In a recent study published in Nature, researchers investigated whether glutamatergic gliotransmission was mediated by specialized astrocytes within the central nervous system.
Study: Specialized astrocytes mediate glutamatergic gliotransmission within the CNS. Image Credit: Kateryna Kon/Shutterstock.com
The role of astrocytes in brain circuitry function, comparable to swift glutamate release, has been questioned resulting from inconsistent data and lack of direct evidence. This mechanism, just like neurons, controls plasticity, excitability, and coordinated activity of synaptic-type networks but in addition contributes to neuropsychiatric conditions.
Concerning the study
In the current study, the astrocyte glutamate exocytosis concept was revised by researchers by considering astrocyte molecular heterogeneity and using bioinformatic, imaging, and molecular methodologies, in addition to cell-specific genetic techniques that interact with glutamine exocytosis within the in vivo settings.
The researchers conducted a study to look at the role of glutamate within the brain and its effects on astrocytes. They used single-cell ribonucleic acid sequencing (scRNA-seq) databases and patch-seq information to perform GluSnFR-based glutamate imaging in situ and in vivo.
A deep neural network classifier was used to annotate the clusters, verifying the right prediction of astrocytes by checking the distribution of known astrocyte markers.
A cross-species evaluation was performed by referencing three hippocampal cell databases. fluorescence in-situ hybridization (FISH) evaluation was performed to investigate hippocampal slices from adult murine cells co-immunostained with astrocytic markers comparable to GS and S100β.
The astrocytes were imaged using two-photon excitation imaging of the dorsal molecular region of the dentate gyrus (DGML) region, which is estimated to comprise significant proportions of glutamate-releasing astrocytes that actively perform synaptic modulatory functions.
To mimic calcium-based glutamatergic glial transmission stimulated by Gq G-protein-coupled receptors (Gq-GPCRs), a designer-type receptor specifically activated by designer drugs (Gq-DREADD) was co-expressed in astrocytes and clozapine-induced chemogenetic stimulation was performed.
To limit probable sources of glutamate release from neuronal cells, hippocampal slices were perfused with synaptic blocker mixtures comprising voltage-gated calcium channel blockers and tetrodotoxin.
The researchers applied clozapine N-oxide (CNO) through temporary puffs locally followed by L-glutamate application as a control. To find out whether astrocyte release occurred through exocytosis, the team sought to impede glutamate filling in vesicles.
The team investigated the possible astrocytic origin of glutamate release by introducing acetylcholine (Ach), a physiologically relevant stimulus for visual cortex astrocytes, and evaluated its effect on the frequency of asynchronous SF-iGluSnFR events observed throughout the astrocytes.
The team also evaluated the impact of astrocyte vesicular glutamate transporter 1 (VGLUT1) deletion on hippocampal memory processing and altered cortico-hippocampal circuitry function, specializing in epileptic seizures. The researchers measured dopamine levels within the dorsal striatum (dST) of VGLUT2GFAP-KO mice and VGLUT2GFAP-WT controls by microdialysis.
Results
The researchers discovered nine different clusters of hippocampus astrocytes, with a major subset expressing synaptic-like glutamine-release machinery and restricted to specific hippocampal locations.
Additionally they discovered an identical astrocyte subgroup that reliably reacted to astrocyte-specific stimulations with sub-second release of glutamate at geographically specific areas of best need, which was inhibited by astrocyte-targeted VGLUT1 ablation.
The synaptic glutamate exocytosis cluster was present in all murine hippocampal databases, in addition to amongst humans.
The 4 neural genes linked to glutamatergic exocytosis in vesicles [(solute carrier family 17 member 7 (slc17a7), slc17a6, (coding for vglut2), synaptosomal-associated protein, 25kda (snap25), and synaptotagmin 1 (Syt1)] were expressed strongly in glutamatergic neurons in addition to S100β/GS-positive cells from the GFAP lineage with isolated nuclei, confirming astrocytic synaptic glutamate exocytosis within the cell population.
The findings showed a hippocampal subpopulation of cells with immunohistochemical, transcriptional, and morphological features of astrocytes comprising transcripts critical to glutamatergic-mediated secretion. The Gq-DREADD stimulation evoked calcium signaling within the astrocytes; nonetheless, only just a few astrocytes had adequate downstream machinery to release glutamate.
Glutamate release responses at all times took place at specific hotspots of an astrocyte, providing direct functional evidence for the existence of a specialized population of glutamatergic astrocytes predicted by transcriptomic studies. The team found a sturdy correlation between the physiological and molecular identification of glutamatergic astrocytes.
Glutamatergic astrocytes exerted a VGLUT1-dependent positive control on theta-burst-evoked long-term potentiation (ϴ-LTP) of the perforant path–granule cell (PP-GC) synapses residing inside their territory. The team observed a protective function of astrocyte VGLUT1-dependent signaling against kainate-induced acute seizures in vivo, opposing the mechanisms causing seizure amplification.
The predominant role of VGLUT2 within the substantia nigra pars compacta (SNpc) circuit was confirmed and indicated an inhibitory role for astrocyte VGLUT2 in controlling the excitatory synaptic input to SNpc dopaminergic neurons.
The findings strongly supported an endogenous regulatory function of astrocyte VGLUT2-dependent signaling in shaping glutamatergic synaptic transmission onto nigral dopaminergic neurons through the activation of presynaptic group III metabotropic glutamate receptors (mGluRs).
Astrocyte VGLUT2-dependent signaling regulated nigrostriatal dopaminergic pathway function in vivo, representing a possible therapeutic goal for Parkinson’s disease.
Overall, the study findings highlighted an atypical subpopulation of specialised astrocytes within the adult brain, providing insights into their roles in central nervous system physiology and diseases. These astrocytes have a molecular signature just like glutamatergic synapses, defining their distribution and functional competence. The findings highlight the functional relevance of those astrocytes, despite their small number, and their potential as therapeutic targets.
Professor Andrea Volterra, honorary professor at UNIL and visiting faculty on the Wyss Center, co-director of the study, told us:
Through advancements in molecular techniques, like single-cell transcriptomics, we have unveiled a newfound complexity inside brain cell classifications. While we traditionally grouped cells into categories like neurons, astrocytes, microglia, and oligodendrocytes, we now know these groups contain subpopulations with unique traits.
What’s particularly intriguing is that some astrocytes possess machinery typically present in neurons, not only at synapses releasing glutamate, but in addition proteins allowing vesicles to fuse with the plasma membrane, enabling the discharge of neurotransmitters. This distinctive combination blends astrocytic features with presynaptic neuronal characteristics, especially those related to glutaminergic neurons.
To evaluate their significance, we conducted experiments involving the knockout of an important protein chargeable for loading vesicles with glutamate. The outcomes were clear: astrocytes lacking this protein could not release glutamate.
Our investigation prolonged to circuit function, notably within the hippocampus, where we observed a discount in long-term potentiation, a significant mechanism for memory formation. Behavioral experiments in mice further demonstrated that while they may learn, memory retrieval became difficult when this specific astrocyte subpopulation was affected.
Within the context of epilepsy, we induced an acute seizure model and located that the absence of glutamate release in these astrocytes worsened the seizures. This means that these cells play a protective role in stopping the initiation of epileptic seizures, underscoring their physiological importance.
Moreover, within the nigrostriatal system, chargeable for controlling movements and crucial in Parkinson’s disease, we made a remarkable discovery. Dopaminergic neurons were found to be under the influence of this family of glutaminergic astrocytes, affecting the regulation of dopamine release. This finding hints at a physiological homeostatic function which may be compromised in Parkinson’s disease.