Neuron Phenotypes And Synaptic Plasticity In Rat Anterior Hypothalamus And Medulla Oblongata Upon Restraint Water-immersion Stress

Restraint water-immersion stress (RWIS), considered to be a mixture of physical and psychological stressors, induces vagally-mediated gastric hypercontractility, hypersecretion of gastric acid and acute gastric erosions within a few hours. It is widely used to study the pathogenesis of stress-induced gastric lesions and to filtrate the medicine that can cure this disease. Using Fos as a marker of the activation of neurons, we found that after different durations of RWIS, the most intense Fos expression was observed in specific brain areas, such as the medullary visceral zone [dorsal motor nucleus of the vagus (DMV), nucleus of solitary tract (NTS), area postrema (AP) and nucleus ambiguous (NA)] and the anterior hypothalamus [paraventricular nucleus (PVN) and supraoptic nucleus (SON)]. These results indicate that the neuronal hyperactivity of the DMV, NTS, AP, NA, PVN and SON may play an important role in the gastric erosions induced by RWIS. Pretreatment with bilateral subdiaphragmatic vagotomy or atropine inhibited gastric hypercontractility, hypersecretion of gastric acid and prominently alleviated gastric erosions under RWIS, while pretreatment with hypophysectomy, adrenalectomy or phenoxybenzamine failed to affect gastric hypercontractility, hypersecretion of gastric acid and gastric erosions under RWIS, which innervates the stomach, and the abnormalities of gastric functions induced by RWIS are not due to the hyperactivity of the hypothalamo-pituitary-adrenal (HPA) axis, but due to the hyperactivity of vagal parasympathetic efferents, which largely originating in the DMV and partly in the NA. It seems that the hyperactivity of the DMV and NA leads to gastric lesions,but, electrical and chemical stimulations of the DMV and NA inhibited gastric motility. So, whether the hyperactivity of the higher centre, mainly the PVN and SON, relieves the inhibition of gastric motility mediated by the primary nerve centre? If so:1. Would the brainstem circuits regulating gastric function change during RWIS? What are the phenotype natures of activated neurons during RWIS?2. The PVN and SON are the main nuclei of the anterior hypothalamus, which might be stimulus-dependent. Vasopressin (AVP) and oxytocin (OT) are two structurally related nonapeptides synthesized mainly in the magnocellular neurons of the PVN and SON, and may act as neurotransmitters and/or neuromodulators which are considered to be related to gastric functions and the regulation of stress response. Whether the activated neurons in the PVN and SON of rats induced by RWIS were vasopressinergic and oxytocinergic neurons, and whether the activated neurons would display certain typical topographic features?3. Supposed that the activated neurons in the anterior hypothalamus not only projected to the candal part of pituitary but also to the medulla oblongata gastric nerve centre, and then may act as neurotransmitters to innervate the activity of the primary nerve centres of regulating gastric functions. If so, whether the phenotypic natures of activated neurons in the medullary visceral zone were AVP sensitive or OT sensitive neurons, where AVP or OT receptors located?4. Synapses are connections between neurons; biological signals are transferred from the presynaptic membrane by the synaptic transmission to the postsynaptic membrane. Synaptophysin (SYN or p38) and SynapsinI are the specific component of presynaptic membrane and the small synaptic vesicles (SSVs). The proteins were clearly found in vesicles in all efferent terminals, in both medial and lateral efferent systems, where they are believed to be involved in synaptic vesicle formation and exocytosis, the exocytosis of stored neurotransmitter. So, SYN and SynapsinI are considered to be the sensitive neuritic markers for synaptic degeneration and regenerative response.Thus, in the present study:(1) Male Wistar rats were randomly divided into 2 groups designated according to their duration of RWIS, respectively 0h and 1h. The rats stressed for 0h were considered as a control group. Additionally, to evaluate the role of catecholaminergic neurons, cholinergic neuron, oxytocinergic neurons ( or OT sensitive neurons) and vasopressinergic (or AVP sensitive neurons) in the anterior hypothalamus and the medulla oblongata gastric nerve centre during RWIS, the phenotype nature of activated neurons was determined by a double immunohistochemical method for collocations of Fos with one of Tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), Vasopressin (AVP), oxytocin (OT) and oxytocin receptor (OTR), V1-type AVP receptors (V1bR). (2) Male Wistar rats were randomly divided into 4 groups designated according to their duration of RWIS, respectively 0h, 1h, 2h and 4h. The rats stressed for 0h were considered as a control group. Additionally, in order to detect whether synaptic plasticity changed, immunohistochemistry staining technique and Western blotting technique were used to determine the expression and amounts of SYN and SynapsinI in the anterior hypothalamus and the medulla oblongata gastric nerve centre during RWIS.The present data demonstrate that:1. The hyperactivity of cholinergic neurons in the DMV and NA but the NTS might be one of the primary central mechanisms of the gastric dysfunctions induced by the RWIS. ChAT-IR neurons were observed evidently from the rostral to the caudal portions of the DVC either in the RWIS rats or the unstressed rats. In the DMV and NA, RWIS for 1h induced a robust decrease in ChAT-IR neurons. In the RWIS 1h rats, the number of Fos+ChAT-IR neurons was evidently increased compared to the unstressed rats, 3.60-fold in the rostral DMV, 16.00-fold in the intermedial DMV, 4.00-fold in the caudal DMV. Meanwhile, the percentage of Fos+ChAT-IR neurons in ChAT-IR neurons was 3% and 21% in the unstressed and RWIS 1h rats, respectively (P<0.01). In the NA, the number of Fos+ChAT-IR neurons was evidently increased 3.29-fold compared to the unstressed rats, and the percentage of Fos+ChAT-IR neurons in ChAT-IR neurons was 36% (i.e. 4.78-fold VS the unsressed rats) (P<0.01). But, in the NTS, the number of ChAT-IR neurons and Fos+ChAT-IR neurons was hardly increased compared to the unstressed rats, and the percentage of Fos+ChAT-IR neurons in ChAT-IR neurons between the RWIS 1h and unstress rats (P>0.05). These data strongly suggest that ChAT-neurons in the DMV and NA, but not the NTS, are related to stress-responsive signal transduction.2. Catecholaminergic neurons in the medulla oblongata gastric nerve centre may be related to the stress-responsive signal transduction, while those in the anterior hypothalamus were not but activated by Catecholaminergic neurons.In the brainstem, the marked activation of catecholaminergic neurons induced by RWIS encompasses mainly the NTS (A2/C2), VLM (A1/C1) cell groups and AP along with a few stained cells were found in the intermediate and the rostral DMV, while no TH-IR neurons were observed, although the CA-nerve ending were relatively abundant in the NA. In RWIS rats and unstressed rats, the percentage of Fos+TH-IR neurons in TH-IR neurons was respectively 38.0% and 14.3% in the DMV (i.e. 2.7-fold), 34.4% and 9.7% in the NTS (i.e. 3.6-fold), 18.6% and 4.5% in the AP (i.e. 4.1-fold), 45.7% and 18.9% in the VLM (i.e. 2.4-fold) (all the P<0.01). These data strongly suggest that CA-neurons in the medullary visceral zone are related to stress-responsive signal transduction.In the anterior hypothalamus, TH-labeled immunoreactive perikarya represented the fairly lowest population of cells in the PVN, and in the SON the presence of TH-immunoreactive perikarya was completely absent either in the control rats or the rats stimulated with RWIS, which indicate that the CA-neurons in the PVN and SON may be not related to the stress-responsive signal transduction, but the neurons may be activated by CA-neurons.3. The hypothalamic oxytocinergic and vasopressinergic neurons were involved in the regulation of a variety of central neural functions, but not the main type neurons activated by RWIS.OT-IR neurons were mostly located in the magnocellular portion of the PVN (including PaMM and PaLM), while mainly in dorsal part of the SON; AVP-IR neurons mainly located in the magnocellular portion of the medial part of the PVN (PaMM), while mainly in dorsal part of the SON. RWIS for 1h results in the activations of a large population of OT- and AVP- containing neurons, 31% and 40% in the PVN, 28% and 53% in the SON, respectively. These data indicate that the hypothalamic oxytocinergic and vasopressinergic neurons may be involved in the regulation of a variety of central neural functions, and more vasopressinergic neurons than the oxytocinergic ons were activated during RWIS.4. The phenotypic natures of activated neurons in the medullary visceral zone were AVP sensitive or OT sensitive neurons, where OTR or V1bR located in the medulla oblongata gastric nerve centre, which indicate that OT and AVP might mediate the activity of these neurons by OTR and V1bR.OTR-IR and V1bR-IR neurons were observed evidently from the rostral to the caudal portions of the DVC either in the RWIS rats or the unstressed rats. In the present study, more than 10% of OTR-IR and V1bR-IR neurons in the DMV were activated in the RWIS rats while less than 3% in the nonstressed rats. In the NTS, the percentages of Fos-IR neurons in the OTR-IR and V1bR-IR neurons were 10% and 8%, while 5% and 4% in the nonstressed rats, respectively. The significant difference of the ratio between the RWIS and nonstressed groups indicates that the OT-sensitive and AVP-sensitive neurons may be involved in the modulation of the gastric dysfunction during the RWIS. In addition, in RWIS 1h rats, the percentage of Fos+OTR-IR and Fos+V1bR-IR neurons in Fos-IR nuclei was more than 58% and 72% in the DMV, 35% and 52% in the NTS, which indicated that OT and AVP mediate the activity of these neurons mainly by OTR and V1bR.5. Amounts and distributions of SYN did not significantly differ in the anterior hypothalamus but the medulla oblongata with different durations of RWIS; and amountsand distribution of SynapsinI in the anterior hypothalamus and the medulla oblongata gastric nerve centre were changedDuring RWIS 0h to 4h, the SYN and SynapsinI immunoreactivity and amounts were examined using immunohistochemistry and Wstern blotting. Amounts and distributions of SYN did not significantly differ in the anterior hypothalamus but the medulla oblongata with different durations of RWIS (P<0.05). Amountsand distribution of SynapsinI in the anterior hypothalamus and the medulla oblongata gastric nerve centre were changed (P<0.05). These data indicate that RWIS effect the distribution and contents of SYN and SynapsinI in the medulla oblongata and/or the anterior hypothalamus. Since changes of SYN and SynapsinI correlated with brain dysfunction, the synaptic plasticity was changed in the anterior hypothalamus and the medulla oblongata gastric nerve centre in rats exposed to RWIS.In conclusion, RWIS for 1h results in the activations of a large population of ChAT-IR neurons in the DMV and NA, which indicates that the hyperactivity of cholinergic neurons in the DMV and NA leads to gastric lesions. OT- and AVP-containing neurons in the PVN and SON, which may be activated by Catecholaminergic neurons, would project to the NTS or DMV, mediated by OTR and V1bR, and then the DMV in turn provide the preganglionic efferent fibers to regulate gastric information. RWIS effects the distribution and contents of SynapsinI but SYN in the anterior hypothalamus, while in the medulla oblongata gastric nerve centre, the distribution and contents of SYN and SynapsinI changed during the RWIS. These data indicate that the synaptic plasticities were changed in the anterior hypothalamus and the medulla oblongata gastric nerve centre in rats exposed to RWIS.