PDGFRα+ subepithelial interstitial cells act as a pacemaker to drive smooth muscle of the guinea pig seminal vesicle.

Smooth muscle cells (SMCs) of the guinea pig seminal vesicle (SV) develop spontaneous phasic contractions, Ca2+ flashes and electrical slow waves in a mucosa-dependent manner, and thus it was envisaged that pacemaker cells reside in the mucosa. Here, we aimed to identify the pacemaker cells in SV mucosa using intracellular microelectrode and fluorescence Ca2+ imaging techniques. Morphological characteristics of the mucosal pacemaker cells were also investigated using focused ion beam/scanning electron microscopy tomography and fluorescence immunohistochemistry. Two populations of mucosal cells developed spontaneous Ca2+ transients and electrical activity, namely basal epithelial cells (BECs) and subepithelial interstitial cells (SICs). Pancytokeratin-immunoreactive BECs were located on the apical side of the basement membrane (BM) and generated asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). The spontaneous Ca2+ transients and STDs were not diminished by 10 µM nifedipine but abolished by 10 µM cyclopiazonic acid (CPA). Platelet-derived growth factor receptor α (PDGFRα)-immunoreactive SICs were distributed just beneath the basal side of the BM and developed synchronous Ca2+ oscillations and electrical slow waves, which were suppressed by 3 µM nifedipine and abolished by 10 µM CPA. In SV mucosal preparations in which some smooth muscle bundles remained attached, SICs and residual SMCs developed temporally correlated spontaneous Ca2+ transients. Neurobiotin injected into SICs spread not only to neighbouring SICs but also to neighbouring SMCs or vice versa. These results suggest that PDGFRα+ SICs electrotonically drive the spontaneous contractions of SV smooth muscle. KEY POINTS: In many visceral smooth muscle organs, spontaneous contractions are electrically driven by non-muscular pacemaker cells. In guinea pig seminal vesicles (SVs), as yet unidentified mucosal cells appear to drive neighbouring smooth muscle cells (SMCs). Two populations of spontaneously active cells are distributed in the SV mucosa. Basal epithelial cells (BECs) generate asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). In contrast, subepithelial interstitial cells (SICs) develop synchronous Ca2+ oscillations and electrical slow waves. Pancytokeratin-immunoreactive (IR) BECs are located on the apical side of the basement membrane (BM), while platelet-derived growth factor receptor α (PDGFRα)-IR SICs are located on the basal side of the BM. Spontaneous Ca2+ transients in SICs are synchronised with those in SV SMCs. Dye-coupling between SICs and SMCs suggests that SICs act as pacemaker cells to drive the spontaneous contractions of SV smooth muscle.

Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle.

KEY POINTS: The mucosa may have neuron-like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+ . These spontaneous events primarily depend on Ca2+ handling by sarco-endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco-endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. ABSTRACT: The role of the mucosa in generating the spontaneous activity of guinea-pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi-fluorescence Ca2+ imaging, respectively. Mucosa-intact but not mucosa-denuded SV preparations generated TTX- (1 µm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 µm). Consistently, SMCs developed mucosa-dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 µm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 µm), a sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) inhibitor. DIDS (300 µm) and niflumic acid (100 µm), blockers for Ca2+ -activated Cl- channels (CACCs), or low Cl- solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L-type voltage-dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco-endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.

Interstitial cell modulation of pyeloureteric peristalsis in the mouse renal pelvis examined using FIBSEM tomography and calcium indicators.

Typical and atypical smooth muscle cells (TSMCs and ASMCs, respectively) and interstitial cells (ICs) within the pacemaker region of the mouse renal pelvis were examined using focused ion beam scanning electron (FIB SEM) tomography, immunohistochemistry and Ca2+ imaging. Individual cells within 500-900 electron micrograph stacks were volume rendered and associations with their neighbours established. 'Ribbon-shaped', Ano1 Cl- channel immuno-reactive ICs were present in the adventitia and the sub-urothelial space adjacent to the TSMC layer. ICs in the proximal renal pelvis were immuno-reactive to antibodies for CaV3.1 and hyperpolarization-activated cation nucleotide-gated isoform 3 (HCN3) channel sub-units, while basal-epithelial cells (BECs) were intensely immuno-reactive to Kv7.5 channel antibodies. Adventitial to the TSMC layer, ASMCs formed close appositions with TSMCs and ICs. The T-type Ca2+channel blocker, Ni2+ (10-200 µM), reduced the frequency while the L-type Ca2+ channel blocker (1 µM nifedipine) reduced the amplitude of propagating Ca2+ waves and contractions in the TSMC layer. Upon complete suppression of Ca2+ entry through TSMC Ca2+ channels, ASMCs displayed high-frequency (6 min-1) Ca2+ transients, and ICs distributed into two populations of cells firing at 1 and 3 min-1, respectively. IC Ca2+ transients periodically (every 3-5 min-1) summed into bursts which doubled the frequency of ASMC Ca2+ transient firing. Synchronized IC bursting and the acceleration of ASMC firing were inhibited upon blockade of HCN channels with ZD7288 or cell-to-cell coupling with carbenoxolone. While ASMCs appear to be the primary pacemaker driving pyeloureteric peristalsis, it was concluded that sub-urothelial HCN3(+), CaV3.1(+) ICs can accelerate ASMC Ca2+ signalling.

En bloc staining with hydroquinone treatment for block face imaging.

IntroductionBecause recent three-dimensional (3D) ultrastructural reconstruction techniques such as serial block face scanning electron microscopy (SBFSEM), obtain their images directly from the flat surface of specimens via material contrast[1], specimens should be strongly stained with heavy metals prior to resin embedding in order to obtain higher material contrast using backscattered electrons (BSEs). To enhance membrane contrast for block face imaging (BFI), we usually stain specimens using the method published by Deerinck[2], and the images obtained show TEM-like contrast.However, recently, our research subjects have required reconstruction of a much larger volume, increasing the total image acquisition time. To reduce the total acquisition time, both high sensitivity detectors and a new specimen preparation method that provides much higher contrast are required. Takahashi et al.[3] have reported that hydroquinone (HQ) treatment during traditional electro-conductive staining increases specimen conductivity and drastically reduces the charge problem for SEM observation. They concluded that HQ treatment might increase the efficiency of secondary electron (SE) generation. Because BFI can be performed using SE as well as BSE, we examined whether addition of HQ treatment to en bloc staining protocols increased the contrast for BFI using SE. Materials & methodsMouse liver tissue was used. Mice were deeply anesthetized by diethyl ether and sodium pentobarbital, and tissues were fixed by transcardial perfusion of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) through the left ventricle, followed by heparin-containing saline. After perfusion, liver tissues were removed and cut into small cubes approximately 1 mm(3) in the fixative, and were further fixed in the same fixative for 2 h at 4°C. Subsequently, en blocstaining was performed as follows: the specimens were treated using a reduced-OTO staining method (1.5% potassium ferrocyanide-2% OsO4, 1% thiocarbohydrazide, and then 2% OsO4). Subsequently, specimens were treated with 1% HQ solution. Some specimens were exempted from this step and used as controls. Specimens were further stained with 4% uranyl acetate and Walton's lead aspartate solution.After staining, specimens were dehydrated using an ethanol series and embedded in epoxy resin (EPON812, TAAB). Surface of specimens block were cut with a diamond knife, and the newly created flat surfaces of the specimens were coated with evaporated carbon (50 Å) and observed using a SEM (Quanta 3D FEG, FEI).ResultsThe HQ-treated specimens generated a larger amount of SEs than control specimens when subjected to irradiation with the same beam, although BSE numbers were not evidently increased by the treatment. The present results suggest that HQ treatment increases SE generation efficiency, but does not enhance the recruitment of heavy metals into specimens. HQ treatment increased the contrast-to-noise ratio of BFI for images obtained using SEs, and may reduce the total image acquisition time of recently developed 3D reconstruction methods based on SEM.

Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy.

IntroductionMitochondrial fission and fusion events are fundamental mechanisms for quality control of mitochondrial functions. Mitochondrial DNA (mtDNA) usually divides in offspring mitochondria after fission and mtDNA dynamics are thought to be coordinated with mitochondrial turnover. Recently, several candidate mechanisms for the relationship between mtDNA division and mitochondrial fission have been suggested ([1], 2012). The dynamics of mtDNA or nucleoids can be observed using fluorescent microscopy, but the ultrastructural aspects of their coordination remain unclear. Although visualization of mtDNA at the electron microscopic level is an important step in understanding how mtDNA division and mitochondrial division are coordinated, it is quite difficult to observe using conventional electron microscopic methods. In the present study, we attempted to establish correlative light and electron microscopy (CLEM) observation to visualize the three-dimensional localization of the mtDNA /nucleoid within mitochondria at electron microscopic resolution using a combination of immuno-electron and focused-ion beam scanning electron microscopy (FIB/SEM) tomography methods. Materials & methodsHeLa cells were fixed using 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer, and then immunohistochemically labeled with anti-TFAM IgG antibody (Abnova, USA) or anti-DNA IgM antibody (Progen, Germany). The cells were then reacted with biotin-labeled secondary antibodies. The immunoreactivities were visualized using two methods: the ABC method and streptavidin Fluoro-Nanogold. Immunohistochemically labeled specimens were then observed using light microscopy. These specimens were then developed using a Gold Enhancement kit (Nanoprobe, USA) for 150 s. Specimens for electron microscopy were stained using the ROTO method, embedded in resin, and subjected to FIB/SEM tomography (Quanta 3D FEG, FEI). 3D reconstruction was performed using the software Amira (FEI). Results & discussionWe were not able to identify a nucleoid-like structure within mitochondria, even in a complete 3D reconstruction using FIB/SEM with conventional staining. In CLEM observations, immunoreaction (IR) products were correlatively observed under LM and EM. Pre-embedding immuno-electron microscopy showed DAB and gold IR in the matrix of some mitochondria. Interestingly, IR products were observed in the globular region of the mitochondrial matrix (approximately 0.4 µm in diameter), frequently localizing in the peripheral end of the mitochondrial matrix, adjacent to the inner membrane. Using the post-embedding immunogold method, gold labels were also observed in a portion of the matrix adjacent to the mitochondrial inner membrane. These immunocytochemical results were concordant with our fluorescent microscopic observations.

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder.

BACKGROUND AND PURPOSE: In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca(2+) transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder. EXPERIMENTAL APPROACH: The morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca(2+) dynamics were visualized using Fluo-4 fluorescence Ca(2+) imaging. KEY RESULTS: A network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca(2+) transients, which were accompanied by phasic venular constrictions. Nicardipine (1 µM) disrupted the synchrony of spontaneous Ca(2+) transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca(2+) transients were suppressed by cyclopiazonic acid (10 µM), 2-aminoethoxydiphenyl borate (10 µM), U-73122 (1 µM), oligomycin (1 µM) and SKF96365 (10 µM), but unaffected by ryanodine (100 µM) or YM-244769 (1 µM), suggesting that pericyte Ca(2+) transients rely on Ca(2+) release from the endoplasmic reticulum via the InsP(3) receptor and also require Ca(2+) influx through store-operated Ca(2+) channels. CONCLUSIONS AND IMPLICATIONS: The pericytes in mouse bladder can generate spontaneous Ca(2+) transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.

Beam deceleration for block-face scanning electron microscopy of embedded biological tissue.

The beam deceleration (BD) method for scanning electron microscopes (SEM) also referred to as "retarding" was applied to back-scattered electron (BSE) imaging of the flat block face of a resin embedded biological specimen under low accelerating voltage and low beam current conditions. BSE imaging was performed with 0-4 kV of BD on en bloc stained rat hepatocyte. BD drastically enhanced the compositional contrast of the specimen and also improved the resolution at low landing energy levels (1.5-3 keV) and a low beam current (10 pA). These effects also functioned in long working distance observation, however, stage tilting caused uncorrectable astigmatism in BD observation. Stage tilting is mechanically required for a FIB/SEM, so we designed a novel specimen holder to minimize the unfavorable tilting effect. The FIB/SEM 3D reconstruction using the new holder showed a reasonable contrast and resolution high enough to analyze individual cell organelles and also the mitochondrial cristae structures (~5 nm) of the hepatocyte. These results indicate the advantages of BD for block face imaging of biological materials such as cells and tissues under low-voltage and low beam current conditions.

Helical arrangement of filaments in microvillar actin bundles.

Actin filament arrays in in vivo microvillar bundles of rat intestinal enterocyte were re-evaluated using electron tomography (ET). Conventional electron microscope observation of semi-thin cross sections (300nm thick) of high-pressure freeze fixed and resin embedded brush border has shown a whirling pattern in the center of the microvilli instead of hexagonally arranged dots, which strongly suggests that the bundle consists of a non-parallel array of filaments. A depth compensation method for the ET was developed to estimate the actual structure of the actin bundle. Specimen shrinkage by beam irradiation during image acquisition was estimated to be 63%, and we restored the original thickness in the reconstruction. The depth compensated tomogram displayed the individual actin filaments within the bundles and it indicated that the actin filaments do not lie exactly parallel to each other: instead, they are twisted in a clockwise coil with a pitch of ∼120°/µm. Furthermore, the lattice of actin filaments was occasionally re-arranged within the bundle. As the microvillar bundle mechanically interacts with the membrane and is thought to be compressed by the membrane's faint tensile force, we removed the shrouding membrane using detergents to eliminate the mechanical interaction. The bared bundles no longer showed the whirling pattern, suggesting that the bundle had released its coiled property. These findings indicate that the bundle has not rigid but elastic properties and a dynamic transformation in its structure caused by a change in the mechanical interaction between the membrane and the bundle.