Specialized astrocyte subpopulation discovery sheds light on brain health and treatment avenues

By Pooja Toshniwal PahariaSep 7 2023Reviewed by Sophia Coveney

In a recent study published in Nature, researchers investigated whether glutamatergic gliotransmission was mediated by specialized astrocytes in the central nervous system.

The role of astrocytes in brain circuitry function, such as swift glutamate release, has been questioned due to inconsistent data and lack of direct evidence. This mechanism, similar to neurons, controls plasticity, excitability, and coordinated activity of synaptic-type networks but also contributes to neuropsychiatric conditions.

About the study

In the present study, the astrocyte glutamate exocytosis concept was revised by researchers by considering astrocyte molecular heterogeneity and using bioinformatic, imaging, and molecular methodologies, as well as cell-specific genetic techniques that interact with glutamine exocytosis in the in vivo settings.

The researchers conducted a study to examine the role of glutamate in 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 correct 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) analysis was performed to analyze hippocampal slices from adult murine cells co-immunostained with astrocytic markers such as 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 brief puffs locally followed by L-glutamate application as a control. To determine 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 within 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, focusing on epileptic seizures. The researchers measured dopamine levels in the dorsal striatum (dST) of VGLUT2^GFAP-KO mice and VGLUT2^GFAP-WT controls by microdialysis.

Results

The researchers discovered nine different clusters of hippocampus astrocytes, with a significant subset expressing synaptic-like glutamine-release machinery and restricted to specific hippocampal locations.

They also discovered a matching astrocyte subgroup that reliably reacted to astrocyte-specific stimulations with sub-second release of glutamate at geographically specific areas of greatest need, which was inhibited by astrocyte-targeted VGLUT1 ablation.

The synaptic glutamate exocytosis cluster was found in all murine hippocampal databases, as well as among humans.

The four 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 as well as S100β/GS-positive cells from the GFAP lineage with isolated nuclei, confirming astrocytic synaptic glutamate exocytosis in 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 in the astrocytes; however, only a few astrocytes had adequate downstream machinery to release glutamate.

Glutamate release responses always 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 robust 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 within 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 in 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 potential therapeutic target for Parkinson's disease.

Overall, the study findings highlighted an atypical subpopulation of specialized astrocytes in the adult brain, providing insights into their roles in central nervous system physiology and diseases. These astrocytes have a molecular signature similar to glutamatergic synapses, defining their distribution and functional competence. The findings highlight the functional relevance of these astrocytes, despite their small number, and their potential as therapeutic targets.

Professor Andrea Volterra, honorary professor at UNIL and visiting faculty at the Wyss Center, co-director of the study, told us: 

Through advancements in molecular techniques, like single-cell transcriptomics, we've unveiled a newfound complexity within 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 found in neurons, not just at synapses releasing glutamate, but also proteins allowing vesicles to fuse with the plasma membrane, enabling the release of neurotransmitters. This distinctive combination blends astrocytic features with presynaptic neuronal characteristics, especially those associated with glutaminergic neurons.

To assess their significance, we conducted experiments involving the knockout of a crucial protein responsible for loading vesicles with glutamate. The outcomes were clear: astrocytes lacking this protein could no longer release glutamate.

Our investigation extended to circuit function, notably in the hippocampus, where we observed a reduction in long-term potentiation, a vital mechanism for memory formation. Behavioral experiments in mice further demonstrated that while they could learn, memory retrieval became challenging when this specific astrocyte subpopulation was affected.

In the context of epilepsy, we induced an acute seizure model and found that the absence of glutamate release in these astrocytes worsened the seizures. This suggests that these cells play a protective role in preventing the initiation of epileptic seizures, underscoring their physiological importance.

Furthermore, in the nigrostriatal system, responsible 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 that may be compromised in Parkinson's disease.