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BioRxiv : the Preprint Server For... Oct 2023Presynaptic voltage-gated Ca2+ channels (CaV) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central...
Presynaptic voltage-gated Ca2+ channels (CaV) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system, most presynaptic terminals are CaV2.1 dominant with a developmental reduction in CaV2.2 and CaV2.3 levels, and CaV2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV2 subtype levels are largely unsolved. Since the CaV2 α1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV2.1 α1subunits containing swapped motifs with the CaV2.2 and CaV2.3 α1 subunit on a CaV2.1/CaV2.2 null background at the calyx of Held presynaptic terminal. We found that expression of CaV2.1 α1 subunit chimeras containing the CaV2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV2.1 α1 subunit. However, the Ca2+ current sensitivity to the CaV2.1 blocker Agatoxin-IVA, was the same between the chimeras and the CaV2.1 α1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV2.1 loop II-III and CT do not individually regulate CaV2.1 preference, but these motifs control CaV2.1 levels and the CaV2.3 CT contains motifs that negatively regulate presynaptic CaV2.3 levels. We propose that the motifs controlling presynaptic CaV2.1 preference are distinct from those regulating CaV2.1 levels and may act synergistically to impact pathways regulating CaV2.1 preference and abundance.
PubMed: 37162941
DOI: 10.1101/2023.04.28.538778 -
Cell Reports Feb 2024Axotomized spinal motoneurons (MNs) lose presynaptic inputs following peripheral nerve injury; however, the cellular mechanisms that lead to this form of synapse loss...
Axotomized spinal motoneurons (MNs) lose presynaptic inputs following peripheral nerve injury; however, the cellular mechanisms that lead to this form of synapse loss are currently unknown. Here, we delineate a critical role for neuronal kinase dual leucine zipper kinase (DLK)/MAP3K12, which becomes activated in axotomized neurons. Studies with conditional knockout mice indicate that DLK signaling activation in injured MNs triggers the induction of phagocytic microglia and synapse loss. Aspects of the DLK-regulated response include expression of C1q first from the axotomized MN and then later in surrounding microglia, which subsequently phagocytose presynaptic components of upstream synapses. Pharmacological ablation of microglia inhibits the loss of cholinergic C boutons from axotomized MNs. Together, the observations implicate a neuronal mechanism, governed by the DLK, in the induction of inflammation and the removal of synapses.
Topics: Animals; Mice; Synapses; Motor Neurons; Signal Transduction; Complement Activation; Presynaptic Terminals; Mice, Knockout
PubMed: 38363678
DOI: 10.1016/j.celrep.2024.113801 -
BioRxiv : the Preprint Server For... Apr 2024Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca influx but exhibit significant heterogeneity in exocytosis...
Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca-triggered vesicle fusion and short-term facilitation. We find that Synaptotagmin-1, Synaptotagmin-7, and Complexin, synergistically restrain SNARE complex assembly, thus preserving vesicles in a stably docked state at rest. Upon Ca activation, Synaptotagmin-1 induces rapid vesicle fusion, while Synaptotagmin-7 mediates delayed fusion. Competitive binding of Synaptotagmin-1 and Synaptotagmin-7 to the same SNAREs, coupled with differential rates of Ca-triggered fusion clamp reversal, govern the kinetics of vesicular fusion. Under conditions mimicking sustained neuronal activity, the Synaptotagmin-7 fusion clamp is destabilized by the elevated basal Ca concentration, thereby enhancing the synchronous component of fusion. These findings provide a direct demonstration that a small set of proteins is sufficient to account for how nerve terminals adapt and regulate the Ca-evoked neurotransmitter exocytosis process to support their specialized functions in the nervous system.
PubMed: 38659918
DOI: 10.1101/2024.04.15.589559 -
Cells Sep 2023This study provides evidence of the existence of presynaptic inhibitory sphingosine-1-phosphate receptor 1 (S1P1R) and facilitatory S1P3R in cortical nerve endings...
This study provides evidence of the existence of presynaptic inhibitory sphingosine-1-phosphate receptor 1 (S1P1R) and facilitatory S1P3R in cortical nerve endings (synaptosomes) of healthy mice. The conclusion relies on the findings that (i) the S1P1R agonist CS-2100 (0.1-30 nM) inhibits the 12 mM KCl-evoked glutamate exocytosis (quantified as the release of [H]D-aspartate) while the S1P3R allosteric agonist CYM-5541 potentiates it and (ii) these effects are inhibited by the S1P1R antagonist Ex 26 (30-300 nM) and the S1P3R antagonist TY-52156 (100-1000 nM), respectively. Confocal microscopy and western blot analysis confirmed the presence of S1P1R and S1P3R proteins in cortical glutamatergic synaptosomes, which were scarcely accessible to biotin in a biotinylation study. Then, we demonstrated that S1P1R and S1P3R densities and their release activity are amplified in cortical synaptosomes of mice suffering from experimental autoimmune encephalomyelitis (EAE), despite receptors maintain their preferential internal distribution. Receptor changes recover following chronic oral therapeutic FTY720 (0.03 mg/Kg/day). These results improve our knowledge of the role of presynaptic release-regulating S1P1Rs and S1P3Rs controlling glutamate transmission in the CNS also unravelling functional adaptations during EAE that recover following chronic FTY720. In a whole, these findings provide new information on the central neuroprotectant activities of FTY720.
Topics: Mice; Animals; Encephalomyelitis, Autoimmune, Experimental; Fingolimod Hydrochloride; Sphingosine-1-Phosphate Receptors; Glutamic Acid
PubMed: 37830557
DOI: 10.3390/cells12192343 -
Ecotoxicology and Environmental Safety Sep 2023Humans are exposed to the common carcinogen benzo[a]pyrene (BaP) by ingesting contaminated foods and water or inhaling polluted air. Given the enriched lipids and...
Humans are exposed to the common carcinogen benzo[a]pyrene (BaP) by ingesting contaminated foods and water or inhaling polluted air. Given the enriched lipids and reduced antioxidative properties in the brain and the accumulation of BaP in the brain due to its high lipophilicity, the brain is susceptible to BaP-induced toxicity. Exposure to BaP leads to impairments in learning and memory, increased anxiety behavior, and neuronal death. It induces protein dysfunctions in neuronal compartments that play essential roles in neuronal activity or physiology. However, the neurotoxicity of BaP on presynaptic terminals, which is crucial to neurotransmission by releasing synaptic vesicles that contain neurotransmitters, has not yet been investigated. In the present study, we investigated the toxicity of BaP at presynaptic terminals in living hippocampal neurons. These neurons were sourced from transgenic mice pups (postnatal 1-day, a total of 12 pups, equal numbers for each sex) that endogenously express synaptic vesicle-fused pHluorin, which is a green fluorescent protein that enables monitoring of synaptic vesicle dynamics. We observed that BaP suppressed synaptic vesicle exocytosis by inhibiting presynaptic Ca entry via P/Q-type Ca channels. Together with molecular docking simulation, we speculate that BaP and metabolites may bind to the P/Q Ca channels. These results suggest the toxic mechanism of BaP exposure-induced abnormal behavior that provides a basis to evaluate the risk assessment of BaP-induced neurotoxicity.
Topics: Mice; Humans; Animals; Calcium Channels, Q-Type; Synaptic Vesicles; Benzo(a)pyrene; Molecular Docking Simulation; Neurons; Synaptic Transmission; Hippocampus; Exocytosis; Mice, Transgenic; Calcium
PubMed: 37506439
DOI: 10.1016/j.ecoenv.2023.115301 -
ENeuro Oct 2023The mushroom body (MB) is an important model system for studying the synaptic mechanisms of associative learning. In this system, coincidence of odor-evoked calcium...
The mushroom body (MB) is an important model system for studying the synaptic mechanisms of associative learning. In this system, coincidence of odor-evoked calcium influx and dopaminergic input in the presynaptic terminals of Kenyon cells (KCs), the principal neurons of the MB, triggers long-term depression (LTD), which plays a critical role in olfactory learning. However, it is controversial whether such synaptic plasticity is accompanied by a corresponding decrease in odor-evoked calcium activity in the KC presynaptic terminals. Here, we address this question by inducing LTD by pairing odor presentation with optogenetic activation of dopaminergic neurons (DANs). This allows us to rigorously compare the changes at the presynaptic and postsynaptic sites in the same conditions. By imaging presynaptic acetylcholine release in the condition where LTD is reliably observed in the postsynaptic calcium signals, we show that neurotransmitter release from KCs is depressed selectively in the MB compartments innervated by activated DANs, demonstrating the presynaptic nature of LTD. However, total odor-evoked calcium activity of the KC axon bundles does not show concurrent depression. We further conduct calcium imaging in individual presynaptic boutons and uncover the highly heterogeneous nature of calcium plasticity. Namely, only a subset of boutons, which are strongly activated by associated odors, undergo calcium activity depression, while weakly responding boutons show potentiation. Thus, our results suggest an unexpected nonlinear relationship between presynaptic calcium influx and the results of plasticity, challenging the simple view of cooperative actions of presynaptic calcium and dopaminergic input.
Topics: Animals; Drosophila; Presynaptic Terminals; Mushroom Bodies; Calcium; Dopamine; Dopaminergic Neurons; Neuronal Plasticity
PubMed: 37848287
DOI: 10.1523/ENEURO.0275-23.2023 -
Cell Reports Feb 2024Inflammation is closely associated with many neurodegenerative disorders. Yet, whether inflammation causes, exacerbates, or responds to neurodegeneration has been...
Inflammation is closely associated with many neurodegenerative disorders. Yet, whether inflammation causes, exacerbates, or responds to neurodegeneration has been challenging to define because the two processes are so closely linked. Here, we disentangle inflammation from the axon damage it causes by individually blocking cytotoxic T cell function and axon degeneration. We model inflammatory damage in mouse skin, a barrier tissue that, despite frequent inflammation, must maintain proper functioning of a dense array of axon terminals. We show that sympathetic axons modulate skin inflammation through release of norepinephrine, which suppresses activation of γδ T cells via the β2 adrenergic receptor. Strong inflammatory stimulation-modeled by application of the Toll-like receptor 7 agonist imiquimod-causes progressive γδ T cell-mediated, Sarm1-dependent loss of these immunosuppressive sympathetic axons. This removes a physiological brake on T cells, initiating a positive feedback loop of enhanced inflammation and further axon damage.
Topics: Animals; Mice; Feedback; Inflammation; Axons; Dermatitis; Presynaptic Terminals
PubMed: 38310514
DOI: 10.1016/j.celrep.2024.113721 -
Cortico-amygdala synaptic structural abnormalities produced by templated aggregation of α-synuclein.BioRxiv : the Preprint Server For... May 2024Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by neuronal α-synuclein (α-syn) inclusions termed Lewy Pathology, which are abundant in...
Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by neuronal α-synuclein (α-syn) inclusions termed Lewy Pathology, which are abundant in the amygdala. The basolateral amygdala (BLA), in particular, receives projections from the thalamus and cortex. These projections play a role in cognition and emotional processing, behaviors which are impaired in α-synucleinopathies. To understand if and how pathologic α-syn impacts the BLA requires animal models of α-syn aggregation. Injection of α-synuclein pre-formed fibrils (PFFs) into the striatum induces robust α-synuclein aggregation in excitatory neurons in the BLA that corresponds with reduced contextual fear conditioning. At early time points after aggregate formation, cortico-amygdala excitatory transmission is abolished. The goal of this project was to determine if α-syn inclusions in the BLA induce synaptic degeneration and/or morphological changes. In this study, we used C57BL/6J mice injected bilaterally with PFFs in the dorsal striatum to induce α-syn aggregate formation in the BLA. A method was developed using immunofluorescence and three-dimensional reconstruction to analyze excitatory cortico-amygdala and thalamo-amygdala presynaptic terminals closely juxtaposed to postsynaptic densities. The abundance and morphology of synapses were analyzed at 6- or 12-weeks post-injection of PFFs. α-Syn aggregate formation in the BLA did not cause a significant loss of synapses, but cortico-amygdala and thalamo-amygdala presynaptic terminals and postsynaptic densities with aggregates of α-synuclein show increased volumes, similar to previous findings in human DLB cortex, and in non-human primate models of PD. Transmission electron microscopy showed that PFF-injected mice showed reduced intervesicular distances similar to a recent study showing phospho-serine-129 α-synuclein increases synaptic vesicle clustering. Thus, pathologic α-synuclein causes major alterations to synaptic architecture in the BLA, potentially contributing to behavioral impairment and amygdala dysfunction observed in synucleinopathies.
PubMed: 38798467
DOI: 10.1101/2024.05.15.594419 -
BioRxiv : the Preprint Server For... Jul 2023The anterior and posterior subregions of the paraventricular thalamus (aPVT and pPVT, respectively) play unique roles in learned behaviors, from fear conditioning to...
The anterior and posterior subregions of the paraventricular thalamus (aPVT and pPVT, respectively) play unique roles in learned behaviors, from fear conditioning to alcohol/drug intake, potentially through differentially organized projections to limbic brain regions including the nucleus accumbens medial shell (mNAcSh). Here, we found that the aPVT projects broadly to the mNAcSh and that the aPVT-mNAcSh circuit encodes positive valence, such that manipulations of the circuit modulated both innately programmed and learned behavioral responses to positively and negatively valenced stimuli, particularly in females. Further, the endogenous activity of aPVT presynaptic terminals in the mNAcSh was greater in response to positively than negatively valenced stimuli, and the probability of synaptic glutamate release from aPVT neurons in the mNAcSh was higher in females than males. In contrast, we found that the pPVT-mNAcSh circuit encodes stimulus salience regardless of valence. While pPVT-mNAcSh circuit inhibition suppressed behavioral responses in both sexes, circuit activation increased behavioral responses to stimuli only in males. Our results point to circuit-specific stimulus feature encoding by parallel PVT-mNAcSh circuits that have sex-dependent biases in organization and function.
PubMed: 37461604
DOI: 10.1101/2023.07.03.547570 -
Frontiers in Cellular Neuroscience 2023Action potentials usually travel orthodromically along a neuron's axon, from the axon initial segment (AIS) toward the presynaptic terminals. Under some circumstances...
INTRODUCTION
Action potentials usually travel orthodromically along a neuron's axon, from the axon initial segment (AIS) toward the presynaptic terminals. Under some circumstances action potentials also travel in the opposite direction, antidromically, after being initiated at a distal location. Given their initiation at an atypical site, we refer to these events as "ectopic action potentials." Ectopic action potentials (EAPs) were initially observed in pathological conditions including seizures and nerve injury. Several studies have described regular-spiking (RS) pyramidal neurons firing EAPs in seizure models. Under nonpathological conditions, EAPs were reported in a few populations of neurons, and our group has found that EAPs can be induced in a large proportion of parvalbumin-expressing interneurons in the neocortex. Nevertheless, to our knowledge there have been no prior reports of ectopic firing in the largest population of neurons in the neocortex, pyramidal neurons, under nonpathological conditions.
METHODS
We performed in vitro recordings utilizing the whole-cell patch clamp technique. To elicit EAPs, we triggered orthodromic action potentialswith either long, progressively increasing current steps, or with trains of brief pulses at 30, 60, or 100 Hz delivered in 3 different ways, varying in stimulus and resting period duration.
RESULTS
We found that a large proportion (72.7%) of neocortical RS cells from mice can fire EAPs after a specific stimulus in vitro, and that most RS cells (56.1%) are capable of firing EAPs across a broad range of stimulus conditions. Of the 37 RS neurons in which we were able to elicit EAPs, it took an average of 863.8 orthodromic action potentials delivered over the course of an average of ~81.4 s before the first EAP was seen. We observed that some cells responded to specific stimulus frequencies while less selective, suggesting frequency tuning in a subset of the cells.
DISCUSSION
Our findings suggest that pyramidal cells can integrate information over long time-scales before briefly entering a mode of self-generated firing that originates in distal axons. The surprising ubiquity of EAP generation in RS cells raises interesting questions about the potential roles of ectopic spiking in information processing, cortical oscillations, and seizure susceptibility.
PubMed: 38034593
DOI: 10.3389/fncel.2023.1267687