Retrograde adenosine/A2A receptor signaling facilitates excitatory synaptic transmission and seizures.

Retrograde signaling at the synapse is a fundamental way by which neurons communicate and neuronal circuit function is fine-tuned upon activity. While long-term changes in neurotransmitter release commonly rely on retrograde signaling, the mechanisms remain poorly understood. Here, we identified adenosine/A2A receptor (A2AR) as a retrograde signaling pathway underlying presynaptic long-term potentiation (LTP) at a hippocampal excitatory circuit critically involved in memory and epilepsy. Transient burst activity of a single dentate granule cell induced LTP of mossy cell synaptic inputs, a BDNF/TrkB-dependent form of plasticity that facilitates seizures. Postsynaptic TrkB activation released adenosine from granule cells, uncovering a non-conventional BDNF/TrkB signaling mechanism. Moreover, presynaptic A2ARs were necessary and sufficient for LTP. Lastly, seizure induction released adenosine in a TrkB-dependent manner, while removing A2ARs or TrkB from the dentate gyrus had anti-convulsant effects. By mediating presynaptic LTP, adenosine/A2AR retrograde signaling may modulate dentate gyrus-dependent learning and promote epileptic activity.

Adeno-associated Virus-mediated Ezh2 Knockdown Reduced the Increment of Newborn Neurons Induced by Forebrain Ischemia in Gerbil Dentate Gyrus.

It is established that neurogenesis of dentate gyrus is increased after ischemic insult, although the regulatory mechanisms have not yet been elucidated. In this study, we focused on Ezh2 which suppresses gene expression through catalyzing trimethylation of lysine 27 of histone 3. Male gerbils were injected with adeno-associated virus (AAV) carrying shRNA targeting to Ezh2 into right dentate gyrus 2 weeks prior to forebrain ischemia. One week after ischemia, animals were injected with thymidine analogue to label proliferating cells. Three weeks after ischemia, animals were killed for histological analysis. AAV-mediated knockdown of Ezh2 significantly decreased the ischemia-induced increment of proliferating cells, and the proliferated cells after ischemia showed significantly longer migration from subgranular zone (SGZ), compared to the control group. Furthermore, the number of neural stem cells in SGZ significantly decreased after ischemia with Ezh2 knockdown group. Of note, Ezh2 knockdown did not affect the number of proliferating cells or the migration from SGZ in the non-ischemic condition. Our data showed that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult dentate gyrus.

Hypothalamic Supramammillary Control of Cognition and Motivation.

The supramammillary nucleus (SuM) is a small region in the ventromedial posterior hypothalamus. The SuM has been relatively understudied with much of the prior focus being on its connection with septo-hippocampal circuitry. Thus, most studies conducted until the 21st century examined its role in hippocampal processes, such as theta rhythm and learning/memory. In recent years, the SuM has been "rediscovered" as a crucial hub for several behavioral and cognitive processes, including reward-seeking, exploration, and social memory. Additionally, it has been shown to play significant roles in hippocampal plasticity and adult neurogenesis. This review highlights findings from recent studies using cutting-edge systems neuroscience tools that have shed light on these fascinating roles for the SuM.

Increased vesicle fusion competence underlies long-term potentiation at hippocampal mossy fiber synapses.

Presynaptic long-term potentiation (LTP) is thought to play an important role in learning and memory. However, the underlying mechanism remains elusive because of the difficulty of direct recording during LTP. Hippocampal mossy fiber synapses exhibit pronounced LTP of transmitter release after tetanic stimulation and have been used as a model of presynaptic LTP. Here, we induced LTP by optogenetic tools and applied direct presynaptic patch-clamp recordings. The action potential waveform and evoked presynaptic Ca2+ currents remained unchanged after LTP induction. Membrane capacitance measurements suggested higher release probability of synaptic vesicles without changing the number of release-ready vesicles after LTP induction. Synaptic vesicle replenishment was also enhanced. Furthermore, stimulated emission depletion microscopy suggested an increase in the numbers of Munc13-1 and RIM1 molecules within active zones. We propose that dynamic changes in the active zone components may be relevant for the increased fusion competence and synaptic vesicle replenishment during LTP.

Subcortical glutamatergic inputs exhibit a Hebbian form of long-term potentiation in the dentate gyrus.

The hippocampus receives glutamatergic and GABAergic inputs from subcortical regions. Despite the important roles of these subcortical inputs in the regulation of hippocampal circuit, it has not been explored whether associative activation of the subcorticohippocampal pathway induces Hebbian plasticity of subcortical inputs. Here, we demonstrate that the hypothalamic supramammillary nucleus (SuM) to the dentate granule cell (GC) synapses, which co-release glutamate and GABA, undergo associative long-term potentiation (LTP) of glutamatergic, but not GABAergic, co-transmission. This LTP is induced by pairing of SuM inputs with GC spikes. We found that this Hebbian LTP is input-specific, requires NMDA receptors and CaMKII activation, and is expressed postsynaptically. By the net increase in excitatory drive of SuM inputs following LTP induction, associative inputs of SuM and the perforant path effectively discharge GCs. Our results highlight the important role of associative plasticity at SuM-GC synapses in the regulation of dentate gyrus activity and for the encoding of SuM-related information.

Excitatory selective LTP of supramammillary glutamatergic/GABAergic cotransmission potentiates dentate granule cell firing.

SignificanceIt is now established that many neurons can release multiple transmitters. Recent studies revealed that fast-acting neurotransmitters, glutamate and GABA, are coreleased from the same presynaptic terminals in some adult brain regions. The dentate gyrus (DG) granule cells (GCs) are innervated by the hypothalamic supramammillary nucleus (SuM) afferents that corelease glutamate and GABA. However, how these functionally opposing neurotransmitters contribute to DG information processing remains unclear. We show that glutamatergic, but not GABAergic, cotransmission exhibits long-term potentiation (LTP) at SuM-GC synapses. By the excitatory selective LTP, the excitation/inhibition balance of SuM inputs increases, and GC firing is enhanced. This study provides evidence that glutamatergic/GABAergic cotransmission balance is rapidly changed in an activity-dependent manner, and such plasticity may modulate DG activity.

Traceable stimulus-dependent rapid molecular changes in dendritic spines in the brain.

Dendritic spines function as microcompartments that can modify the efficiency of their associated synapses. Here, we analyzed stimulus-dependent molecular changes in spines. The F-actin capping protein CapZ accumulates in parts of dendritic spines within regions where long-term potentiation has been induced. We produced a transgenic mouse line, AiCE-Tg, in which CapZ tagged with enhanced green fluorescence protein (EGFP-CapZ) is expressed. Twenty minutes after unilateral visual or somatosensory stimulation in AiCE-Tg mice, relative EGFP-CapZ signal intensification was seen in a subset of dendritic spines selectively in stimulated-side cortices; this right-left difference was abolished by NMDA receptor blockade. Immunolabeling of α-actinin, a PSD-95 binding protein that can recruit AMPA receptors, showed that the α-actinin signals colocalized more frequently in spines with the brightest EGFP-CapZ signals (top 100) than in spines with more typical EGFP-CapZ signal strength (top 1,000). This stimulus-dependent in vivo redistribution of EGFP-CapZ represents a novel molecular event with plasticity-like characteristics, and bright EGFP-CapZ in AiCE-Tg mice make high-CapZ spines traceable in vivo and ex vivo. This mouse line has the potential to be used to reveal sequential molecular events, including synaptic tagging, and to relate multiple types of plasticity in these spines, extending knowledge related to memory mechanisms.

Supramammillary Nucleus Afferents to the Dentate Gyrus Co-release Glutamate and GABA and Potentiate Granule Cell Output.

The supramammillary nucleus (SuM) of the hypothalamus projects to the dentate gyrus (DG) and the CA2 region of the hippocampus. Although the SuM-to-hippocampus circuits have been implicated in spatial and emotional memory formation, little is known about precise neural connections between the SuM and hippocampus. Here, we report that axons of SuM neurons make monosynaptic connections to granule cells (GCs) and GABAergic interneurons, but not to hilar mossy cells, in the DG and co-release glutamate and γ-aminobutyric acid (GABA) at these synapses. Although inputs from the SuM can excite some interneurons, the inputs alone fail to generate spikes in GCs. However, despite the insufficient excitatory drive and GABAergic co-transmission, SuM inputs have net excitatory effects on GCs and can potentiate GC firing when temporally associated with perforant path inputs. Our results indicate that the SuM influences DG information processing by modulating GC outputs.

Near-infrared deep brain stimulation via upconversion nanoparticle-mediated optogenetics.

Optogenetics has revolutionized the experimental interrogation of neural circuits and holds promise for the treatment of neurological disorders. It is limited, however, because visible light cannot penetrate deep inside brain tissue. Upconversion nanoparticles (UCNPs) absorb tissue-penetrating near-infrared (NIR) light and emit wavelength-specific visible light. Here, we demonstrate that molecularly tailored UCNPs can serve as optogenetic actuators of transcranial NIR light to stimulate deep brain neurons. Transcranial NIR UCNP-mediated optogenetics evoked dopamine release from genetically tagged neurons in the ventral tegmental area, induced brain oscillations through activation of inhibitory neurons in the medial septum, silenced seizure by inhibition of hippocampal excitatory cells, and triggered memory recall. UCNP technology will enable less-invasive optical neuronal activity manipulation with the potential for remote therapy.

LTP at Hilar Mossy Cell-Dentate Granule Cell Synapses Modulates Dentate Gyrus Output by Increasing Excitation/Inhibition Balance.

Excitatory hilar mossy cells (MCs) in the dentate gyrus receive inputs from dentate granule cells (GCs) and project back to GCs locally, contralaterally, and along the longitudinal axis of the hippocampus, thereby establishing an associative positive-feedback loop and connecting functionally diverse hippocampal areas. MCs also synapse with GABAergic interneurons that mediate feed-forward inhibition onto GCs. Surprisingly, although these circuits have been implicated in both memory formation (e.g., pattern separation) and temporal lobe epilepsy, little is known about activity-dependent plasticity of their synaptic connections. Here, we report that MC-GC synapses undergo a presynaptic, NMDA-receptor-independent form of long-term potentiation (LTP) that requires postsynaptic brain-derived neurotrophic factor (BDNF)/TrkB and presynaptic cyclic AMP (cAMP)/PKA signaling. This LTP is input specific and selectively expressed at MC-GC synapses, but not at the disynaptic inhibitory loop. By increasing the excitation/inhibition balance, MC-GC LTP enhances GC output at the associative MC-GC recurrent circuit and may contribute to dentate-dependent forms of learning and epilepsy.

Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders.

Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders.

Acute inhibition of diacylglycerol lipase blocks endocannabinoid-mediated retrograde signalling: evidence for on-demand biosynthesis of 2-arachidonoylglycerol.

The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase α (DGLα) is one of the best-characterized retrograde messengers at central synapses. It has been thought that 2-AG is produced 'on demand' upon activation of postsynaptic neurons. However, recent studies propose that 2-AG is pre-synthesized by DGLα and stored in neurons, and that 2-AG is released from such 'pre-formed pools' without the participation of DGLα. To address whether the 2-AG source for retrograde signalling is the on-demand biosynthesis by DGLα or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca(2+) elevation, Gq/11 protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca(2+)-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre-Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca(2+) elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGLα rather than mobilization from presumptive pre-formed pools.

Endocannabinoid signaling and synaptic function.

Endocannabinoids are key modulators of synaptic function. By activating cannabinoid receptors expressed in the central nervous system, these lipid messengers can regulate several neural functions and behaviors. As experimental tools advance, the repertoire of known endocannabinoid-mediated effects at the synapse, and their underlying mechanism, continues to expand. Retrograde signaling is the principal mode by which endocannabinoids mediate short- and long-term forms of plasticity at both excitatory and inhibitory synapses. However, growing evidence suggests that endocannabinoids can also signal in a nonretrograde manner. In addition to mediating synaptic plasticity, the endocannabinoid system is itself subject to plastic changes. Multiple points of interaction with other neuromodulatory and signaling systems have now been identified. In this Review, we focus on new advances in synaptic endocannabinoid signaling in the mammalian brain. The emerging picture not only reinforces endocannabinoids as potent regulators of synaptic function but also reveals that endocannabinoid signaling is mechanistically more complex and diverse than originally thought.

Endocannabinoids and retrograde modulation of synaptic transmission.

Since the first reports of endocannabinoid-mediated retrograde signaling in 2001, great advances have been made toward understanding the molecular basis and functions of the endocannabinoid system. Electrophysiological studies have revealed that the endocannabinoid system is functional at various types of synapses throughout the brain. Basic mechanisms have been clarified as to how endocannabinoids are produced and released from postsynaptic neurons and regulate neurotransmitter release through activating presynaptic cannabinoid CB(1) receptors, although there remain unsolved questions and some discrepancies. In addition to this major function, recent studies suggest diverse functions of endocannabinoids, including control of other endocannabinoid-independent forms of synaptic plasticity, regulation of neuronal excitability, stimulation of glia-neuron interaction, and induction of CB(1)R-independent plasticity. Using recently developed pharmacological and genetic tools, behavioral studies have elucidated the roles of the endocannabinoid system in various aspects of neural functions. In this review, we make a brief overview of molecular mechanisms underlying the endocannabinoid-mediated synaptic modulation and also summarize recent findings, which shed new light on a diversity of functional roles of endocannabinoids.

[Lipid mediator endocannabinoids in modulating synaptic transmission].

Marijuana smoking elicits various psychoactive effects through type 1 cannabinoid receptors (CB(1)Rs) in the brain. CB(1)R is a seven-transmembrane domain. G(i/o)-protein coupled receptors, and is expressed throughout the central nervous system including the hippocampus, cerebellum, striatum and cerebral cortex. Endogenous ligands for CB(1)R (endocannabinoids) are lipid in nature, and anandamide and 2-arachidonoylglycerol (2-AG) are considered to be the two major endocannabinoids. Endocannabinoids are known to function as retrograde messengers at synapses. Endocannabinoids are released from postsynaptic neurons in activity-dependent manners, and retrogradely activate presynaptic CB(1)Rs, resulting in short-term or long-term suppression of synaptic transmission. Endocannabinoid-mediated retrograde signaling is observed at various brain regions and considered as a general mechanism of synaptic modulation in the brain. Endocannabinoid release is triggered by postsynaptic Ca2+ elevation or activation of G(q/11)-protein coupled receptors. Recent studies have demonstrated that 2-AG mediates retrograde signaling at synapses in the brain. Endocannabinoid-mediated retrograde signaling is involved in long-term synaptic plasticity in several brain regions. At behavioral level, endocannabinoid signaling is known to be involved in hippocampus-, amygdala- and cerebellum-dependent learning and memory.

Neuronal protease-activated receptor 1 drives synaptic retrograde signaling mediated by the endocannabinoid 2-arachidonoylglycerol.

Protease-activated receptor 1 (PAR1) is a member of the G-protein coupled receptors that are proteolytically activated by serine proteases. Recent studies suggest a definite contribution of PAR1 to brain functions, including learning and memory. However, cellular mechanisms by which PAR1 activation influences neuronal activity are not well understood. Here we show that PAR1 activation drives retrograde endocannabinoid signaling and thereby regulates synaptic transmission. In cultured hippocampal neurons from rat, PAR1 activation by thrombin or PAR1-specific peptide agonists transiently suppressed inhibitory transmission at cannabinoid-sensitive, but not cannabinoid-insensitive, synapses. The PAR1-induced suppression of synaptic transmission was accompanied by an increase in paired-pulse ratio, and was blocked by a cannabinoid CB(1) receptor antagonist. The PAR1-induced suppression was blocked by pharmacological inhibition of postsynaptic diacylglycerol lipase (DGL), a key enzyme for biosynthesis of the major endocannabinoid 2-arachidonoylglycerol (2-AG), and was absent in knock-out mice lacking the α isoform of DGL. The PAR1-induced IPSC suppression remained intact under the blockade of metabotropic glutamate receptors and was largely resistant to the treatment that blocked Ca(2+) elevation in glial cells following PAR1 activation, which excludes the major contribution of glial PAR1 in IPSC suppression. We conclude that activation of neuronal PAR1 triggers retrograde signaling mediated by 2-AG, which activates presynaptic CB(1) receptors and suppresses transmitter release at hippocampal inhibitory synapses.

The endocannabinoid 2-arachidonoylglycerol produced by diacylglycerol lipase alpha mediates retrograde suppression of synaptic transmission.

Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGLalpha) and beta (DGLbeta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLalpha knockout mice, whereas the retrograde suppression was intact in DGLbeta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLalpha knockout brains, whereas the 2-AG content was normal in DGLbeta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLalpha, but not by DGLbeta, mediates retrograde suppression at central synapses.

Endocannabinoid-mediated control of synaptic transmission.

The discovery of cannabinoid receptors and subsequent identification of their endogenous ligands (endocannabinoids) in early 1990s have greatly accelerated research on cannabinoid actions in the brain. Then, the discovery in 2001 that endocannabinoids mediate retrograde synaptic signaling has opened up a new era for cannabinoid research and also established a new concept how diffusible messengers modulate synaptic efficacy and neural activity. The last 7 years have witnessed remarkable advances in our understanding of the endocannabinoid system. It is now well accepted that endocannabinoids are released from postsynaptic neurons, activate presynaptic cannabinoid CB(1) receptors, and cause transient and long-lasting reduction of neurotransmitter release. In this review, we aim to integrate our current understanding of functions of the endocannabinoid system, especially focusing on the control of synaptic transmission in the brain. We summarize recent electrophysiological studies carried out on synapses of various brain regions and discuss how synaptic transmission is regulated by endocannabinoid signaling. Then we refer to recent anatomical studies on subcellular distribution of the molecules involved in endocannabinoid signaling and discuss how these signaling molecules are arranged around synapses. In addition, we make a brief overview of studies on cannabinoid receptors and their intracellular signaling, biochemical studies on endocannabinoid metabolism, and behavioral studies on the roles of the endocannabinoid system in various aspects of neural functions.