerable work has measured the average connectivity of several interneuron classes, there remains a fundamental lack of understanding of the connectivity distribution of distinct inhibitory cell types with synaptic resolution, how it relates to properties of target cells and how it affects function. Here, we used large-scale electron microscopy and functional imaging to address these questions for chandelier cells in layer 2/3 of the mouse visual cortex. With dense reconstructions from electron microscopy, we mapped the complete chandelier input onto 153 pyramidal neurons. We found that synapse number is highly variable across the population and is correlated with several structural features of the target neuron. This variability in the number of axo-axonic ChC synapses is higher than the variability seen in perisomatic inhibition. Biophysical simulations show that the observed pattern of axo-axonic inhibition is particularly effective in controlling excitatory output when excitation and inhibition are co-active. Finally, we measured chandelier cell activity in awake animals using a cell-type specific calcium imaging approach and saw highly correlated activity across chandelier cells. In the same experiments, in vivo chandelier population activity correlated with pupil dilation, a proxy for arousal. Together these results suggest that chandelier cells provide a circuit-wide signal whose strength is adjusted relative to the properties of target neurons.

Mammalian cortex features a large diversity of neuronal cell types, each with characteristic anatomical, molecular and functional properties. Synaptic connectivity rules powerfully shape how each cell type participates in the cortical circuit, but comprehensively mapping connectivity at the resolution of distinct cell types remains difficult. Here, we used millimeter-scale volumetric electron microscopy to investigate the connectivity of inhibitory neurons across a dense neuronal population spanning all layers of mouse visual cortex with synaptic resolution. We classified all 1183 excitatory neurons within a 100 micron column into anatomical subclasses using quantitative morphological and synapse features based on full dendritic reconstructions, finding both familiar subclasses corresponding to axonal projections and novel intralaminar distinctions based on synaptic properties. To relate these subclasses to single-cell connectivity, we reconstructed all 164 inhibitory interneurons in the same column, producing a wiring diagram of inhibition with more than 70,000 synapses. We found widespread cell-type-specific inhibition, including interneurons selectively targeting certain excitatory subpopulations among spatially intermingled neurons in layer 2/3, layer 5, and layer 6. Globally, inhibitory connectivity was organized into "motif groups," heterogeneous collections of cells that collectively target both perisomatic and dendritic compartments of the same combinations of excitatory subtypes. We also discovered a novel category of disinhibitory-specialist interneurons that preferentially targets basket cells. Collectively, our analysis revealed new organizing principles for cortical inhibition and will serve as a powerful foundation for linking modern multimodal neuronal atlases with the cortical wiring diagram.

Avian song learning shares striking similarities with human speech acquisition and requires a basal ganglia (BG)-thalamo-cortical circuit. Information processing and transmission speed in the BG is thought to be limited by synaptic architecture of two serial inhibitory connections. Propagation speed may be critical in the avian BG circuit given the temporally precise control of musculature during vocalization. We used electrical stimulation of the cortical inputs to the BG to study, with fine time resolution, the functional connectivity within this network. We found that neurons in thalamic and cortical nuclei that are not directly connected with the stimulated area can respond to the stimulation with extremely short latencies. Through pharmacological manipulations, we trace this property back to the BG and show that the cortical stimulation triggers fast disinhibition of the thalamic neurons. Surprisingly, feedforward inhibition mediated by striatal inhibitory neurons onto BG output neurons sometimes precedes the monosynaptic excitatory drive from cortical afferents. The fast feedforward inhibition lengthens a single interspike interval in BG output neurons by just a few milliseconds. This short delay is sufficient to drive a strong, brief increase in firing probability in the target thalamic neurons, evoking short-latency responses. By blocking glutamate receptors in vivo, we show that thalamic responses do not appear to rely on excitatory drive, and we show in a theoretical model that they could be mediated by postinhibitory rebound properties. Such fast signaling through disinhibition and rebound may be a crucial specialization for learning of rapid and temporally precise motor acts such as vocal communication.