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1.
Nature ; 628(8008): 590-595, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38480889

RESUMO

Distinct brain and behavioural states are associated with organized neural population dynamics that are thought to serve specific cognitive functions1-3. Memory replay events, for example, occur during synchronous population events called sharp-wave ripples in the hippocampus while mice are in an 'offline' behavioural state, enabling cognitive mechanisms such as memory consolidation and planning4-11. But how does the brain re-engage with the external world during this behavioural state and permit access to current sensory information or promote new memory formation? Here we found that the hippocampal dentate spike, an understudied population event that frequently occurs between sharp-wave ripples12, may underlie such a mechanism. We show that dentate spikes are associated with distinctly elevated brain-wide firing rates, primarily observed in higher order networks, and couple to brief periods of arousal. Hippocampal place coding during dentate spikes aligns to the mouse's current spatial location, unlike the memory replay accompanying sharp-wave ripples. Furthermore, inhibiting neural activity during dentate spikes disrupts associative memory formation. Thus, dentate spikes represent a distinct brain state and support memory during non-locomotor behaviour, extending the repertoire of cognitive processes beyond the classical offline functions.


Assuntos
Ondas Encefálicas , Cognição , Hipocampo , Animais , Camundongos , Hipocampo/fisiologia , Consolidação da Memória/fisiologia , Nível de Alerta/fisiologia , Potenciais de Ação , Inibição Neural , Cognição/fisiologia , Ondas Encefálicas/fisiologia , Masculino , Feminino
2.
J Neurosci ; 35(27): 10039-57, 2015 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-26157003

RESUMO

Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic intracellular membrane cisternae at perisomatic GABAergic symmetrical synapses. Interestingly, neither AM251, JZL184, nor PF3845 affected CB1-positive dendritic interneuron synapses. Together, these findings are consistent with the possibility that constitutively active CB1 receptors substantially influence perisomatic GABA release probability and indicate that the synaptic effects of tonic 2-AG release are tightly controlled by presynaptic MGL activity and also by postsynaptic endovanilloid signaling and FAAH activity. SIGNIFICANCE STATEMENT: Tonic cannabinoid signaling plays a critical role in the regulation of synaptic transmission. However, the mechanistic details of how persistent CB1 cannabinoid receptor activity inhibits neurotransmitter release have remained elusive. Therefore, electrophysiological recordings, lipid measurements, and super-resolution imaging were combined to elucidate those signaling molecules and mechanisms that underlie tonic cannabinoid signaling. The findings indicate that constitutive CB1 activity has pivotal function in the tonic control of hippocampal GABA release. Moreover, the endocannabinoid 2-arachidonoylglycerol (2-AG) is continuously generated postsynaptically, but its synaptic effect is regulated strictly by presynaptic monoacylglycerol lipase activity. Finally, anandamide signaling antagonizes tonic 2-AG signaling via activation of postsynaptic transient receptor potential vanilloid TRPV1 receptors. This unexpected mechanistic diversity may be necessary to fine-tune GABA release probability under various physiological and pathophysiological conditions.


Assuntos
Endocanabinoides/metabolismo , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPV/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Ácidos Araquidônicos/farmacologia , Moduladores de Receptores de Canabinoides/farmacologia , Endocanabinoides/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Glicerídeos/farmacologia , Hipocampo/citologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Piperidinas/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptor CB1 de Canabinoide/fisiologia , Sinapses/metabolismo , Sinapses/ultraestrutura , Canais de Cátion TRPV/genética
3.
Science ; 383(6686): 967-970, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38422134

RESUMO

Endocannabinoid (eCB)-mediated suppression of inhibitory synapses has been hypothesized, but this has not yet been demonstrated to occur in vivo because of the difficulty in tracking eCB dynamics and synaptic plasticity during behavior. In mice navigating a linear track, we observed location-specific eCB signaling in hippocampal CA1 place cells, and this was detected both in the postsynaptic membrane and the presynaptic inhibitory axons. All-optical in vivo investigation of synaptic responses revealed that postsynaptic depolarization was followed by a suppression of inhibitory synaptic potentials. Furthermore, interneuron-specific cannabinoid receptor deletion altered place cell tuning. Therefore, rapid, postsynaptic, activity-dependent eCB signaling modulates inhibitory synapses on a timescale of seconds during behavior.


Assuntos
Região CA1 Hipocampal , Endocanabinoides , Potenciais Pós-Sinápticos Inibidores , Sinapses , Transmissão Sináptica , Animais , Camundongos , Endocanabinoides/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Sinalização do Cálcio , Região CA1 Hipocampal/fisiologia , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/fisiologia , Masculino , Feminino , Camundongos Knockout
4.
Nat Commun ; 15(1): 601, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38238329

RESUMO

Epilepsy is a prevalent disorder involving neuronal network hyperexcitability, yet existing therapeutic strategies often fail to provide optimal patient outcomes. Chemogenetic approaches, where exogenous receptors are expressed in defined brain areas and specifically activated by selective agonists, are appealing methods to constrain overactive neuronal activity. We developed BARNI (Bradanicline- and Acetylcholine-activated Receptor for Neuronal Inhibition), an engineered channel comprised of the α7 nicotinic acetylcholine receptor ligand-binding domain coupled to an α1 glycine receptor anion pore domain. Here we demonstrate that BARNI activation by the clinical stage α7 nicotinic acetylcholine receptor-selective agonist bradanicline effectively suppressed targeted neuronal activity, and controlled both acute and chronic seizures in male mice. Our results provide evidence for the use of an inhibitory acetylcholine-based engineered channel activatable by both exogenous and endogenous agonists as a potential therapeutic approach to treating epilepsy.


Assuntos
Epilepsia , Receptores Nicotínicos , Camundongos , Masculino , Humanos , Animais , Receptores Colinérgicos , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptores Nicotínicos/genética , Agonistas Nicotínicos/farmacologia , Acetilcolina/farmacologia , Convulsões/genética
5.
Sci Adv ; 10(22): eado0077, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38809980

RESUMO

While our understanding of the nanoscale architecture of anterograde synaptic transmission is rapidly expanding, the qualitative and quantitative molecular principles underlying distinct mechanisms of retrograde synaptic communication remain elusive. We show that a particular form of tonic cannabinoid signaling is essential for setting target cell-dependent synaptic variability. It does not require the activity of the two major endocannabinoid-producing enzymes. Instead, by developing a workflow for physiological, anatomical, and molecular measurements at the same unitary synapse, we demonstrate that the nanoscale stoichiometric ratio of type 1 cannabinoid receptors (CB1Rs) to the release machinery is sufficient to predict synapse-specific release probability. Accordingly, selective decrease of extrasynaptic CB1Rs does not affect synaptic transmission, whereas in vivo exposure to the phytocannabinoid Δ9-tetrahydrocannabinol disrupts the intrasynaptic nanoscale stoichiometry and reduces synaptic variability. These findings imply that synapses leverage the nanoscale stoichiometry of presynaptic receptor coupling to the release machinery to establish synaptic strength in a target cell-dependent manner.


Assuntos
Receptor CB1 de Canabinoide , Transdução de Sinais , Sinapses , Transmissão Sináptica , Animais , Transmissão Sináptica/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Sinapses/metabolismo , Terminações Pré-Sinápticas/metabolismo , Camundongos , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Dronabinol/farmacologia
6.
J Neurosci Methods ; 367: 109451, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34921843

RESUMO

The endocannabinoid (eCB) system is one of the most widespread neuromodulatory systems in the mammalian brain, with a multifaceted role in functions ranging from development to synaptic plasticity. Endocannabinoids are synthesized on demand from membrane lipid precursors, and act primarily on a single G-protein coupled receptor type, CB1, to carry out diverse functions. Despite the importance of the eCB system both in healthy brain function and in disease, critically important details of eCB signaling remained unknown. How eCBs are released from the membrane, how these lipid molecules are transported between cells, and how the distribution of their receptors is controlled, remained elusive. Recent advances in optical microscopy methods and biosensor engineering may open up new avenues for studying eCB signaling. We summarize applications of superresolution microscopy using single molecule localization to reveal distinct patterns of nanoscale CB1 distribution in neuronal axons and axon terminals. We review single particle tracking studies using quantum dots that allowed visualizing CB1 trajectories. We highlight the recent development of fluorescent eCB biosensors, that revealed spatiotemporally specific eCB release in live cells and live animals. Finally, we discuss future directions where method development may help to advance a precise understanding of eCB signaling.


Assuntos
Endocanabinoides , Transdução de Sinais , Animais , Encéfalo , Mamíferos , Plasticidade Neuronal/fisiologia , Neurônios , Receptor CB1 de Canabinoide
7.
Epilepsy Curr ; 22(1): 54-60, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35233202

RESUMO

Epileptic seizures are associated with excessive neuronal spiking. Perisomatic γ-aminobutyric acid (GABA)ergic interneurons specifically innervate the subcellular domains of postsynaptic excitatory cells that are critical for spike generation. With a revolution in transcriptomics-based cell taxonomy driving the development of novel transgenic mouse lines, selectively monitoring and modulating previously elusive interneuron types is becoming increasingly feasible. Emerging evidence suggests that the three types of hippocampal perisomatic interneurons, axo-axonic cells, along with parvalbumin- and cholecystokinin-expressing basket cells, each follow unique activity patterns in vivo, suggesting distinctive roles in regulating epileptic networks.

8.
Neuron ; 110(12): 1959-1977.e9, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35489331

RESUMO

Ripples are brief high-frequency electrographic events with important roles in episodic memory. However, the in vivo circuit mechanisms coordinating ripple-related activity among local and distant neuronal ensembles are not well understood. Here, we define key characteristics of a long-distance projecting GABAergic cell group in the mouse hippocampus that selectively exhibits high-frequency firing during ripples while staying largely silent during theta-associated states when most other GABAergic cells are active. The high ripple-associated firing commenced before ripple onset and reached its maximum before ripple peak, with the signature theta-OFF, ripple-ON firing pattern being preserved across awake and sleep states. Controlled by septal GABAergic, cholinergic, and CA3 glutamatergic inputs, these ripple-selective cells innervate parvalbumin and cholecystokinin-expressing local interneurons while also targeting a variety of extra-hippocampal regions. These results demonstrate the existence of a hippocampal GABAergic circuit element that is uniquely positioned to coordinate ripple-related neuronal dynamics across neuronal assemblies.


Assuntos
Hipocampo , Interneurônios , Animais , Hipocampo/fisiologia , Interneurônios/fisiologia , Camundongos , Neurônios/fisiologia , Parvalbuminas , Vigília
9.
Nat Biotechnol ; 40(5): 787-798, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34764491

RESUMO

Endocannabinoids (eCBs) are retrograde neuromodulators with important functions in a wide range of physiological processes, but their in vivo dynamics remain largely uncharacterized. Here we developed a genetically encoded eCB sensor called GRABeCB2.0. GRABeCB2.0 consists of a circular-permutated EGFP and the human CB1 cannabinoid receptor, providing cell membrane trafficking, second-resolution kinetics with high specificity for eCBs, and shows a robust fluorescence response at physiological eCB concentrations. Using GRABeCB2.0, we monitored evoked and spontaneous changes in eCB dynamics in cultured neurons and acute brain slices. We observed spontaneous compartmentalized eCB transients in cultured neurons and eCB transients from single axonal boutons in acute brain slices, suggesting constrained, localized eCB signaling. When GRABeCB2.0 was expressed in the mouse brain, we observed foot shock-elicited and running-triggered eCB signaling in the basolateral amygdala and hippocampus, respectively. In a mouse model of epilepsy, we observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. GRABeCB2.0 is a robust probe for eCB release in vivo.


Assuntos
Endocanabinoides , Neurônios , Animais , Encéfalo/metabolismo , Endocanabinoides/metabolismo , Hipocampo/fisiologia , Camundongos , Neurônios/metabolismo , Transdução de Sinais
10.
Science ; 374(6574): 1492-1496, 2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34914519

RESUMO

Locomotor speed is a basic input used to calculate one's position, but where this signal comes from is unclear. We identified neurons in the supramammillary nucleus (SuM) of the rodent hypothalamus that were highly correlated with future locomotor speed and reliably drove locomotion when activated. Robust locomotion control was specifically identified in Tac1 (substance P)­expressing (SuMTac1+) neurons, the activation of which selectively controlled the activity of speed-modulated hippocampal neurons. By contrast, Tac1-deficient (SuMTac1−) cells weakly regulated locomotion but potently controlled the spike timing of hippocampal neurons and were sufficient to entrain local network oscillations. These findings emphasize that the SuM not only regulates basic locomotor activity but also selectively shapes hippocampal neural activity in a manner that may support spatial navigation.


Assuntos
Hipocampo/fisiologia , Hipotálamo Posterior/fisiologia , Locomoção , Neurônios/fisiologia , Potenciais de Ação , Animais , Hipocampo/citologia , Hipotálamo Posterior/citologia , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/fisiologia , Ratos , Navegação Espacial , Substância P/genética , Ritmo Teta
11.
Nat Commun ; 12(1): 6505, 2021 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-34764251

RESUMO

Immunolabeling and autoradiography have traditionally been applied as the methods-of-choice to visualize and collect molecular information about physiological and pathological processes. Here, we introduce PharmacoSTORM super-resolution imaging that combines the complementary advantages of these approaches and enables cell-type- and compartment-specific nanoscale molecular measurements. We exploited rational chemical design for fluorophore-tagged high-affinity receptor ligands and an enzyme inhibitor; and demonstrated broad PharmacoSTORM applicability for three protein classes and for cariprazine, a clinically approved antipsychotic and antidepressant drug. Because the neurobiological substrate of cariprazine has remained elusive, we took advantage of PharmacoSTORM to provide in vivo evidence that cariprazine predominantly binds to D3 dopamine receptors on Islands of Calleja granule cell axons but avoids dopaminergic terminals. These findings show that PharmacoSTORM helps to quantify drug-target interaction sites at the nanoscale level in a cell-type- and subcellular context-dependent manner and within complex tissue preparations. Moreover, the results highlight the underappreciated neuropsychiatric significance of the Islands of Calleja in the ventral forebrain.


Assuntos
Ínsulas Olfatórias/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperazinas/farmacologia , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo
12.
Neuron ; 109(23): 3838-3850.e8, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34648750

RESUMO

The axon initial segment of hippocampal pyramidal cells is a key subcellular compartment for action potential generation, under GABAergic control by the "chandelier" or axo-axonic cells (AACs). Although AACs are the only cellular source of GABA targeting the initial segment, their in vivo activity patterns and influence over pyramidal cell dynamics are not well understood. We achieved cell-type-specific genetic access to AACs in mice and show that AACs in the hippocampal area CA1 are synchronously activated by episodes of locomotion or whisking during rest. Bidirectional intervention experiments in head-restrained mice performing a random foraging task revealed that AACs inhibit CA1 pyramidal cells, indicating that the effect of GABA on the initial segments in the hippocampus is inhibitory in vivo. Finally, optogenetic inhibition of AACs at specific track locations induced remapping of pyramidal cell place fields. These results demonstrate brain-state-specific dynamics of a critical inhibitory controller of cortical circuits.


Assuntos
Interneurônios , Ácido gama-Aminobutírico , Animais , Axônios/fisiologia , Hipocampo/fisiologia , Interneurônios/fisiologia , Camundongos , Células Piramidais/fisiologia , Sinapses/fisiologia , Ácido gama-Aminobutírico/fisiologia
13.
Neuron ; 109(6): 997-1012.e9, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33529646

RESUMO

Interneurons expressing cholecystokinin (CCK) and parvalbumin (PV) constitute two key GABAergic controllers of hippocampal pyramidal cell output. Although the temporally precise and millisecond-scale inhibitory regulation of neuronal ensembles delivered by PV interneurons is well established, the in vivo recruitment patterns of CCK-expressing basket cell (BC) populations has remained unknown. We show in the CA1 of the mouse hippocampus that the activity of CCK BCs inversely scales with both PV and pyramidal cell activity at the behaviorally relevant timescales of seconds. Intervention experiments indicated that the inverse coupling of CCK and PV GABAergic systems arises through a mechanism involving powerful inhibitory control of CCK BCs by PV cells. The tightly coupled complementarity of two key microcircuit regulatory modules demonstrates a novel form of brain-state-specific segregation of inhibition during spontaneous behavior.


Assuntos
Região CA1 Hipocampal/fisiologia , Interneurônios/fisiologia , Células Piramidais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Colecistocinina/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parvalbuminas/metabolismo
14.
Neuron ; 109(15): 2398-2403.e4, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34352214

RESUMO

The brain's endocannabinoid system is a powerful controller of neurotransmitter release, shaping synaptic communication under physiological and pathological conditions. However, our understanding of endocannabinoid signaling in vivo is limited by the inability to measure their changes at timescales commensurate with the high lability of lipid signals, leaving fundamental questions of whether, how, and which endocannabinoids fluctuate with neural activity unresolved. Using novel imaging approaches in awake behaving mice, we now demonstrate that the endocannabinoid 2-arachidonoylglycerol, not anandamide, is dynamically coupled to hippocampal neural activity with high spatiotemporal specificity. Furthermore, we show that seizures amplify the physiological endocannabinoid increase by orders of magnitude and drive the downstream synthesis of vasoactive prostaglandins that culminate in a prolonged stroke-like event. These results shed new light on normal and pathological endocannabinoid signaling in vivo.


Assuntos
Região CA1 Hipocampal/metabolismo , Endocanabinoides/metabolismo , Convulsões/metabolismo , Transmissão Sináptica/fisiologia , Animais , Camundongos , Ratos
15.
Sci Rep ; 10(1): 14992, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929133

RESUMO

Long-lasting confusion and memory difficulties during the postictal state remain a major unmet problem in epilepsy that lacks pathophysiological explanation and treatment. We previously identified that long-lasting periods of severe postictal hypoperfusion/hypoxia, not seizures per se, are associated with memory impairment after temporal lobe seizures. While this observation suggests a key pathophysiological role for insufficient energy delivery, it is unclear how the networks that underlie episodic memory respond to vascular constraints that ultimately give rise to amnesia. Here, we focused on cellular/network level analyses in the CA1 of hippocampus in vivo to determine if neural activity, network oscillations, synaptic transmission, and/or synaptic plasticity are impaired following kindled seizures. Importantly, the induction of severe postictal hypoperfusion/hypoxia was prevented in animals treated by a COX-2 inhibitor, which experimentally separated seizures from their vascular consequences. We observed complete activation of CA1 pyramidal neurons during brief seizures, followed by a short period of reduced activity and flattening of the local field potential that resolved within minutes. During the postictal state, constituting tens of minutes to hours, we observed no changes in neural activity, network oscillations, and synaptic transmission. However, long-term potentiation of the temporoammonic pathway to CA1 was impaired in the postictal period, but only when severe local hypoxia occurred. Lastly, we tested the ability of rats to perform object-context discrimination, which has been proposed to require temporoammonic input to differentiate between sensory experience and the stored representation of the expected object-context pairing. Deficits in this task following seizures were reversed by COX-2 inhibition, which prevented severe postictal hypoxia. These results support a key role for hypoperfusion/hypoxia in postictal memory impairments and identify that many aspects of hippocampal network function are resilient during severe hypoxia except for long-term synaptic plasticity.


Assuntos
Amnésia/fisiopatologia , Hipocampo/fisiopatologia , Convulsões/fisiopatologia , Acetaminofen/farmacologia , Animais , Região CA1 Hipocampal/fisiopatologia , Hipocampo/efeitos dos fármacos , Hipóxia/fisiopatologia , Potenciação de Longa Duração , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Células Piramidais/fisiologia , Ratos Long-Evans , Convulsões/induzido quimicamente , Convulsões/complicações , Transmissão Sináptica , Vasoconstrição
16.
J Neurosci ; 28(5): 1058-63, 2008 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-18234884

RESUMO

Endocannabinoids are regarded as retrograde signaling molecules at various types of synapses throughout the CNS. The lipid derivatives anandamide and 2-arachidonoylglycerol (2-AG) are generally thought to be the key molecular players in this process. Previous anatomical and electrophysiological studies provided compelling evidence that the biosynthetic enzyme of 2-AG is indeed localized in the postsynaptic plasma membrane, whereas its target, the CB1 cannabinoid receptor, and the enzyme responsible for its inactivation are both found presynaptically. This molecular architecture of 2-AG signaling is a conserved feature of most synapses and supports the retrograde signaling role of 2-AG. Conversely, the molecular and neuroanatomical organization of synaptic anandamide signaling remains largely unknown. In contrast to its predicted role in retrograde signaling, here we show that N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), a biosynthetic enzyme of anandamide and its related bioactive congeners, the N-acylethanolamines (NAEs), is concentrated presynaptically in several types of hippocampal excitatory axon terminals. Furthermore, high-resolution quantitative immunogold labeling demonstrates that this calcium-sensitive enzyme is localized predominantly on the intracellular membrane cisternae of axonal calcium stores. Finally, the highest density of NAPE-PLD is found in mossy terminals of granule cells, which do not express CB1 receptors. Together, these findings suggest that anandamide and related NAEs are also present at glutamatergic synapses, but the sites of their synthesis and action are remarkably different from 2-AG, indicating distinct physiological roles for given endocannabinoids in the regulation of synaptic neurotransmission and plasticity.


Assuntos
Cálcio/metabolismo , Moduladores de Receptores de Canabinoides/biossíntese , Endocanabinoides , Ácido Glutâmico/fisiologia , Terminações Pré-Sinápticas/enzimologia , Aciltransferases/biossíntese , Aciltransferases/metabolismo , Aciltransferases/fisiologia , Animais , Cálcio/análise , Moduladores de Receptores de Canabinoides/genética , Moduladores de Receptores de Canabinoides/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipase D/biossíntese , Fosfolipase D/metabolismo , Fosfolipase D/fisiologia , Terminações Pré-Sinápticas/química , Terminações Pré-Sinápticas/ultraestrutura , Receptor CB1 de Canabinoide/biossíntese , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Sinapses/química , Sinapses/enzimologia , Sinapses/ultraestrutura
17.
Brain Struct Funct ; 222(5): 2345-2357, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27905022

RESUMO

In the not too distant future, humankind will embark on one of its greatest adventures, the travel to distant planets. However, deep space travel is associated with an inevitable exposure to radiation fields. Space-relevant doses of protons elicit persistent disruptions in cognition and neuronal structure. However, whether space-relevant irradiation alters neurotransmission is unknown. Within the hippocampus, a brain region crucial for cognition, perisomatic inhibitory control of pyramidal cells (PCs) is supplied by two distinct cell types, the cannabinoid type 1 receptor (CB1)-expressing basket cells (CB1BCs) and parvalbumin (PV)-expressing interneurons (PVINs). Mice subjected to low-dose proton irradiation were analyzed using electrophysiological, biochemical and imaging techniques months after exposure. In irradiated mice, GABA release from CB1BCs onto PCs was dramatically increased. This effect was abolished by CB1 blockade, indicating that irradiation decreased CB1-dependent tonic inhibition of GABA release. These alterations in GABA release were accompanied by decreased levels of the major CB1 ligand 2-arachidonoylglycerol. In contrast, GABA release from PVINs was unchanged, and the excitatory connectivity from PCs to the interneurons also underwent cell type-specific alterations. These results demonstrate that energetic charged particles at space-relevant low doses elicit surprisingly selective long-term plasticity of synaptic microcircuits in the hippocampus. The magnitude and persistent nature of these alterations in synaptic function are consistent with the observed perturbations in cognitive performance after irradiation, while the high specificity of these changes indicates that it may be possible to develop targeted therapeutic interventions to decrease the risk of adverse events during interplanetary travel.


Assuntos
Células Piramidais/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Hipocampo/metabolismo , Interneurônios/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Parvalbuminas/metabolismo
18.
Nat Protoc ; 11(1): 163-83, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26716705

RESUMO

Single-molecule localization microscopy (SMLM) is rapidly gaining popularity in the life sciences as an efficient approach to visualize molecular distribution with nanoscale precision. However, it has been challenging to obtain and analyze such data within a cellular context in tissue preparations. Here we describe a 5-d tissue processing and immunostaining procedure that is optimized for SMLM, and we provide example applications to fixed mouse brain, heart and kidney tissues. We then describe how to perform correlated confocal and 3D-superresolution imaging on these sections, which allows the visualization of nanoscale protein localization within labeled subcellular compartments of identified target cells in a few minutes. Finally, we describe the use of VividSTORM (http://katonalab.hu/index.php/vividstorm), an open-source software for correlated confocal and SMLM image analysis, which facilitates the measurement of molecular abundance, clustering, internalization, surface density and intermolecular distances in a cell-specific and subcellular compartment-restricted manner. The protocol requires only basic skills in tissue staining and microscopy.


Assuntos
Microscopia/métodos , Imagem Molecular/métodos , Software , Algoritmos , Animais , Transporte Biológico , Análise por Conglomerados , Imageamento Tridimensional , Camundongos , Microscopia Confocal , Razão Sinal-Ruído , Coloração e Rotulagem , Interface Usuário-Computador
19.
Neuron ; 92(2): 479-492, 2016 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-27764673

RESUMO

Long-term changes of neurotransmitter release are critical for proper brain function. However, the molecular mechanisms underlying these changes are poorly understood. While protein synthesis is crucial for the consolidation of postsynaptic plasticity, whether and how protein synthesis regulates presynaptic plasticity in the mature mammalian brain remain unclear. Here, using paired whole-cell recordings in rodent hippocampal slices, we report that presynaptic protein synthesis is required for long-term, but not short-term, plasticity of GABA release from type 1 cannabinoid receptor (CB1)-expressing axons. This long-term depression of inhibitory transmission (iLTD) involves cap-dependent protein synthesis in presynaptic interneuron axons, but not somata. Translation is required during the induction, but not maintenance, of iLTD. Mechanistically, CB1 activation enhances protein synthesis via the mTOR pathway. Furthermore, using super-resolution STORM microscopy, we revealed eukaryotic ribosomes in CB1-expressing axon terminals. These findings suggest that presynaptic local protein synthesis controls neurotransmitter release during long-term plasticity in the mature mammalian brain.


Assuntos
Axônios/metabolismo , Interneurônios/metabolismo , Depressão Sináptica de Longo Prazo , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/metabolismo , Biossíntese de Proteínas , Receptor CB1 de Canabinoide/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Benzoxazinas/farmacologia , Corpo Celular/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Simulação de Dinâmica Molecular , Morfolinas/farmacologia , Naftalenos/farmacologia , Inibição Neural , Neurônios/metabolismo , Imagem Óptica , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Células Piramidais/metabolismo , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Ribossomos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
20.
Front Cell Neurosci ; 9: 100, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25859182

RESUMO

Fragile X is the most common cause of inherited intellectual disability and a leading cause of autism. The disease is caused by mutation of a single X-linked gene called fmr1 that codes for the Fragile X mental retardation protein (FMRP), a 71 kDa protein, which acts mainly as a translation inhibitor. Fragile X patients suffer from cognitive and emotional deficits that coincide with abnormalities in dendritic spines. Changes in spine morphology are often associated with altered excitatory transmission and long-term plasticity, the most prominent deficit in fmr1-/y mice. The nucleus accumbens, a central part of the mesocortico-limbic reward pathway, is now considered as a core structure in the control of social behaviors. Although the socio-affective impairments observed in Fragile X suggest dysfunctions in the accumbens, the impact of the lack of FMRP on accumbal synapses has scarcely been studied. Here we report for the first time a new spike timing-dependent plasticity paradigm that reliably triggers NMDAR-dependent long-term potentiation (LTP) of excitatory afferent inputs of medium spiny neurons (MSN) in the nucleus accumbens core region. Notably, we discovered that this LTP was completely absent in fmr1-/y mice. In the fmr1-/y accumbens intrinsic membrane properties of MSNs and basal excitatory neurotransmission remained intact in the fmr1-/y accumbens but the deficit in LTP was accompanied by an increase in evoked AMPA/NMDA ratio and a concomitant reduction of spontaneous NMDAR-mediated currents. In agreement with these physiological findings, we found significantly more filopodial spines in fmr1-/y mice by using an ultrastructural electron microscopic analysis of accumbens core medium spiny neuron spines. Surprisingly, spine elongation was specifically due to the longer longitudinal axis and larger area of spine necks, whereas spine head morphology and postsynaptic density size on spine heads remained unaffected in the fmr1-/y accumbens. These findings together reveal new structural and functional synaptic deficits in Fragile X.

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