VAMP7 knockdown in secretory granules impairs CCL2 secretion in mast cells.

Mast cells (MCs) possess numerous potent inflammatory mediators and undergo differential regulation in response to antigen (Ag) stimulation. Among the regulatory systems governing secretory responses, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in facilitating granule-plasma membrane fusion and subsequent secretion. Our previous investigation documented the involvement of vesicle-associated membrane protein 3 (VAMP3) in regulating cytokine secretions in RBL-2H3 cells, a model for MC IgE-mediated responses. In addition to VAMP3, VAMP7 is expressed in MCs, but its functional role remains elusive. The present study seeks to explore VAMP7-specific regulatory mechanisms in MCs, shedding light on one of the mechanisms governing heterogeneous secretory responses in these cells. Murine bone marrow-derived mast cells (BMMCs) were examined to analyze the subcellular distribution of inflammatory mediators, specifically TNFα, CCL2, and histamine, and VAMPs (i.e., VAMP3, VAMP7, and VAMP8). Immunocytochemistry and the transient expression of fluorescent protein-conjugated target proteins were used to discern the distribution of various inflammatory mediators and VAMP7 through confocal laser scanning microscopy. Each inflammatory mediator (TNFα, CCL2, and histamine) was found in secretory granules of different sizes within BMMCs. VAMP7 exhibited a distinct distribution compared to VAMP3 in these granules. Notably, an overlapping distribution was observed between VAMP7 and CCL2, but not between VAMP7 and TNFα or VAMP7 and histamine. This suggests that CCL2 resides within VAMP7-expressing granules and is subject to VAMP7-dependent secretory regulation. Consistently, BMMCs with VAMP7 knockdown showed markedly reduced CCL2 secretion after Ag stimulation. These observations underscore the heterogeneity of MC secretory responses and unveil a novel VAMP7-dependent CCL2 secretion mechanism within MCs. This discovery might pave the way for the development of more precise therapeutic strategies to modulate MC secretion in allergic conditions.

Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase ß phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase ß. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

Calcineurin B1 Deficiency Reduces Proliferation, Increases Apoptosis, and Alters Secretion in Enteric Glial Cells of Mouse Small Intestine in Culture.

To investigate the roles of calcineurin (CN) in glial cells, we previously generated conditional knockout (CKO) mice lacking CNB1 in glial cells. Because these CKO mice showed dysfunction and inflammation of the small intestine in addition to growth impairment and postweaning death, we have focused on enteric glial cells (EGCs) in the small intestine. In this study, we examined the effects of CNB1 deficiency on the proliferation and survival of EGCs and the expression and secretion of EGC-derived substances in culture to reveal the mechanisms of how CNB1 deficiency leads to dysfunction and inflammation of the small intestine. In primary myenteric cultures of the small intestine, EGCs from the CKO mice showed reduced proliferation and increased apoptosis compared with EGCs from control mice. In purified EGC cultures from the CKO mice, Western blot analysis showed increased expression of S100B, iNOS, GFAP, and GDNF, and increased phosphorylation of NF-κB p65. In the supernatants of purified EGC cultures from the CKO mice, ELISA showed reduced secretion of TGF-ß1. In contrast, GDNF secretion was not altered in purified EGC cultures from the CKO mice. Furthermore, treatment with an S100B inhibitor partially rescued the CKO mice from growth impairment and postweaning death in vivo. In conclusion, CNB1 deficiency leads to reduced proliferation and increased apoptosis of EGCs and abnormal expression and secretion of EGC-derived substances, which may contribute to dysfunction and inflammation of the small intestine.

A Simple Method for Purified Primary Culture of Enteric Glial Cells from Mouse Small Intestine.

Enteric glial cells (EGCs) have been recognized as an important cell type constituting the enteric nervous system. EGCs control intestinal function and homeostasis through interactions with enteric neurons, epithelial cells and immune cells. To clarify the roles of EGCs in intestinal function and homeostasis, especially through secretion of and response to physiologically active substances, purified EGCs in primary culture have great advantages as an experimental tool. However, contamination by other cell types, fibroblasts in particular, is problematic in conventional primary myenteric culture. Previous methods to purify primary EGCs take a long time (over one month), are expensive, and are labor intensive. In the present study, we sought to purify primary EGCs from mouse small intestine by a simpler method than previous ones. After trying various protocols, we have established a method combining serum-free treatment and scraping fibroblasts off with a pipette tip. With our method, a purity of more than 90% EGCs was achieved after 14-d primary culture. Thus, our method is useful for investigating the roles of EGCs in intestinal function and homeostasis in detail in vitro.

Surface Plasmon Resonance of Two-Dimensional Gold Colloidal Crystals Formed on Gold Plates.

The free electrons inside precious metals such as Au vibrate when the surface of the metal is irradiated with an electromagnetic wave of an appropriate frequency. This oscillation is referred to as surface plasmon resonance (SPR), and the resonance frequency varies with permittivity of the medium around the metal. SPR sensors are widely applied in the fields of bioscience and pharmaceutical sciences, including biosensing for drug discovery, biomarker screening, virus detection, and testing for food safety. Here, we fabricated a metal-insulator-metal (MIM) SPR sensor by constructing two-dimensional (2D) regular array of Au colloidal particles (2D colloidal crystals) on an insulator layer over a thin Au film coated on a glass substrate surface. The 2D crystals were fabricated by electrostatically adsorbing negatively charged three-dimensional crystals onto a positively charged thin insulator formed on Au film. The plasmon peaks/dips from the MIM structure were measured in aqueous solutions of ethylene glycol (EG) at various concentrations. Multiple plasmon peaks/dips were observed due to the localized SPR (LSPR) of the Au particles and the Fano resonance between the Au particles and thin film. The plasmon peaks/dips shifted to higher wavelengths on increasing EG concentrations due to an increase in the refractive index of the media. The observed peak/dip shift was approximately twice that of LSPR from an isolated Au particle. We expect the present MIM substrate will be useful as a highly sensitive sensor in the pharmaceutical field.

Promotion of Dendritic Differentiation of Cerebellar Purkinje Cells by Ca2+/calmodulin-dependent Protein Kinase IIα, IIß and IV and Possible Involvement of CREB Phosphorylation.

Cerebellar Purkinje cells develop the most elaborate dendritic trees among neurons in the brain. To examine the role of Ca2+/calmodulin-dependent protein kinase (CaMK) IIα, IIß and IV in the dendritic differentiation of Purkinje cells, we introduced siRNA against these CaMKs into Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. Single-cell electroporation enables us to transfer siRNA into specific cells within a heterogeneous cell population. In addition, we can easily and reliably transfer multiple types of siRNA into a cell simply by loading them together in one micropipette. Any one of the siRNA against CaMKIIα, IIß and IV (single knockdown) or any combinations of two of the siRNA against these CaMKs (double knockdown) had no significant effects on the dendritic differentiation of Purkinje cells. However, the combination of all three siRNA against these CaMKs (triple knockdown) inhibited the branching of Purkinje cell dendrites. Furthermore, the triple knockdown reduced the phosphorylation of CREB in Purkinje cells. These findings suggest the promotion of dendritic differentiation of Purkinje cells by CaMKIIα, IIß and IV and the possible involvement of phosphorylation of CREB as a common substrate of these CaMKs.

Receptor dynamics regulates actin polymerization state through phosphorylation of cofilin in mast cells.

Aggregation of IgE bound to the high-affinity IgE receptor (FcεRI) by a multivalent antigen induces mast cell activation, while disaggregation of aggregated FcεRI by monomer hapten immediately terminates degranulation mediated by dephosphorylation of Syk and mediates a decrease in intracellular Ca2+ concentration ([Ca2+]i). The actin polymerization state is intimately involved in mast cell activation mediated by FcεRI aggregation. However, the relation between aggregation-disaggregation of FcεRI and actin rearrangement in mast cells is not well understood. The addition of a multivalent antigen rapidly depolymerized actin filaments, while the subsequent addition of monomer hapten rapidly recovered actin polymerization. Whereas cofilin, an actin-severing protein, was temporally dephosphorylated several minutes after a multivalent antigen stimulation and the addition of monomer hapten rapidly increased cofilin phosphorylation level within 30 s. The removal of extracellular Ca2+ instead of monomer hapten addition did not restore cofilin phosphorylation, suggesting that the significant decrease in [Ca2+]i by monovalent hapten was not a critical reason for the actin rearrangement. Additionally, monovalent hapten did not completely reduce [Ca2+]i in mast cells pretreated with jasplakinolide, an inhibitor of actin depolymerization. These results suggest that the multivalent antigen-induced actin depolymerization mediated by cofilin dephosphorylation, and the subsequent addition of monovalent hapten in the F-actin severing state efficiently elicited actin re-polymerization by cofilin phosphorylation.

Monomer hapten and hapten-specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms.

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

Calcineurin B1 Deficiency in Glial Cells Reduces Gastrointestinal Motility and Results in Maldigestion and/or Malabsorption in Mice.

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase abundantly expressed in the nervous system. To investigate the roles of calcineurin in glial cells, we previously generated glial calcineurin B1-conditional knockout (CKO) mice and found that these mice displayed dysfunction of enteric glial cells, mucosal degeneration and inflammation in the small intestine, and growth retardation and postweaning death, suggesting a novel role of calcineurin in enteric glial cells. Although these findings raised a possibility that abnormalities in calcineurin B1-deficient enteric glial cells may cause dysregulation of gastrointestinal motility and result in maldigestion and/or malabsorption in the CKO mice, these issues remain to be elucidated. In the present study, we showed that gastrointestinal motility was reduced in the CKO mice. Degeneration of mucosal epithelium was observed in the small intestine. Glucose levels were decreased in serum, whereas starch, glucose, and lipid levels were increased in feces. Thus, calcineurin B1 deficiency in glial cells reduces gastrointestinal motility, induces mucosal epithelium degeneration in the small intestine, and results in maldigestion and/or malabsorption in mice, further supporting the important role of calcineurin in enteric glial cells.

Calcineurin B1 Deficiency in Glial Cells Induces Mucosal Degeneration and Inflammation in Mouse Small Intestine.

Although calcineurin is abundantly expressed in the nervous system and involved in neurite extension and synaptic plasticity in neurons, little is known about its roles in glial cells. To investigate the roles of calcineurin in glial cells, we generated glial calcineurin B1-conditional knockout (CKO) mice and analyzed the abnormalities in the small intestine. The CKO mice were generated by crossing floxed calcineurin B1 mice with glial fibrillary acidic protein (GFAP)-Cre mice. The CKO mice exhibited growth retardation approximately from the third postnatal week and died mostly within the fourth postnatal week. The small intestine of the CKO mice was thin and hemorrhagic. The mucosal layer was degenerated and GFAP expression was reduced in the CKO small intestine. These pathological changes were associated with inflammation and increased intestinal permeability. In contrast, no apparent abnormalities were observed in the large intestine of the CKO mice. Nuclear factor of activated T cells failed to translocate into the nucleus after stimulation in enteric glial cells of the CKO small intestine. In conclusion, the calcineurin B1 deficiency in glial cells impairs the small intestine and leads to malnutrition and eventual death in mice, suggesting that calcineurin plays a novel and important role in enteric glial cells.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development.

Expression of the GluA2 subunit of glutamate receptors is required for the normal dendritic differentiation of cerebellar Purkinje cells.

Cerebellar Purkinje cells differentiate the most elaborate dendritic trees among neurons in the brain and constitute the principal part of cerebellar neuronal circuitry. In the present study, we examined the role of the GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in the dendritic differentiation of Purkinje cells. Since mature Purkinje cells express the GluA2 subunit, AMPA receptors on them exhibit a low Ca2+ permeability. Does this expression of GluA2, leading to the loss of Ca2+ permeability of AMPA receptors, have a positive significance in the dendritic differentiation of Purkinje cells? To answer this question, we introduced GluA2 siRNA into immature Purkinje cells in cerebellar cell cultures using a single-cell electroporation technique. The dendritic elongation and branching, as well as spine formation, were inhibited by GluA2 knockdown in Purkinje cells. GluA2 knockdown augmented the elevation of intracellular Ca2+ concentrations and a higher incidence of oscillation of intracellular Ca2+ concentrations in response to glutamate. These findings suggest that excessive elevation of intracellular Ca2+ concentrations has a negative effect on the dendritic differentiation of Purkinje cells and that the expression of GluA2 inhibits this negative effect in the development of Purkinje cells.

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions.

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion.

Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 µm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 µm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins.

Mast cells are involved in allergic responses and undergo exocytotic release of inflammatory mediators in response to antigen stimulation. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this membrane fusion process; some SNARE-binding proteins regulate SNARE-dependent liposome membrane fusion. SNARE-binding protein complexin II is expressed in mast cells, where it positively regulates exocytotic release after antigen stimulation. We found that complexin II suppressed SNARE-dependent membrane fusion between mast cell SNARE-containing liposomes. This inhibitory effect of complexin II was abolished when we used a structurally divergent mutant (R59H) complexin II, where Arg59 is substituted with histidine. These results suggest that complexin II negatively regulates SNARE-dependent exocytotic membrane fusion in mast cells, and this inhibitory effect is dependent upon Arg59.

Phosphorylation of syntaxin-3 at Thr 14 negatively regulates exocytosis in RBL-2H3 mast cells.

Recent studies have revealed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin-3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin-3. We found that Thr 14 of syntaxin-3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin-3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin-3 at Thr 14 (T14E) induced structural changes in syntaxin-3, and this mutation inhibited binding of syntaxin-3 to Munc18-2. These results suggest that phosphorylated syntaxin-3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin-3 and Munc18-2.

Effects of PIP2 on membrane fusion between mast cell SNARE liposomes mediated by synaptotagmin 2.

Recent studies have revealed that SNARE proteins are involved in exocytotic release in mast cells. Previously, we reported that mast cell SNARE proteins induce membrane fusion between liposomes. Moreover, we found that synaptotagmin 2, a candidate Ca2+ sensor for mast cell exocytosis, enhanced SNARE-mediated membrane fusion via Ca2+ and phosphatidylserine. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an acidic phospholipid like phosphatidylserine. In the present study, we investigated whether PIP2 is involved in the enhancement effect of synaptotagmin 2 on SNARE-mediated membrane fusion. PIP2 did not show any significant effect on SNARE-mediated membrane fusion by itself. In the presence of Ca2+, synaptotagmin 2 enhanced SNARE-mediated membrane fusion between liposomes containing PIP2. However, even in the presence of Ca2+, when we used 100% PC liposomes, synaptotagmin 2 did not show any significant effect on SNARE-mediated membrane fusion. These results indicated that PIP2 is involved in the enhancement effect of synaptotagmin 2 on membrane fusion between liposomes containing mast cell SNARE proteins.

Orai-2 is localized on secretory granules and regulates antigen-evoked Ca²âº mobilization and exocytosis in mast cells.

The increase in intracellular Ca(2+) through the Ca(2+) channel is an indispensable step for the secretion of inflammatory mediators by mast cells. It was recently reported that Orai-1 is responsible for the Ca(2+) influx that is activated by depletion of stored Ca(2+). There are three isoforms of Orai: Orai-1, Orai-2, and Orai-3; however, isoforms other than Orai-1 are poorly understood. We found that Orai-2 is expressed and localized on secretory granules in RBL-2H3. Ca(2+) release from Ca(2+) store, induced by antigen stimulation, was significantly attenuated by knockdown of Orai-2, while that induced by thapsigargin was not affected. Furthermore, exocytotic release induced by antigen stimulation was inhibited in knockdown cells. This observation suggests a new role of Orai isoforms in secretory cells.

ZIP2 protein, a zinc transporter, is associated with keratinocyte differentiation.

Zinc is essential for the proper functioning of various enzymes and transcription factors, and its homeostasis is rigorously controlled by zinc transporters (SLC39/ZIP, importers; SLC30/ZnT, exporters). Skin disease is commonly caused by a zinc deficiency. Dietary and inherited zinc deficiencies are known to cause alopecia and the development of vesicular or pustular dermatitis. A previous study demonstrated that zinc played crucial roles in the survival of keratinocytes and their unique functions. High levels of zinc have been detected in the epidermis. Epidermal layers are considered to use a mechanism that preferentially takes in zinc, which is involved with the unique functions of keratinocytes. However, few studies have investigated the ZIP (Zrt- and Irt-like protein) proteins specifically expressed in keratinocytes and their functions. We explored the ZIP proteins specifically expressed in the epidermis and analyzed their functions. Gene expression analysis showed that the expression of ZIP2 was consistently higher in the epidermis than in the dermis. Immunohistochemistry analysis confirmed the expression of ZIP2 in differentiating keratinocytes. The expression of ZIP2 was found to be up-regulated by the differentiation induction of cultured keratinocytes. Intracellular zinc levels were decreased in keratinocytes when ZIP2 was knocked down by siRNA, and this subsequently inhibited the differentiation of keratinocytes. Moreover, we demonstrated that ZIP2 knockdown inhibited the normal formation of a three-dimensional cultured epidermis. Taken together, the results of this study suggest that ZIP2, a zinc transporter expressed specifically in the epidermis, and zinc taken up by ZIP2 are necessary for the differentiation of keratinocytes.

Optimization of single-cell electroporation protocol for forced gene expression in primary neuronal cultures.

The development and function of the central nervous system (CNS) are realized through interactions between many neurons. To investigate cellular and molecular mechanisms of the development and function of the CNS, it is thus crucial to be able to manipulate the gene expression of single neurons in a complex cell population. We recently developed a technique for gene silencing by introducing small interfering RNA into single neurons in primary CNS cultures using single-cell electroporation. However, we had not succeeded in forced gene expression by introducing expression plasmids using single-cell electroporation. In the present study, we optimized the experimental conditions to enable the forced expression of green fluorescent protein (GFP) in cultured cerebellar Purkinje neurons using single-cell electroporation. We succeeded in strong GFP expression in Purkinje neurons by increasing the inside diameter of micropipettes or by making the size of the original plasmid smaller by digestion and cyclizing it by ligation. Strong GFP expression in Purkinje neurons electroporated under the optimal conditions continued to be observed for more than 25 days after electroporation. Thus, this technique could be used for forced gene expression in single neurons to investigate cellular and molecular mechanisms of the development, function, and disease of the CNS.