Experimentally induced diabetes causes glial activation, glutamate toxicity and cellular damage leading to changes in motor function

Behavioral impairments are the most empirical consequence of diabetes mellitus documented in both humans and animal models, but the underlying causes are still poorly understood. As the cerebellum plays a major role in coordination and execution of the motor functions, we investigated the possible involvement of glial activation, cellular degeneration and glutamate transportation in the cerebellum of rats, rendered diabetic by a single injection of streptozotocin (STZ; 45 mg/kg body weight; intraperitoneally). Motor function alterations were studied using Rotarod test (motor coordination) and grip strength (muscle activity) at 2nd, 4th, 6th, 8th, 10th, and 12th week post-diabetic confirmation. Scenario of glial (astroglia and microglia) activation, cell death and glutamate transportation was gaged using immunohistochemistry, histological study and image analysis. Cellular degeneration was clearly demarcated in the diabetic cerebellum. Glial cells were showing sequential and marked activation following diabetes in terms of both morphology and cell number. Bergmann glial cells were hypertrophied and distorted. Active caspase-3 positive apoptotic cells were profoundly present in all three cerebellar layers. Reduced co-labeling of GLT-1 and GFAP revealed the altered glutamate transportation in cerebellum following diabetes. These results, exclusively derived from histology, immunohistochemistry and cellular quantification, provide first insight over the associative reciprocity between the glial activation, cellular degeneration and reduced glutamate transportation, which presumably lead to the behavioral alterations following STZ-induced diabetes

Iba1 expressing microglia in the dorsal root ganglia become activated following peripheral nerve injury in rats

110-116The presence of microglia in dorsal root ganglia (DRG) has not been reported earlier. The dorsal root ganglia contain satellite glial cells (SGCs) and macrophages, which are considered to have infiltrated from the systemic blood. An attempt was made to investigate whether microglia as found in the central nervous system are also present in the dorsal root ganglia of untreated rats and following experimental peripheral nerve injury. Female adult Wistar rats were subjected to sciatic nerve transection injury on the right hand side. The DRGs of the right side were studied with the contralateral DRGs of the left side serving as controls. The tissues, harvested at different time points after injury, following intracardial perfusion fixation, and frozen sections were immunolabeled with anti-GFAP as a marker for SGCs and anti-Iba1 and OX-6 as markers for microglia and activated macrophagic microglia, respectively. These antibodies were also used in combination to ascertain if Iba1+ cells are the SGCs or otherwise and also if macrophagic OX-6+ cells are Iba1 positive microglia. The results indicate that Iba1 positive microglial cells are different from the SGCs in the DRGs. The Iba1 positive microglial cells respond to the sciatic nerve injury becoming activated and macrophagic and express MHCII molecules. Such activated microglia apparently may serve as neurosupportive cells, providing neuroprotection and scavenging cellular debris in response to the injury

Delivery of different genes into pre- and post-synaptic neocortical interneurons connected by GABAergic synapses.

Local neocortical circuits play critical roles in information processing, including synaptic plasticity, circuit physiology, and learning, and GABAergic inhibitory interneurons have key roles in these circuits. Moreover, specific neurological disorders, including schizophrenia and autism, are associated with deficits in GABAergic transmission in these circuits. GABAergic synapses represent a small fraction of neocortical synapses, and are embedded in complex local circuits that contain many neuron and synapse types. Thus, it is challenging to study the physiological roles of GABAergic inhibitory interneurons and their synapses, and to develop treatments for the specific disorders caused by dysfunction at these GABAergic synapses. To these ends, we report a novel technology that can deliver different genes into pre- and post-synaptic neocortical interneurons connected by a GABAergic synapse: First, standard gene transfer into the presynaptic neurons delivers a synthetic peptide neurotransmitter, containing three domains, a dense core vesicle sorting domain, a GABAA receptor-binding domain, a single-chain variable fragment anti-GABAA ß2 or ß3, and the His tag. Second, upon release, this synthetic peptide neurotransmitter binds to GABAA receptors on the postsynaptic neurons. Third, as the synthetic peptide neurotransmitter contains the His tag, antibody-mediated, targeted gene transfer using anti-His tag antibodies is selective for these neurons. We established this technology by expressing the synthetic peptide neurotransmitter in GABAergic neurons in the middle layers of postrhinal cortex, and the delivering the postsynaptic vector into connected GABAergic neurons in the upper neocortical layers. Targeted gene transfer was 61% specific for the connected neurons, but untargeted gene transfer was only 21% specific for these neurons. This technology may support studies on the roles of GABAergic inhibitory interneurons in circuit physiology and learning, and support gene therapy treatments for specific disorders associated with deficits at GABAergic synapses

Utilizing human cerebral organoids to model breast cancer brain metastasis in culture

Abstract Background Metastasis, the spread, and growth of malignant cells at secondary sites within a patient’s body, accounts for over 90% of cancer-related mortality. Breast cancer is the most common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10–16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses, which represents a critical barrier for the development of efficient therapies for affected breast cancer patients. Methods Previous research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM, we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP, other cell lines of MCF-7, HCC-1806, and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid. Results We found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover, MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids. Conclusions Our co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture

An identified ensemble within a neocortical circuit encodes essential information for genetically-enhanced visual shape learning

Advanced cognitive tasks are encoded in distributed neocortical circuits that span multiple forebrain areas. Nonetheless, synaptic plasticity and neural network theories hypothesize that essential information for performing these tasks is encoded in specific ensembles within these circuits. Relatively simpler subcortical areas contain specific ensembles that encode learning, suggesting that neocortical circuits contain such ensembles. Previously, using localized gene transfer of a constitutively active protein kinase C (PKC), we established that a genetically-modified circuit in rat postrhinal cortex, part of the hippocampal formation, can encode some essential information for performing specific visual shape discriminations. However, these studies did not identify any specific neurons that encode learning; the entire circuit might be required. Here, we show that both learning and recall require fast neurotransmitter release from an identified ensemble within this circuit, the transduced neurons; we blocked fast release from these neurons by coexpressing a Synaptotagmin I siRNA with the constitutively active PKC. During learning or recall, specific signaling pathways required for learning are activated in this ensemble; during learning, calcium/calmodulin-dependent protein kinase II, MAP kinase, and CREB are activated; and, during recall, dendritic protein synthesis and CREB are activated. Using activity-dependent gene imaging, we showed that during learning, activity in this ensemble is required to recruit and activate the circuit. Further, after learning, during image presentation, blocking activity in this ensemble reduces accuracy, even though most of the rest of the circuit is activated. Thus, an identified ensemble within a neocortical circuit encodes essential information for performing an advanced cognitive task