Zhi-Ren Rao

Key points: Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca²⁺‐dependent release of gliotransmitters includes glutamate and ATP. Following an ‘on‐cell‐like’ mechanical stimulus to a single astrocyte, Ca²⁺ independent single, large, non‐quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca²⁺‐dependent vesicular release, larger non‐quantal ATP release depends on P2X7 channels following mechanical stretch. Astrocytic ATP release is essential for brain functions such as synaptic long‐term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non‐vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped ‘ATP‐sniffer cells’, we re‐investigated astrocytic ATP release at single‐vesicle resolution in hippocampal astrocytes. Following an ‘on‐cell‐like’ mechanical stimulus of a single astrocyte, a Ca²⁺ independent single large non‐quantal ATP release occurred, in contrast to the Ca²⁺‐dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation‐induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non‐quantal ATP release depends on P2X7 channels in astrocytes.

Growing evidences have revealed that the proforms of several neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT3), by binding to p75 neurotrophin receptor and sortilin, could induce neuronal apoptosis and are implicated in the pathogenesis of various neurodegenerative diseases. The glial cell line-derived neurotrophic factor (GDNF), one of the most potent useful neurotrophic factors for the treatment of Parkinson’s disease (PD), is firstly synthesized as the proform (proGDNF) like other neurotrophin NGF, BDNF, and NT3. However, little is known about proGDNF expression and secretion under physiological as well as pathological states in vivo or in vitro. In this study, we investigated the expression profile and dynamic changes of proGDNF in brains of aging and PD animal models, with the interesting finding that proGDNF was a predominant form of GDNF with molecular weight of about 36 kDa by reducing and nonreducing immunoblots in adult brains and was unregulated in the aging, lipopolysaccharide (LPS), and 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) insult. We further provided direct evidence that accompanied activation of primary astrocytes as well as C6 cell line induced by LPS stimulation, proGDNF was increasingly synthesized and released as the uncleaved form in cell culture. Taken together, our results strongly suggest that proGDNF may be a biologically active protein and has specific effects on the cells close to its secreting site, and a potentially important role of proGDNF signaling in the brains, in the glia–neuronal interaction or in the pathogenesis of PD, should merit further investigation.

The response to hyperosmotic stresses in the abdominal cavity is regulated, in part, by vasopressin (VP)‐secreting neurons in the supraoptic nucleus (SON). How osmotic stress signals are transmitted to the brain is incompletely understood, and whether the transmission routes for osmotic stress signals differ between acute and chronic stresses is unknown. Here we investigated the role of the vagus, splanchnic nerves, and astrocytes in the SON in transducing acute hyperosmotic‐stress signals from the abdominal cavity. We found that acute administration of hyperosmotic saline triggered the activation of neurons as well as astrocytes in the SON and the adjoining ventral glia limitans (SON‐VGL). Severing the subdiaphragmatic vagal nerve (SDV) prevented the normal response of cells in the SON to HS treatment and attenuated the release of VP into the bloodstream. Lesioning the splanchnic nerves (SNL) diminished HS‐induced release of VP, but to a much lesser extent than SDV. Furthermore, SNL did not significantly affect the up‐regulation of Fos in SON neurons or the up‐regulation of Fos and GFAP in SON and SON‐VGL astrocytes that normally occurred in response to HS and did not affect HS‐induced expansion of the SON‐VGL. Inhibiting astrocytes with fluorocitrate (FCA) prevented the response of the SON to HS and attenuated the release of VP, similarly to SDV surgery. These results suggest that the vagus is the principle route for the transmission of hyperosmotic signals to the brain and that astrocytes in the SON region are necessary for the activation of SON neurons and the release of VP into the bloodstream.

Acute hyperosmolarity induced a time‐dependent expression of Fos protein in both neurons and astrocytes of the rat supraoptic nucleus, with peak Fos expresion occurring at 45 min in astrocytes and at 90 min in neurons after hypertonic stimulation in vivo. To determine whether the two cell types were activated separately or in an integrated manner, animals were pretreated with fluorocitrate, a glial metabolic blocker or carbenoxolone, a gap junction blocker followed by an acute hypertonic stimulation similar to that of the controls. Antibodies against glial fibrillary acidic protein, connexin 43, vasopressin, and oxytocin were used in serial sections to identify the cellular elements of the supraoptic nucleus. It was found that interruption of astrocyte metabolism with fluorocitrate significantly reduced Fos protein expression in both astrocytes and neurons, whereas blockage of gap junctions with carbenoxolone clearly reduced Fos protein expression in neurons, but not in astrocytes. These results indicate that both neurons and astrocytes in the rat supraoptic nucleus are involved in regulating osmolarity. Astrocytes are activated first, whereas connexin 43 functional hemichannels in SON astrocytes are required for the subsequent activation of the neurons.

To investigate whether hypertonic saline (HS) can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line.

Ultrastructure of junction areas between neurons and astrocytes in rat supraoptic nuclei

Responses of the neurons in medulla oblongata and C8-T1 spinal cord lateral horn of rats induced by simulated weightlessness were investigated using anti-Fos protein and anti-tyrosine hydroxylase (TH) double staining immunohistochemical methods, and Nissl-staining technique respectively. After four weeks of tail-suspension, many Fos-like positive neurons were localized in the medullary visceral zone (MVZ), predominantly in the nucleus of tractus solitarii and ventrolateral medulla, and some of them showed TH-like immunoreactivity. Sizes of the cell bodies of the lateral horn neurons in C8-T1 segment were significantly increased in 4-wk tail-suspended rats (P<0.05) as compared with that in controls. The results suggest that the neurons in MVZ and the spinal lateral horn may be involved in the adaptation of central cardiovascular regulation during weightlessness.