Glial Cell Types Research Articles

Single-cell transcriptome identifies molecular subtype of autism spectrum disorder impacted by de novo loss-of-function variants regulating glial cells

BackgroundIn recent years, several hundred autism spectrum disorder (ASD) implicated genes have been discovered impacting a wide range of molecular pathways. However, the molecular underpinning of ASD, particularly from the point of view of ‘brain to behaviour’ pathogenic mechanisms, remains largely unknown.MethodsWe undertook a study to investigate patterns of spatiotemporal and cell type expression of ASD-implicated genes by integrating large-scale brain single-cell transcriptomes (> million cells) and de novo loss-of-function (LOF) ASD variants (impacting 852 genes from 40,122 cases).ResultsWe identified multiple single-cell clusters from three distinct developmental human brain regions (anterior cingulate cortex, middle temporal gyrus and primary visual cortex) that evidenced high evolutionary constraint through enrichment for brain critical exons and high pLI genes. These clusters also showed significant enrichment with ASD loss-of-function variant genes (p < 5.23 × 10–11) that are transcriptionally highly active in prenatal brain regions (visual cortex and dorsolateral prefrontal cortex). Mapping ASD de novo LOF variant genes into large-scale human and mouse brain single-cell transcriptome analysis demonstrate enrichment of such genes into neuronal subtypes and are also enriched for subtype of non-neuronal glial cell types (astrocyte, p < 6.40 × 10–11, oligodendrocyte, p < 1.31 × 10–09).ConclusionAmong the ASD genes enriched with pathogenic de novo LOF variants (i.e. KANK1, PLXNB1), a subgroup has restricted transcriptional regulation in non-neuronal cell types that are evolutionarily conserved. This association strongly suggests the involvement of subtype of non-neuronal glial cells in the pathogenesis of ASD and the need to explore other biological pathways for this disorder.

Gliogenic Potential of Single Pallial Radial Glial Cells in Lower Cortical Layers.

During embryonic development, progenitor cells are progressively restricted in their potential to generate different neural cells. A specific progenitor cell type, the radial glial cells, divides symmetrically and then asymmetrically to produce neurons, astrocytes, oligodendrocytes, and NG2-glia in the cerebral cortex. However, the potential of individual progenitors to form glial lineages remains poorly understood. To further investigate the cell progeny of single pallial GFAP-expressing progenitors, we used the in vivo genetic lineage-tracing method, the UbC-(GFAP-PB)-StarTrack. After targeting those progenitors in embryonic mice brains, we tracked their adult glial progeny in lower cortical layers. Clonal analyses revealed the presence of clones containing sibling cells of either a glial cell type (uniform clones) or two different glial cell types (mixed clones). Further, the clonal size and rostro-caudal cell dispersion of sibling cells differed depending on the cell type. We concluded that pallial E14 neural progenitors are a heterogeneous cell population with respect to which glial cell type they produce, as well as the clonal size of their cell progeny.

Microglia and immunotherapy in Alzheimer’s disease

Microglia are a type of glial cells that play a key role in the repair of damage to the central nervous system (CNS). In the pathological condition of Alzheimer's disease (AD), β-amyloid peptide and a variety of pro-inflammatory factors can activate microglia, resulting in the secretion of a variety of inflammatory factors and neurotoxins. This leads to neuronal damage and even apoptosis, thus triggering AD. In contrast, microglia can protect the CNS by phagocytizing Aβ to slow down AD development. In this review, the roles of microglia in AD neuroinflammation and the scope of immunotherapy for AD are summarized to provide a theoretical basis for AD prevention and treatment.

Estimation of the density of neural, glial, and endothelial lineage cells in the adult mouse dentate gyrus.

The dentate gyrus subregion of the mammalian hippocampus is an adult neural stem cell niche and site of lifelong neurogenesis. Hypotheses regarding the role of adult-born neuron synaptic integration in hippocampal circuit function are framed by robust estimations of adult-born versus pre/perinatally-born neuron number. In contrast, the non-neurogenic functions of adult neural stem cells and their immediate progeny, such as secretion of bioactive growth factors and expression of extracellular matrix-modifying proteins, lack similar framing due to few estimates of their number versus other prominent secretory cells. Here, we apply immunohistochemical methods to estimate cell density of neural stem/progenitor cells versus other major classes of glial and endothelial cell types that are potentially secretory in the dentate gyrus of adult mice. Of the cell types quantified, we found that GFAP+SOX2+ stellate astrocytes were the most numerous, followed by CD31+ endothelia, GFAP-SOX2+ intermediate progenitors, Olig2+ oligodendrocytes, Iba1+ microglia, and GFAP+SOX2+ radial glia-like neural stem cells. We did not observe any significant sex differences in density of any cell population. Notably, neural stem/progenitor cells were present at a similar density as several cell types known to have potent functional roles via their secretome. These findings may be useful for refining hypotheses regarding the contributions of these cell types to regulating hippocampal function and their potential therapeutic uses. All experimental protocols were approved by the Ohio State University Institutional Animal Care and Use Committee (protocol# 2016A00000068) on July 14, 2016.

Proteomic Profiling of Astrocytic O-GlcNAc Transferase-Related Proteins in the Medial Prefrontal Cortex.

The medial prefrontal cortex (mPFC), a key part of the brain networks that are closely related to the regulation of behavior, acts as a key regulator in emotion, social cognition, and decision making. Astrocytes are the majority cell type of glial cells, which play a significant role in a number of processes and establish a suitable environment for the functioning of neurons, including the brain energy metabolism. Astrocyte’s dysfunction in the mPFC has been implicated in various neuropsychiatric disorders. Glucose is a major energy source in the brain. In glucose metabolism, part of glucose is used to convert UDP-GlcNAc as a donor molecule for O-GlcNAcylation, which is controlled by a group of enzymes, O-GlcNAc transferase enzyme (OGT), and O-GlcNAcase (OGA). However, the role of O-GlcNAcylation in astrocytes is almost completely unknown. Our research showed that astrocytic OGT could influence the expression of proteins in the mPFC. Most of these altered proteins participate in metabolic processes, transferase activity, and biosynthetic processes. GFAP, an astrocyte maker, was increased after OGT deletion. These results provide a framework for further study on the role of astrocytic OGT/O-GlcNAcylation in the mPFC.

The function of Scox in glial cells is essential for locomotive ability in Drosophila

Synthesis of cytochrome c oxidase (Scox) is a Drosophila homolog of human SCO2 encoding a metallochaperone that transports copper to cytochrome c, and is an essential protein for the assembly of cytochrome c oxidase in the mitochondrial respiratory chain complex. SCO2 is highly conserved in a wide variety of species across prokaryotes and eukaryotes, and mutations in SCO2 are known to cause mitochondrial diseases such as fatal infantile cardioencephalomyopathy, Leigh syndrome, and Charcot-Marie-Tooth disease, a neurodegenerative disorder. These diseases have a common symptom of locomotive dysfunction. However, the mechanisms of their pathogenesis remain unknown, and no fundamental medications or therapies have been established for these diseases. In this study, we demonstrated that the glial cell-specific knockdown of Scox perturbs the mitochondrial morphology and function, and locomotive behavior in Drosophila. In addition, the morphology and function of synapses were impaired in the glial cell-specific Scox knockdown. Furthermore, Scox knockdown in ensheathing glia, one type of glial cell in Drosophila, resulted in larval and adult locomotive dysfunction. This study suggests that the impairment of Scox in glial cells in the Drosophila CNS mimics the pathological phenotypes observed by mutations in the SCO2 gene in humans.

Heterogeneity and Molecular Markers for CNS Glial Cells Revealed by Single-Cell Transcriptomics.

Glial cells, including astrocytes, oligodendrocytes, and microglia, are the major components in the central nervous system (CNS). Studies have revealed the heterogeneity of each glial cell type and that they each may play distinct roles in physiological processes and/or neurological diseases. Single-cell sequencing (scRNA-seq) technology developed in recent years has extended our understanding of glial cell heterogeneity from the perspective of transcriptome profiling. This review summarizes the marker genes of major glial cells in the CNS and reveals their heterogeneity in different species, CNS regions, developmental stages, and pathological states (Alzheimer's disease and spinal cord injury), expanding our knowledge of glial cell heterogeneity on both molecular and functional levels.

Spinal glial cells in itch modulation.

Glial cells are non‐neuronal cells in the nervous system that are electrically non‐excitable and outnumber neurons in humans. Glial cells have attracted attention in recent years for their active involvement in the regulation of neuronal activity, suggesting their contribution to the pathogenesis and progression of neurological diseases. Studies have shown that astrocytes, a type of glial cell, are activated in the spinal cord in response to skin inflammation and contribute to the exacerbation of chronic itch. This review summarizes the current knowledge about the role of astrocytes and other glial cells in the modulation of itch processing and the mechanism of their activation under itch conditions.

SoxE group transcription factor Sox8 promotes astrocytic differentiation of neural stem/precursor cells downstream of Nfia.

The brain consists of three major cell types: neurons and two glial cell types (astrocytes and oligodendrocytes). Although they are generated from common multipotent neural stem/precursor cells (NS/PCs), embryonic NS/PCs cannot generate all of the cell types at the beginning of brain development. NS/PCs first undergo extensive self‐renewal to expand their pools, and then acquire the potential to produce neurons, followed by glial cells. Astrocytes are the most frequently found cell type in the central nervous system (CNS), and play important roles in brain development and functions. Although it has been shown that nuclear factor IA (Nfia) is a pivotal transcription factor for conferring gliogenic potential on neurogenic NS/PCs by sequestering DNA methyltransferase 1 (Dnmt1) from astrocyte‐specific genes, direct targets of Nfia that participate in astrocytic differentiation have yet to be completely identified. Here we show that SRY‐box transcription factor 8 (Sox8) is a direct target gene of Nfia at the initiation of the gliogenic phase. We found that expression of Sox8 augmented leukemia inhibitory factor (LIF)‐induced astrocytic differentiation, while Sox8 knockdown inhibited Nfia‐enhanced astrocytic differentiation of NS/PCs. In contrast to Nfia, Sox8 did not induce DNA demethylation of an astrocyte‐specific marker gene, glial fibrillary acidic protein (Gfap), but instead associated with LIF downstream transcription factor STAT3 through transcriptional coactivator p300, explaining how Sox8 expression further facilitated LIF‐induced Gfap expression. Taken together, these results suggest that Sox8 is a crucial Nfia downstream transcription factor for the astrocytic differentiation of NS/PCs in the developing brain.

Interventional Strategies for Parkinson Disease: Can Neural Precursor Cells Forge a Path Ahead?

Neural precursor cells (NPCs), derived from pluripotent stem cells (PSCs), with their unique ability to generate multiple neuronal and glial cell types are extremely useful for understanding biological mechanisms in normal and diseased states. However, generation of specific neuronal subtypes (mature) from NPCs in large numbers adequate for cell therapy is challenging due to lack of a thorough understanding of the cues that govern their differentiation. Interestingly, neural stem cells (NSCs) themselves are in consideration for therapy given their potency to form different neural cell types, release of trophic factors, and immunomodulatory effects that confer neuroprotection. With the recent COVID-19 outbreak and its accompanying neurological indications, the immunomodulatory role of NSCs may gain additional significance in the prevention of disease progression in vulnerable populations. In this regard, small-molecule mediated NPC generation from PSCs via NSC formation has become an important strategy that ensures consistency and robustness of the process. The development of the mammalian brain occurs along the rostro-caudal axis, and the establishment of anterior identity is an early event. Wnt signaling, along with fibroblast growth factor and retinoic acid, acts as a caudalization signal. Further, the increasing amount of epigenetic data available from human fetal brain development has enhanced both our understanding of and ability to experimentally manipulate these developmental regulatory programs in vitro. However, the impact on homing and engraftment after transplantation and subsequently on therapeutic efficacy of NPCs based on their derivation strategy is not yet clear. Another formidable challenge in cell replacement therapy for neurodegenerative disorders is the mode of delivery. In this Perspective, we discuss these core ideas with insights from our preliminary studies exploring the role of PSC-derived NPCs in rat models of MPTP-induced Parkinson's disease following intranasal injections.

An atlas of gene regulatory elements in adult mouse cerebrum

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures1. Recent surveys of mouse and human brains with single-cell transcriptomics2–6 and high-throughput imaging technologies7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans.

Spinal Cord Stimulation Reduces Ventricular Arrhythmias by Attenuating Reactive Gliosis and Activation of Spinal Interneurons.

This study investigated spinal cord neuronal and glial cell activation during cardiac ischemia-reperfusion (IR)-triggered ventricular arrhythmias and neuromodulation therapy by spinal cord stimulation (SCS). Myocardial ischemia induces changes in cardiospinal neural networks leading to sudden cardiac death. Neuromodulation with SCS decreases cardiac sympathoexcitation; however, the molecular mechanisms remain unknown. Yorkshire pigs (n=16) were randomized to Control, IR, or IR+SCS groups. A 4-pole SCS lead was placed in the T1-T4 epidural space with stimulation for 30minutes before IR (50Hz, 0.4-ms duration, 90% motor threshold). Cardiac electrophysiological mapping and Ventricular Arrhythmia Score (VAS) were recorded. Immunohistochemistry of thoracic spinal sections was used to map and identify Fos-positive neuronal and glial cell types during IR with and without SCS. IR increased cardiac sympathoexcitation and arrhythmias (VAS=6.2 ± 0.9) that were attenuated in IR+ SCS (VAS=2.8 ± 0.5; P=0.017). IR increased spinal cellular Fos expression (#Fos+ cells Control=23 ± 2 vs IR=88 ± 5; P< 0.0001) in T1-T4, with the greatest increase localized to T3, and the greatest %Fos+ cells being microglia and astrocytes. Fos expression was attenuated by IR+ SCS (62 ± 4; P< 0.01), primarily though a reduction in Fos+ microglia and astrocytes, as SCS also led to increase in Fos+ neurons in deep dorsal laminae. In a porcine model, cardiac IR was associated with astrocyte and microglial cell activation. Our results suggest that preemptive thoracic SCS decreased IR-induced cardiac sympathoexcitation and ventricular arrhythmias through attenuation of reactive gliosis and activation of inhibitory interneurons in the dorsal horn of spinal cord.

Inflammatory cytokines TNFα, IL-1β, and IL-6 are induced in endotoxin- stimulated microglia through different signaling cascades.

By using an animal model in which inflammatory cytokines are induced in lipopolysaccharide (LPS)-injected rat brain, we investigated the induction of tumor necrosis factor alpha (TNFα), interleukin-1beta (IL-1β), and IL-6. Immunoblotting and immunohistochemistry revealed that all three cytokines were transiently induced in the cerebral cortex at about 12 h after LPS injection. To clarify which glial cell type induced the cytokines, we examined the respective abilities of astrocytes and microglia in vitro. Primary microglia largely induced TNFα, IL-1β and IL-6 in response to LPS, but primary astrocytes induced only limited levels of TNFα. Thus, we used specific inhibitors to focus on microglia in surveying signaling molecules involved in the induction of TNFα, IL-1β, and IL-6. The experiments using mitogen-activated protein kinases (MAPK) inhibitors revealed that c-Jun N-terminal kinase (JNK)/p38, external signal regulated kinase (ERK)/JNK, and ERK/JNK/p38 are necessary for the induction of TNFα, IL-1β, and IL-6, respectively. The experiments using protein kinase C (PKC) inhibitor clarified that PKCα is required for the induction of all these cytokines in LPS-stimulated microglia. Furthermore, LPS-dependent IL-1β/IL-6 induction was suppressed by pretreatment with a nitric oxide (NO) scavenger, suggesting that NO is involved in the signaling cascade of IL-1β/IL-6 induction. Thus, an inducible NO synthase induced in the LPS-injected cerebral cortex might be related to the induction of IL-1β/IL-6 through the production of NO in vivo. Taken together, these results demonstrated that microglia induce different kinds of inflammatory cytokine through specific combinations of MAPKs and by the presence or absence of NO.

Primary Cilia in Glial Cells: An Oasis in the Journey to Overcoming Neurodegenerative Diseases.

Many neurodegenerative diseases have been associated with defects in primary cilia, which are cellular organelles involved in diverse cellular processes and homeostasis. Several types of glial cells in both the central and peripheral nervous systems not only support the development and function of neurons but also play significant roles in the mechanisms of neurological disease. Nevertheless, most studies have focused on investigating the role of primary cilia in neurons. Accordingly, the interest of recent studies has expanded to elucidate the role of primary cilia in glial cells. Correspondingly, several reports have added to the growing evidence that most glial cells have primary cilia and that impairment of cilia leads to neurodegenerative diseases. In this review, we aimed to understand the regulatory mechanisms of cilia formation and the disease-related functions of cilia, which are common or specific to each glial cell. Moreover, we have paid close attention to the signal transduction and pathological mechanisms mediated by glia cilia in representative neurodegenerative diseases. Finally, we expect that this field of research will clarify the mechanisms involved in the formation and function of glial cilia to provide novel insights and ideas for the treatment of neurodegenerative diseases in the future.

Molecular Expression Profile of Changes in Rat Acute Spinal Cord Injury.

Background: Spinal cord injury (SCI) is a highly lethal and debilitating disease with a variety of etiologies. To date, there is no effective therapeutic modality for a complete cure. The pathological mechanisms of spinal cord injury at the molecular gene and protein expression levels remain unclear.Methods: This study used single-cell transcriptomic analysis and protein microarray analysis to analyzes changes in the gene expression profiles of cells and secretion of inflammatory factors respectively, around the lesion site in a rat SCI model.Results: Single-cell transcriptomic analysis found that three types of glial cells (microglia, astrocyte, and oligodendrocyte) becomes activated after acute injury, with GO exhibiting a variety of inflammatory-related terms after injury, such as metabolic processes, immune regulation, and antigen presentation. Protein microarray results showed that the levels of four inflammatory cytokines favoring SCI repair decreased while the levels of nine inflammatory cytokines hindering SCI repair increased after injury.Conclusion: These findings thus reveal the changes in cellular state from homeostatic to reactive cell type after SCI, which contribute to understand the pathology process of SCI, and the potential relationship between glial cells and inflammatory factors after SCI, and provides new theoretical foundation for further elucidating the molecular mechanisms of secondary SCI.

NG2 glia-derived GABA release tunes inhibitory synapses and contributes to stress-induced anxiety

NG2 glia, also known as oligodendrocyte precursor cells (OPCs), play an important role in proliferation and give rise to myelinating oligodendrocytes during early brain development. In contrast to other glial cell types, the most intriguing aspect of NG2 glia is their ability to directly sense synaptic inputs from neurons. However, whether this synaptic interaction is bidirectional or unidirectional, or its physiological relevance has not yet been clarified. Here, we report that NG2 glia form synaptic complexes with hippocampal interneurons and that selective photostimulation of NG2 glia (expressing channelrhodopsin-2) functionally drives GABA release and enhances inhibitory synaptic transmission onto proximal interneurons in a microcircuit. The mechanism involves GAD67 biosynthesis and VAMP-2 containing vesicular exocytosis. Further, behavioral assays demonstrate that NG2 glia photoactivation triggers anxiety-like behavior in vivo and contributes to chronic social defeat stress.

Extracellular Vesicles Taken up by Astrocytes.

Extracellular vesicles (EVs) are composed of lipid bilayer membranes and contain various molecules, such as mRNA and microRNA (miRNA), that regulate the functions of the recipient cell. Recent studies have reported the importance of EV-mediated intercellular communication in the brain. The brain contains several types of cells, including neurons and glial cells. Among them, astrocytes are the most abundant glial cells in the mammalian brain and play a wide range of roles, from structural maintenance of the brain to regulation of neurotransmission. Furthermore, since astrocytes can take up EVs, it is possible that EVs originating from inside and outside the brain affect astrocyte function, which in turn affects brain function. However, it has not been fully clarified whether the specific targeting mechanism of EVs to astrocytes as recipient cells exists. In recent years, EVs have attracted attention as a cell-targeted therapeutic approach in various organs, and elucidation of the targeting mechanism of EVs to astrocytes may pave the way for new therapies for brain diseases. In this review, we focus on EVs in the brain that affect astrocyte function and discuss the targeting mechanism of EVs to astrocytes.

Lineage-specific control of convergent differentiation by a Forkhead repressor.

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell type-specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

Glial Chloride Homeostasis Under Transient Ischemic Stress.

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl–]int) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl–]int are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl–]int in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl–]int, ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na+-K+-2Cl– cotransporter (NKCC1) and chloride outward transport (efflux) through K+-Cl– cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl–]int to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl–]int, whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl–]int was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl–]int in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl–]int in any of the tested glial cell types. However, [Cl–]int changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.

The role of hippocampal glial glutamate transporter (GLT-1) in morphine-induced behavioral responses.

Opioid abuse modifies synaptic plasticity, which leads to behavioral changes, such as morphine dependence, but the mechanism remains poorly understood. Glial cells play an important role in the modulation of synaptic plasticity and are involved in addictive‐like behaviors. The indisputable role of glutamate in opiate addiction has been shown. Astrocytes, a type of glial cells, which are integral functional elements of synapses, modulate the concentration of glutamate in the synaptic space. One of the most important mechanisms for glutamate concentration regulation is its uptake from the synaptic cleft. In this study, we evaluated the role of hippocampal glial glutamate transporter (GLT‐1) in morphine dependence. Male rats received subcutaneous (s.c.) morphine sulfate (10 mg/kg) at an interval of 12 h for 9 days. In order to activate GLT‐1, animals received an intrahippocampal injection of ceftriaxone (0.5 mmol/0.5 μl) in the CA1 region of the hippocampus, 30 min before each morphine administration. Rats were assessed for morphine dependence by monitoring naloxone hydrochloride‐induced morphine withdrawal. Our results showed that hippocampal microinjection of ceftriaxone, as an activator of GLT‐1, reduced some signs of morphine withdrawal, such as activity, diarrhea, head tremor, freezing, and ptosis. It seems that hippocampal GLT‐1 can be affected by chronic morphine administration and involved in morphine dependence. Therefore, its activation may reduce morphine side effects by reducing hippocampal glutamate.