Doctor of Philosophy, Semmelweis University (2017)
Master of Science, Eotvos Lorand University (2009)
A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type- and subcellular compartment-specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic ?(9)-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of ?(9)-tetrahydrocannabinol treatment. These findings indicate that cell type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.
View details for DOI 10.1038/nn.3892
View details for Web of Science ID 000346995000014
View details for PubMedID 25485758
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.
View details for DOI 10.1038/s41598-020-71935-6
View details for PubMedID 32929133
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.
View details for PubMedID 27905022
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.
View details for DOI 10.1016/j.neuron.2016.09.040
View details for Web of Science ID 000386762000028
View details for PubMedID 27764673
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.
View details for DOI 10.1038/nprot.2016.002
View details for Web of Science ID 000367462100011
View details for PubMedID 26716705
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.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.
View details for DOI 10.1523/JNEUROSCI.4112-14.2015
View details for Web of Science ID 000358253400023
View details for PubMedID 26157003
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.
View details for DOI 10.3389/fncel.2015.00100
View details for Web of Science ID 000352443700001
View details for PubMedID 25859182
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.
View details for DOI 10.1523/JNEUROSCI.5102-07.2008
View details for Web of Science ID 000252778000006
View details for PubMedID 18234884