Astrocytes are an important part of the nervous system, they play a vital role in the maintenance and protection of neural networks, and they have complex and diverse functions. Whether astrocytes are involved in electrical signaling is a long-standing question, and previous in vitro experiments have given conflicting results. A recent study published in Nature is the first to confirm the existence of a specific subset of astrocytes that are able to release glutamate through exocytosis, thereby participating in electrical signaling in the nervous system.
Written by |Veronica (Tsinghua University School of Medicine).
The nervous system is made up of two main types of cells: neurons and glial cells (glial cells). For a long time, it was generally believed that neurons were responsible for the functional activities of the nervous system, and glial cells were regarded as "background cells" that only had auxiliary roles such as support, nutrition and protection for neurons. However, as research has progressed, this view has been challenged – the role of glial cells is much more than that.
In September 2023, an article published in the journal Nature confirmed for the first time that there is a specific subset of astrocytes that are able to participate in the electrical signaling of the nervous system by releasing glutamate through exocytosis. This discovery subverts the traditional cognition, reveals that astrocytes have an important physiological role in the nervous system, and also opens up new ideas for the development of complex neurological diseases.
Astrocytes: the largest number and most complex glial cells.
Glial cells were first described in 1856 by the German pathologist Rudolf Virchow (1821-1902) as the connective tissue within the brain and spinal cord that connects neurons together. The word glia is derived from the Greek word for glue, which also reflects scientists' original understanding of the function of glial cells – to "glue" neurons together and weave them into a tight neural network. Within the human central nervous system, glial cells are 10 50 times more numerous than neurons, up to 1 5x10 12 [2]. Similar to neurons, glial cells have protrusions on the surface, but instead of dendrites and axons, they cannot form chemical synapses with each other, but are connected by gap junctions. If neurons and their protrusions are compared to a forest, glial cells are fungi in the forest, wrapping around tree trunks and intertwining into a web.
In fact, glial cells are not a single type of cell, but include a variety of cell types. In the central nervous system, glial cells mainly include astrocyte, oligodendrocyte and microglia. In the peripheral nervous system, it mainly includes Schwann cells and satellite cells.
Figure 1Fluorescence microscopy of the central nervous system. In the figure, neurons (blue) are surrounded by a large number of glial cells, including astrocytes (red) and oligodendrocytes (green).Credit: Jonathan Cohen Nih
Our protagonists today are astrocytes. Astrocytes are the largest number of glial cells in the central nervous system and have the most complex functions, which are indispensable components for maintaining the homeostasis of the nervous system, and can be said to be the "star" of glial cells.
In 1871, the Italian neuroanatomist and pathologist Camillo Golgi (1843-1926, the discoverer of the famous Golgi apparatus) invented the famous chromate-silver nitrate staining technique [3], which observed the morphology of astrocytes through a microscope and divided them into two basic subtypes: protoplasmic and fibrous. In the traditional view, the former is mainly distributed in gray matter, with short and thick protrusions and numerous branches; The latter is mainly found in the white matter, with long straight protrusions and few branches. However, this classification method significantly underestimates the heterogeneity of astrocytes. In fact, there is a high degree of heterogeneity in astrocytes at the transcriptional and functional levels in different regions of the brain and in different cortical layers. However, there is no conclusive jury as to how this heterogeneity develops.
Among the different species, astrocytes account for about 20-50% of the central nervous system [4]. A large number of astrocytes are closely adjacent to and glued together with neurons, and their long protrusions weave together in the brain and spinal cord to form a scaffold that supports neurons. The protruding ends of astrocytes are enlarged, forming perivascular footings and participating in the formation of the blood-brain barrier (BBB). These protrusions enclose the nerve endings of neurons while avoiding the interference of different afferent fibers, and play a role in isolating various areas within the central nervous system.
In addition to these basic functions, scientists have discovered that astrocytes have more complex functions. For example, astrocytes are able to take up glutamate and -aminobutyric acid (GABA), transmitters released by neurons, and convert them into glutamine. These neurotransmitters can activate receptors on the surface of neurons, excitating neurons, enabling the transmission of electrical signals between adjacent neurons. However, glutamine cannot activate receptors, avoid the continuous excitation of neurons, and can also be transported back to neurons for recycling, providing raw materials for neurons to synthesize new transmitters.
The human brain accounts for about 2% of total body weight but consumes 20% of the body's glucose, with neurons having the highest demand for energy and sustained glucose**. Astrocytes can take up glucose from the blood and convert it into glycogen stores, or into lactose to power active neurons. This metabolic process is closely related to the astrocyte-neuronal antioxidant exchange system, which helps to alleviate oxidative stress damage in neurons. In addition, astrocytes can also produce a variety of neurotrophic factors, which play an important role in the growth, development, survival, and functional integrity of neurons.
During development, astrocytes play a role in guiding neuronal migration and synaptic pruning, regulating synaptic formation and function. They themselves can also act as antigen-presenting cells in the central nervous system, presenting antigens to T lymphocytes and exerting an immune response.
Unlike neurons, glial cells have the ability to proliferate throughout their lives. When the brain and spinal cord are damaged and degenerated, they rely mainly on the proliferation of astrocytes to fill the tissue defects. However, hyperplasia may lead to the formation of glial cell tumors and may also become a focal point for seizures. Studies have shown that in vitro glial cell differentiation into neurons can be achieved, which provides hope for the development of a variety of neurodegenerative diseases [5]. However, some scholars hold the opposite view, arguing that glial-neuronal transformation is not yet possible. They used lineage tracing techniques to confirm that glial cells were not transformed into neurons, but that some endogenous neurons were mislabeled [6].
Figure 2Fluorescence microscopy of astrocytesSource: D**ID Robertson, LCR Science Photo Library
Debate: Can astrocytes be involved in electrical signaling?
From the above introduction, it can be seen that the study of astrocyte function is one of the important frontier topics, and there are still many unknowns to be explored. One of these questions has been around for decades: whether astrocytes are involved in electrical signaling in the nervous system.
Electrical signaling is fundamental to the proper functioning of the nervous system and is essential for sustaining life activities, adapting to environmental changes, and fulfilling the complex functions of living organisms. Abnormalities in electrical signaling can lead to a variety of diseases, including neurodegenerative diseases, epilepsy, and pain disorders. In the past understanding, neurons are the only cells in the nervous system that have the function of electrical signaling. Some scholars believe that astrocytes may be involved in electrical signaling, but there is always a lack of conclusive evidence.
In 1990, a research team from Yale University School of Medicine in the United States found [7] that glutamate could induce an increase in the level of free calcium ions in hippocampal astrocytes under in vitro culture conditions. The study confirmed the presence of glutamate receptors on the surface of astrocytes, suggesting that they may be involved in neuroelectrical signaling.
In 1994, a research team from the College of Zoology and Genetics at Iowa State University in the United States constructed an in vitro co-culture system of astrocytes and neurons [8], and found that the addition of bradykinin increased the concentration of calcium ions in astrocytes, which in turn induced glutamate release. The released glutamate triggers an increase in the concentration of calcium ions in neurons by binding to glutamate receptors on the surface of neurons. In the isolated neuronal culture system without astrocytes, the addition of bradykinin did not cause a change in the concentration of calcium ions within the neurons. This suggests that astrocytes can transmit electrical signals to neurons by releasing glutamate under in vitro culture conditions.
In 1997, Andrea Volterra's team from the Institute of Pharmacology at the University of Milan in Italy found the opposite [9], that astrocytes respond to electrical signals from neurons. They used fluorescence confocal microscopy to observe rat brain slices and found that stimulation of neuronal afferent fibers caused fluctuations in calcium concentration in astrocytes (oscillation), and the frequency of calcium concentration fluctuations was related to the stimulation pattern received by nerve fibers.
Figure 3Schematic diagram of the "triple synapse" theory. presynaptic neuron: presynaptic neuron; postsynaptic neuron: postsynaptic neuron; astrocyte: astrocytes; Ca2+: calcium ion concentration; NT (neurotransmitters): neurotransmitters released by neurons; GT (gliotransmitters): neurotransmitters released by glial cellsSource: Ref. [10].
The general theory is that the transmission process of signals between neurons is that presynaptic neurons release neurotransmitters that activate receptors on the surface of postsynaptic neurons, triggering fluctuations in intracellular calcium ion concentration, thereby excitating postsynaptic neurons. After the discovery that astrocytes may be involved in electrical signaling, the "tripartite synapse" theory was proposed [10]. It is theorized that the integration and conduction of electrical signals at synapses involves not only the presynaptic and postsynaptic ends, but also the adjacent perisynaptic astrocytes.
Between 2000 and 2012, more than 100 publications** were published in this field, supporting the involvement of astrocytes in the transmission of nerve electrical signals through synapses. However, there are also opposing voices, questioning the rationality of data collection and interpretation. The opposing argument is that most experiments are conducted in vitro cultured astrocytes and cannot prove that the process of neurotransmitter release by astrocytes actually occurs in vivo.
In vivo experiments, the strongest evidence comes from a transgenic mouse model in which vesicle release from astrocytes was inhibited. However, in 2014, a mouse model widely used in astrocyte research was found to be flawed [11], leading to doubts about the reliability of all studies using this mouse model. In this mouse model, the glial fibrillary acidic protein (GFAP) promoter was used to knock out a key protein (SNARE) during vesicle trafficking and release, thereby inhibiting transmitter release. Previous studies suggested that GFAP was only specifically expressed in astrocytes, but it was later found that some neurons could also express GFAP. As a result, there is an "off-target effect" in this mouse model. The biological effect observed after SNARE knockout does not prove that the astrocyte transmitter release process exists in vivo and has a physiological function, as this effect may be related to the inhibition of transmitter release in some neurons.
As described above on the function of astrocytes, most scholars agree that astrocytes can take up glutamate released by neurons, thereby eliminating the ongoing effects of neurotransmitters on neurons. However, more direct evidence is needed to confirm whether astrocytes can participate in the conduction of neuronal electrical signals through the release of glutamate.
Nature's latest research confirms that astrocytes are involved in electrical nerve signaling.
Since the discovery in 1997 that neurons can transmit electrical signals to astrocytes, Andrea Volterra's team has been working on astrocyte-neuron signaling and has made outstanding contributions to this field. In September 2023, the journal Nature published a study by Volterra's team** [12], titled "Specialized Astrocytes Mediate Glutamatergic Gliotransmission in the CNS", which provides strong evidence for the involvement of astrocytes in neuroelectrical signaling.
Figure 4The latest research from the Volterra team**. Source: Ref. [12].
Through the integrated analysis of single-cell RNA sequencing data from eight mouse hippocampal single-cell cells and patch-seq sequencing data from mouse hippocampus, the researchers divided mouse hippocampal astrocytes into nine subsets with different molecular characteristics. It was found that only one subset was selectively expressed and exocytosis (the process by which intracellular vesicles fuse with cell membranes to transport substances in vesicles to the outside of cells, which is an important mechanism for neurotransmitter release), calcium-ion-regulated exocytosis, and regulation of neurotransmitter secretion secretion) and regulation of glutamate secretion. This suggests that this subset of astrocytes is theoretically capable of being involved in electrical signaling. This subset of astrocytes is unevenly distributed in mouse brain regions, even in specific neural circuits.
To verify the presence of this subset of astrocytes in the human brain, the researchers searched for specific molecular markers they found in three human hippocampal single-cell transcriptome sequencing open-source data. The results confirmed that a subset of astrocytes capable of releasing glutamate was also present in the human hippocampus.
Figure 5Schematic diagram of in vitro experiments. Six to eight weeks after viral injection into mouse brain slices, the researchers used two-photon confocal microscopy to take images of astrocytesSource: Ref. [12].
Single-cell transcriptomic sequencing results are compelling, but still only circumstantial. To directly confirm that specific astrocytes are able to release glutamate, Volterra's team used two-photon confocal microscopy to observe transmitter release in the dorsal molecular layer of dentate gyrus (data** glutamate-secreting astrocytes in this region) of the mouse brain. Glutamate receptors selectively expressed in mouse astrocytes were imaged, and synaptic release blockers were added to the experimental system to rule out interference with neuronal transmitter release. To mimic the concentration-dependent release of calcium ions mediated by G protein-coupled receptors (GPCRs) in vivo, the research team expressed a GPCR receptor activated by clozapine n-oxide (CNO) in astrocytes of experimental mice. After local addition of CNO to mouse brain slices, they observed the release of glutamate transmitters in a subset of astrocytes and found that this subset of astrocytes was clustered in a specific area, known as the glutamate release "hotspot" region. By injecting a viral vector into mouse brain slices, the researchers specifically knocked out vesicular glutamate transporter 1 (VGLUT1) in mouse astrocytes. It was found that the CNO-induced release of glutamate transmitters could not be observed in the "hot spot" region after VGLUT1 knockout, demonstrating that astrocyte transmitter release was mediated by exocytosis.
Figure 6Schematic diagram of in vivo experiments. two-photon: two-photon confocal microscopy; head bar: a pole used to hold the head of the mouse; cranial window: cranial window, used to inject drugs; drug: Experimental medicationSource: Ref. [12].
In order to confirm that the process of astrocyte transmitter release can occur in vivo, the researchers opened the skull of mice and used two-photon confocal microscopy to observe glutamate release in the primary visual cortex of awake mice. In the absence of drug stimulation, they recorded endogenous astrocytes glutamate release signals, saying that astrocytes were able to sense fluctuations in glutamate concentrations in the extracellular space in their natural state. The frequency of glutamate release from astrocytes was significantly increased under the stimulation of CNO.
In addition, the researchers also conducted functional experiments to demonstrate that VGLUT1-dependent astrocyte transmitter release has a protective effect on acute seizures; The VGLUT2-dependent astrocyte signaling pathway has the function of regulating the substantia-striatum circuit, which is a potential target of Parkinson's disease.
The Volterra team found out the molecular characteristics of astrocytes that can release glutamate through single-cell sequencing technology, directly observed the process of astrocyte transmitter release through in vivo and in vitro experiments, and demonstrated the potential protective effect of astrocyte transmitter release in neurological diseases with functional experiments. The field of electrical conduction function of astrocytes has finally ushered in the final evidence. At the same time, the findings provide an explanation for the conflicting studies of the past three decades. Since only a certain subset of astrocytes is able to release glutamate, previous studies have concluded that glutamate release is closely related to astrocyte material: glutamate release cannot be observed if the astrocytes used by the investigator are not of that particular subpopulation.
Figure 7A recent photo of Volterra. Twenty-five years have passed since Volterra first discovered that astrocytes cultured in vitro can respond to neuronal electrical signals. This time, he brought new blockbuster evidenceSource: Andrea Voltera
In an interview, Volterra said: "We are right, there are astrocytes that release glutamate. But we were also wrong, because we thought that all astrocytes would release glutamate. [13] Dimitri Rusakov, a professor of neuroscience at University College London in the United Kingdom, commented: "It is almost certain to say that these findings have upended the current understanding of how brain signaling is conducted, but the question of how exactly it will be subverted remains an open question." ”
Good research leads to more problems.
Confirming that astrocytes can release glutamate transmitters is only the first step, and there are still many questions waiting to be answered in the future. What are the effects of glutamate transmitters released by astrocytes on synapses? What brain functions require the involvement of astrocytes? Why are there only certain areas of the brain that are rich in glutamatergic astrocytes?
Of course, there are some technical questions to be answered – how to better label astrocytes. The ideal astrocyte marker (molecule) should be stable, specifically labeled for that type of cell, and expressed at similar levels in each cell. Existing markers have their own shortcomings, such as GFAP (a protein involved in cytoskeletal assembly) that can vary greatly from cell to cell and can vary dramatically in disease or injury. However, the expression level of ALDH1L1 (a metabolic enzyme) is relatively stable but very low, which is difficult to detect by immunofluorescence immunohistochemistry, and the expression level of ALDH1L1 (a metabolic enzyme) is also high in liver cells. The lack of perfect cell-specific markers has brought great obstacles to the study of astrocytes.
A great scientific study can not only answer questions, but also raise countless new questions. The sheer number of astrocytes offers endless possibilities that have attracted a number of scientists. As Lusakov puts it, "We have accumulated a lot of evidence, and what we need now is a theory that can bring it all together." ”
References. 1] virchow, r. (1856). gesammelte abhandlungen zur wissenschaftlichen medizin. meidinger sohn & co.
2] Wu Jiang et al. Neurology, People's Medical Publishing House, 3rd edition, June 2015.
3] golgi, c. (1871). contribuzione alla fina anatomia degli organi centrali del sistema nervosos. tipi f**a e garagnani.
4] hasel, p. (2021). astrocytes. current biology, 31(7):r326-r327.
5] wu, z. (2020). gene therapy conversion of striatal astrocytes into gabaergic neurons in mouse models of huntington's disease. nature communications, 27;11(1):1105.
6] wang, ll. (2021). revisiting astrocyte to neuron conversion with lineage tracing in vivo. cell, 184(21):5465-5481.e16.
7] cornell-bell, ah. (1990). glutamate induces calcium w**es in cultured astrocytes: long-range glial signaling. science, 247(4941):470-3.
8] parpura, v. (1994). glutamate-mediated astrocyte–neuron signalling. nature, 369, 744–747.
9] pasti, l. (1997). intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ. journal of neuroscience, 17(20):7817-30.
10] perea, g. (2009). tripartite synapses: astrocytes process and control synaptic information. trends in neuroscience, 32(8):421-31.
11] sloan, sa. (2014). looks can be deceiving: reconsidering the evidence for gliotransmission. neuron, 17;84(6):1112-5.
12] de ceglia, r. (2023). specialized astrocytes mediate glutamatergic gliotransmission in the cns. nature, 622(7981), 120-129.
This article is supported by the Science Popularization China Star Program Project, produced by the Science Popularization Department of the China Association for Science and Technology, supervised by the China Science and Technology Press, Beijing Zhongke Galaxy Culture Media***