Selection of maize hybrids based on genotype × yield × trait (GYT) in different environments.

This study aimed to identify the best genotypes using the genotype × yield × trait (GYT) method. To investigate the relationships was performed between yield × traits in four regions of Karaj, Birjand, Shiraz and Arak in two cropping years in a randomized complete block design (RCBD) with three replications. The average grain yield in four regions and two years of the experiment was calculated as 5966 kg/ha, and GYT was obtained based on the multiplication of grain yield with different traits. Comparing the average effect of genotype × year in different environments showed that KSC703 and KSC707 hybrids are among the most productive hybrids among the studied genotypes in grain yield. By examining the correlation coefficients between yield × traits in the tested areas, Y × TWG with Y × GW, Y × NRE, Y × NGR and Y × EL, Y × ED with Y × NGR, Y × NRE with Y × GW and the combination of Y × GW with Y × GL had a positive and significant correlation in all regions. The correlation diagrams were drawn on the evaluated areas' data and showed the correlation of most compounds except Y × GT with each other. Based on the analysis of the main components, the first three components explained the greatest diversity in the population. They were named the component ear grain profile, grain thickness component and plant height profile component.

Interrelationships Between Spinal Sympathetic Preganglionic Neurons, Autonomic Systems and Electrical Synapses Formed by Connexin36-containing Gap Junctions.

Spinal sympathetic preganglionic neurons (SPNs) are among the many neuronal populations in the mammalian central nervous system (CNS) where there is evidence for electrical coupling between cell pairs linked by gap junctions composed of connexin36 (Cx36). Understanding the organization of this coupling in relation to autonomic functions of spinal sympathetic systems requires knowledge of how these junctions are deployed among SPNs. Here, we document the distribution of immunofluorescence detection of Cx36 among SPNs identified by immunolabelling of their various markers, including choline acetyltransferase, nitric oxide and peripherin in adult and developing mouse and rat. In adult animals, labelling of Cx36 was exclusively punctate and dense concentrations of Cx36-puncta were distributed along the entire length of the spinal thoracic intermediolateral cell column (IML). These puncta were also seen in association with SPN dendritic processes in the lateral funiculus, the intercalated and central autonomic areas and those within and extending medially from the IML. All labelling for Cx36 was absent in spinal cords of Cx36 knockout mice. High densities of Cx36-puncta were already evident among clusters of SPNs in the IML of mouse and rat at postnatal days 10-12. In Cx36BAC::eGFP mice, eGFP reporter was absent in SPNs, thus representing false negative detection, but was localized to some glutamatergic and GABAergic synaptic terminals. Some eGFP+ terminals were found contacting SPN dendrites. These results indicate widespread Cx36 expression in SPNs, further supporting evidence of electrical coupling between these cells, and suggest that SPNs are innervated by neurons that themselves may be electrically coupled.

Heat shock-induced enhanced susceptibility of barley to Bipolaris sorokiniana is associated with elevated ROS production and plant defence-related gene expression.

Heat stress alters plant defence responses to pathogens. Short-term heat shock promotes infections by biotrophic pathogens. However, little is known about how heat shock affects infection by hemibiotrophic pathogens like Bipolaris sorokiniana (teleomorph: Cochliobolus sativus). We assessed the effect of heat shock in B. sorokiniana-susceptible barley (Hordeum vulgare cv. Ingrid) by monitoring leaf spot symptoms, B. sorokiniana biomass, ROS and plant defence-related gene expression following pre-exposure to heat shock. For heat shock, barley plants were kept at 49 °C for 20 s. B. sorokiniana biomass was assessed by qPCR, ROS levels determined by histochemical staining, while gene expression was assayed by RT-qPCR. Heat shock suppressed defence responses of barley to B. sorokiniana, resulting in more severe necrotic symptoms and increased fungal biomass, as compared to untreated plants. Heat shock-induced increased susceptibility was accompanied by significant increases in ROS (superoxide, H2 O2 ). Transient expression of plant defence-related antioxidant genes and a barley programmed cell death inhibitor (HvBI-1) were induced in response to heat shock. However, heat shock followed by B. sorokiniana infection caused further transient increases in expression of HvSOD and HvBI-1 correlated with enhanced susceptibility. Expression of the HvPR-1b gene encoding pathogenesis-related protein-1b increased several fold 24 h after B. sorokiniana infection, however, heat shock further increased transcript levels along with enhanced susceptibility. Heat shock induces enhanced susceptibility of barley to B. sorokiniana, associated with elevated ROS levels and expression of plant defence-related genes encoding antioxidants, a cell death inhibitor, and PR-1b. Our results may contribute to elucidating the influence of heat shock on barley defence responses to hemibiotrophic pathogens.

Characteristics of Electrical Synapses, C-terminals and Small-conductance Ca2+ activated Potassium Channels in the Sexually Dimorphic Cremaster Motor Nucleus in Spinal Cord of Mouse and Rat.

Sexually dimorphic motoneurons (MNs) located in lower lumbar spinal cord are involved in mating and reproductive behaviours and are known to be coupled by electrical synapses. The cremaster motor nucleus in upper lumbar spinal cord has also been suggested to support physiological processes associated with sexual behaviours in addition to its thermoregulatory and protective role in maintaining testes integrity. Using immunofluorescence approaches, we investigated whether cremaster MNs also exhibit features reflecting their potential for electrical synaptic communication and examined some of their other synaptic characteristics. Both mice and rats displayed punctate immunolabelling of Cx36 associated with cremaster MNs, indicative of gap junction formation. Transgenic mice with enhanced green fluorescent protein (eGFP) reporter for connexin36 expression showed that subpopulations of cremaster MNs in both male and female mice express eGFP, with greater proportions of those in male mice. The eGFP+ MNs within the cremaster nucleus vs. eGFP- MNs inside and outside this nucleus displayed a 5-fold greater density of serotonergic innervation and exhibited a paucity of innervation by C-terminals arising from cholinergic V0c interneurons. All MNs within the cremaster motor nucleus displayed prominent patches of immunolabelling for SK3 (K+) channels around their periphery, suggestive of their identity as slow MNs, many though not all of which were in apposition to C-terminals. The results provide evidence for electrical coupling of a large proportion of cremaster MNs and suggest the existence of two populations of these MNs with possibly differential innervation of their peripheral target muscles serving different functions.

Stability yield indices on different sweet corn hybrids based on AMMI analysis.

Currently, sweet corn is considered an important crop due to its high sugar content and low starch content. Important sugars in sweet corn include sucrose, fructose, glucose, and maltose. The purpose of the present study was to use the yield indices of the eight examined sweet corn hybrids and the correlation of the yield indices together. Concentration is important for consumers in terms of yield indices. The research site was located at the Látókép Experimental Station of the University of Debrecen. The small plot experiment had a strip plot design with four replications. The previous crop was sweet corn; the plant density was 64 thousand/ha. The obtained result indicates that Biplot AMMI based on IPCA1 showed that the DB, NO, GS, and GB hybrids had stability and high performance in terms of yield indices. At the same time, fructose and glucose had stable parameters for the hybrids involved in the study. IPCA1 AMMI biplot showed that the ME hybrid had stability and high performance in terms of iron and zinc as well. IPCA2 AMMI biplot showed that DE, GB, and GS hybrids had stability and the highest performance on yield parameters in the scope of the research. Fructose, glucose, and sucrose had stable parameters on hybrids based on IPCA2. The DB and SE hybrids had desirable performance in Lutein and Zeaxanthin based on the biplot. The DE hybrid had a maximum performance on iron and zinc parameters.

Expression of the gap junction protein connexin36 in small intensely fluorescent (SIF) cells in cardiac parasympathetic ganglia of rodents.

In mammals, several endocrine cell types are electrically coupled by connexin36 (Cx36)-containing gap junctions, which mediate intercellular communication and allow regulated and synchronized cellular activity through exchange of ions and small metabolites via formation of intercellular channels that link plasma membranes of apposing cells. One cell type thought to be endocrine-like in nature are small intensely fluorescent (SIF) cells that store catecholamines in their dense-core vesicles and reside in autonomic ganglia. Here, using immunofluorescence approaches, we examined whether SIF cells located specifically in cardiac parasympathetic ganglia of adult and neonatal mice and adult rats follow patterns of Cx36 expression seen in other endocrine cells. In these ganglia, SIF cells were identified by their distinct small soma size, autofluorescence at 475 nm, and immunolabelling for their markers tyrosine hydroxylase and vesicular monoamine transporter-1. SIF cells were often found in pairs or clusters among principal cholinergic neurons. Immunofluorescence labelling of Cx36 occurred exclusively as fine puncta that appeared at contacts between SIF cell processes and somata or at somato-somatic appositions of SIF cells. These puncta were absent in cardiac parasympathetic ganglia of Cx36 null mice. Transgenic mice expressing enhanced green fluorescent protein reporter for Cx36 expression displayed labelling for the reporter in SIF cells. The results suggest that Cx36-containing gap junctions electrically couple SIF cells, which is consistent with previous suggestions that these may be classified as endocrine-type cells that secrete catecholamines into the bloodstream in a regulated manner.

Quantitative and qualitative yield in sweet maize hybrids.

Today, sweet corn is considered an important vegetable due to its high sugar content and low starch content. Cluster analysis and variance analysis showed that hybrids had variations in yield indices. GB, DE and GS hybrids had similar performance on indices. SE hybrid that has significant performance on zeaxanthin. Biplot showed that fructose, glucose, sucrose and potassium had stability value on hybrids. All the hybrids had the best performance on fructose, glucose, sucrose and potassium factors. Factor biplot positively correlated with yield indices, including calcium, iron, zinc, magnesium, α-Carotene, 9Z-ß-Carotene, phosphorus, and ß-carotene. On the other hand, there is a positive correlation with fructose, glucose, potassium, lutein, sucrose, ß-Cryptoxanthin, and zeaxanthin. So, to evaluate or increase lutein and zeaxanthin, the other parameters like sugar content (fructose, glucose, and sucrose) are important factors and have an effect together. Factor analysis and biplot showed that ME hybrid had a maximum performance on the first factor of yield indices. Also, the second factor of yield indices had a maxi-mum effect on NO hybrids. SE hybrids had maximum performance in zeaxanthin and GS hybrid had maximum performance in zinc, phosphorus, and iron. The dry matter had stability on DB hybrid.

On the Organization of Connexin36 Expression in Electrically Coupled Cholinergic V0c Neurons (Partition Cells) in the Spinal Cord and Their C-terminal Innervation of Motoneurons.

Large cholinergic neurons (V0c neurons; aka, partition cells) in the spinal cord project profusely to motoneurons on which they form C-terminal contacts distinguished by their specialized postsynaptic subsurface cisterns (SSCs). The V0c neurons are known to be rhythmically active during locomotion and release of acetylcholine (ACh) from their terminals is known to modulate the excitability of motoneurons in what appears to be a task-dependent manner. Here, we present evidence that a subpopulation of V0c neurons express the gap junction forming protein connexin36 (Cx36), indicating that they are coupled by electrical synapses. Based on immunofluorescence imaging and the use of Cx36BAC-enhanced green fluorescent protein (eGFP) mice in which C-terminals immunolabelled for their marker vesicular acetylcholine transporter (vAChT) are also labelled for eGFP, we found a heterogeneous distribution of eGFP+ C-terminals on motoneurons at cervical, thoracic and lumber spinal levels. The density of C-terminals on motoneurons varied as did the proportion of those that were eGFP+ vs. eGFP-. We present evidence that fast vs. slow motoneurons have a greater abundance of these terminals and fast motoneurons also have the highest density that were eGFP+. Thus, our results indicate that a subpopulation of V0c neurons projects preferentially to fast motoneurons, suggesting that the capacity for synchronous activity conferred by electrical synapses among networks of coupled V0c neurons enhances their dynamic capabilities for synchronous regulation of motoneuron excitability during high muscle force generation. The eGFP+ vs. eGFP- V0c neurons were more richly innervated by serotonergic terminals, suggesting their greater propensity for regulation by descending serotonergic systems.

Approximate complex electrical potential distribution in the monodomain model with unequal conductivity and relative permittivity anisotropy ratios.

OBJECTIVE: Electrical conductivity and relative permittivity are properties that indicate muscle health and they have different values parallel and perpendicular to the direction of the myofiber, a concept known as anisotropy. When the intrinsic electrical properties of muscle have ratios of anisotropy that are different then there is no analytical solution that can describe the electrical potential distribution in the tissue. APPROACH: Here, we present approximate analytical solutions to monodomain equations with unequal anisotropy ratios. For this, we base our analysis on perturbation theory where the electrical potential is approximated by the sum of the zeroth- and first-order terms of an infinite series. MAIN RESULTS: The validity of the approach is confirmed using experimental data for healthy and diseased muscle available online. SIGNIFICANCE: A better understanding of electrical potential distribution in anisotropic skeletal muscle tissue will allow the development of improved diagnostic tools for neuromuscular diseases.

Permittivity of ex vivo healthy and diseased murine skeletal muscle from 10 kHz to 1 MHz.

A better understanding of the permittivity property of skeletal muscle is essential for the development of new diagnostic tools and approaches for neuromuscular evaluation. However, there remain important knowledge gaps in our understanding of this property in healthy and diseased skeletal muscle, which hinder its translation into clinical application. Here, we report the permittivity of gastrocnemius muscle in healthy wild type mice and murine models of spinal muscular atrophy, muscular dystrophy, diabetes, amyotrophic lateral sclerosis and in a model of myofiber hypertrophy. Data were measured ex vivo from 10 kHz to 1 MHz using the four-electrode impedance technique. Additional quantitative histology information were obtained. Ultimately, the normative data reported will offer the scientific community the opportunity to develop more accurate models for the validation and prediction of experimental observations in both pre-clinical and clinical neuromuscular disease research.

New electrical impedance methods for the in situ measurement of the complex permittivity of anisotropic skeletal muscle using multipolar needles.

This paper provides a rigorous analysis on the measurement of the permittivity of two-dimensional anisotropic biological tissues such as skeletal muscle using the four-electrode impedance technique. The state-of-the-art technique requires individual electrodes placed at the same depth in contact with the anisotropic material, e.g. using monopolar needles. In this case, the minimum of measurements in different directions needed to estimate the complex permittivity and its anisotropy direction is 3, which translates into 12 monopolar needle insertions (i.e. 3 directions × 4 electrodes in each direction). Here, we extend our previous work and equip the reader with 8 new methods for multipolar needles, where 2 or more electrodes are spaced along the needle's shaft in contact with the tissue at different depths. Using multipolar needles, the new methods presented reduce the number of needle insertions by a factor of 2 with respect to the available methods. We illustrate the methods with numerical simulations and new experiments on ex vivo ovine skeletal muscle (n = 3). Multi-frequency longitudinal and transverse permittivity data from 30 kHz to 1 MHz is made publicly available in the supplementary material. The methods presented here for multipolar needles bring closer the application of needle electrical impedance to patients with neuromuscular diseases.

A brief review on the rising incidence of chronic kidney diseases and non-alcoholic fatty liver disease.

Chronic kidney diseases (CKDs) are the most common forms of kidney disease all around the world. The incidence of CKD is rising, which is mainly driven by population aging as well as by a global rise in hypertension, metabolic syndrome, and metabolic risk factors, particularly obesity and type-2 diabetes. The high mortality, morbidity of CKD, and the health care costs of the renal replacement therapy have led investigators to seek recent and potentially modifiable risk factors such as non-alcoholic fatty liver disease (NAFLD). NAFLD is the hepatic manifestation of metabolic syndrome and the most common cause of chronic liver disease. It incorporates a spectrum of liver diseases ranging from simple steatosis to steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. On the basis of recent publications, the prevalence of CKD is significantly increased among patients with NAFLD, and the prevalence of NAFLD is also higher in CKD patients than in patients without NAFLD. These findings suggest that patients with NAFLD should be screened for CKD and patients with CKD and metabolic syndrome should be screened for NAFLD. Patients with NAFLD and CKD should be treated and followed up by a multidisciplinary team that involves specialists in hepatology, nephrology, diabetes, and cardiology.

Arterial stiffness may predict renal and cardiovascular prognosis in autosomal-dominant polycystic kidney disease.

Background and aims Autosomal-dominant polycystic kidney disease (ADPKD) is one of the most common causes of end-stage renal disease (ESRD). The most important cause of death among ADPKD patients is cardiovascular (CV). The aim of this study was to examine the prognostic significance of arterial stiffness on CV and renal outcomes in ADPKD. Methods A total of 55 patients with ADPKD were examined. Pulse wave velocity was determined and stiffness index (SIDVP) was calculated. Combined primary endpoints (CV and renal) were major CV events (myocardial infarction, stroke, and CV intervention) as CV endpoints, and attaining of ESRD or start of renal replacement therapy as renal endpoints. Secondary endpoints were CV or renal endpoints separately. Results The mean age of those 55 ADPKD patients was 45 ± 12 years, 21 patients were male. The average value of the SIDVP was 11.11 ± 2.22 m/s. The patients were divided into two groups by the cutoff value of 11 m/s of SIDVP and then outcomes were analyzed. In the higher arterial stiffness group (SIDVP > 11 m/s), occurrence of combined primary endpoint (CV and renal) was significantly higher than in the group with more elastic arteries (p = 0.033). A statistically significant difference was found in the renal endpoints (p = 0.018), but not in the CV endpoints (p = 0.952) between the two groups. Conclusions Increased arterial stiffness predicts the onset of ESRD in ADPDK. Assessment of SIDVP appears to be a useful method for estimating the renal and CV prognosis in ADPKD.

Structural and Intermolecular Associations Between Connexin36 and Protein Components of the Adherens Junction-Neuronal Gap Junction Complex.

Intimate structural and functional relationships between gap junctions and adherens junctions have been demonstrated in peripheral tissues, but have not been thoroughly examined in the central nervous system, where adherens junctions are often found in close proximity to neuronal gap junctions. Here, we used immunofluorescence approaches to document the localization of various protein components of adherens junctions in relation to those that we have previously reported to occur at electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36). The adherens junction constituents N-cadherin and nectin-1 were frequently found to localize near or overlap with Cx36-containing gap junctions in several brain regions examined. This was also true of the adherens junction-associated proteins α-catenin and ß-catenin, as well as the proteins zonula occludens-1 and AF6 (aka, afadin) that were reported constituents of both adherens junctions and gap junctions. The deployment of the protein constituents of these junctions was especially striking at somatic contacts between primary afferent neurons in the mesencephalic trigeminal nucleus (MesV), where the structural components of adherens junctions appeared to be maintained in connexin36 null mice. These results support emerging views concerning the multi-molecular composition of electrical synapses and raise possibilities for various structural and functional protein-protein interactions at what now can be considered the adherens junction-neuronal gap junction complex. Further, the results point to intracellular signaling pathways that could potentially contribute to the assembly, maintenance and turnover of this complex, as well as to the dynamic nature of neuronal communication at electrical synapses.

Connexin36 Expression in Primary Afferent Neurons in Relation to the Axon Reflex and Modality Coding of Somatic Sensation.

Electrical coupling mediated by connexin36-containing gap junctions that form electrical synapses is known to be prevalent in the central nervous system, but such coupling was long ago reported also to occur between cutaneous sensory fibers. Here, we provide evidence supporting the capability of primary afferent fibers to engage in electrical coupling. In transgenic mice with enhanced green fluorescent protein (eGFP) serving as a reporter for connexin36 expression, immunofluorescence labeling of eGFP was found in subpopulations of neurons in lumbar dorsal root and trigeminal sensory ganglia, and in fibers within peripheral nerves and tissues. Immunolabeling of connexin36 was robust in the sciatic nerve, weaker in sensory ganglia than in peripheral nerve, and absent in these tissues from Cx36 null mice. Connexin36 mRNA was detected in ganglia from wild-type mice, but not in those from Cx36 null mice. Labeling of eGFP was localized within a subpopulation of ganglion cells containing substance P and calcitonin gene-releasing peptide, and in peripheral fibers containing these peptides. Expression of eGFP was also found in various proportions of sensory ganglion neurons containing transient receptor potential (TRP) channels, including TRPV1 and TRPM8. Ganglion cells labeled for isolectin B4 and tyrosine hydroxylase displayed very little co-localization with eGFP. Our results suggest that previously observed electrical coupling between peripheral sensory fibers occurs via electrical synapses formed by Cx36-containing gap junctions, and that some degree of selectivity in the extent of electrical coupling may occur between fibers belonging to subpopulations of sensory neurons identified according to their sensory modality responsiveness.

New electrical impedance methods for the in situ measurement of the complex permittivity of anisotropic biological tissues.

The capability of measuring the complex permittivity of tissues has the potential to provide valuable new insights to inform medical assessment and diagnosis. However, existing electrical impedance approaches have practical limitations when aiming to measure tissues' anisotropy with accuracy. Here we present new methods that overcome the limitations of previous approaches by modeling the anisotropy in both the resistivity and reactivity of tissues measured in three or more different directions. These new methods are validated with numerical simulations and in situ experiments on healthy ovine skeletal muscle. The obtained data between 3 kHz and 1 MHz are also made publicly available in the supplementary information.

Altered neurite morphology and cholinergic function of induced pluripotent stem cell-derived neurons from a patient with Kleefstra syndrome and autism.

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD.

Cx36, Cx43 and Cx45 in mouse and rat cerebellar cortex: species-specific expression, compensation in Cx36 null mice and co-localization in neurons vs. glia.

Electrical synapses formed by connexin36 (Cx36)-containing gap junctions between interneurons in the cerebellar cortex have been well characterized, including those formed between basket cells and between Golgi cells, and there is gene reporter-based evidence for the expression of connexin45 (Cx45) in the cerebellar molecular layer. Here, we used immunofluorescence approaches to further investigate expression patterns of Cx36 and Cx45 in this layer and to examine localization relationships of these connexins with each other and with glial connexin43 (Cx43). In mice, strain differences were found, such that punctate labelling for Cx36 was differentially distributed in the molecular layer of C57BL/6 vs. CD1 mice. In mice with EGFP reporter representing Cx36 expression, Cx36-puncta were localized to processes of stellate cells and other cerebellar interneurons. Punctate labelling of Cx45 was faint in the molecular layer of wild-type mice and was increased in intensity in mice with Cx36 gene ablation. The vast majority of Cx36-puncta co-localized with Cx45-puncta, which in turn was associated with the scaffolding protein zonula occludens-1. In rats, Cx45-puncta were also co-localized with Cx36-puncta and additionally occurred along Bergmann glial processes adjacent to Cx43-puncta. The results indicate strain and species differences in Cx36 as well as Cx45 expression, possible compensatory processes after loss of Cx36 expression and localization of Cx45 to both neuronal and Bergmann glial gap junctions. Further, expression of both Cx43 and Cx45 in Bergmann glia of rat may contribute to the complex properties of junctional coupling between these cells and perhaps to their reported coupling with Purkinje cells.

Connexin36 localization to pinealocytes in the pineal gland of mouse and rat.

Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36-containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase-positive and 5-hydroxytryptamine-positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens-1 (ZO-1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36-containing gap junctions that also incorporate the scaffolding protein ZO-1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36-containing gap junctions, which may now be tested by the use of Cx36 null mice.