GABA And ENK Have Effect On Gustatory Input In The Rostral Portion Of The Nucleus Of The Solitary Tract

The rostral portion of the nucleus of the solitary tract (rNST) is closely related to process and regulate effects on gustatory input. rNST receives visceral afferent fibers consisting of the primary afferent fibers from the facial, glossopharyngeal and vagus nerve, and gustatory information from NST is further converyed to the parabrachial nuclei,the hypothalamus, the thalamus, central nucleus of the amygdale and insular cortex.The NST is rich in neuroaction substance. Some experiments have demonstrated the excitatory influence of glutamate, SP and the inhibitory effect of GABA, ENK on cells in gustatory region of the NST.The NST is a region known to express GABA. Previous studies have shown an extensive network of ENK-immunoreactive fibers and terminal within the gustatory zone of NST,μ-opoid receptors andδ- opoid receptors have also shown intense expression in NST.Malanga proposed opoids inhibit GABA-mediated synaptic transmission .But Echo confirmed there is synergism between opoids and GABA. Both the modulatory effects of opioid peptides on GABA-induced inhibition and the studies that GABA-immunopositive nearons also exhibited MOR in NST are still deficient of morphological eviedence.According to these hints, the present study was principally designed to observe the change of food intake following administration of GABA and ENK into rNST rigion by stereotaxic apparatus and investigate the connection among ENK-ir terminals, GABA-ir neurons and MOR-ir nearons in NST by using immunoflouorescence histochemical double-staining and immuno-electronmicroscrope double- staining technique. Part 1 Alterations in food intake elicited by GABA and ENK administered into the rostral portion of the nucleus tractus solitarious of ratsObjectiveTo observed the change of food intake following administration of GABA and ENK into rNST rigion by stereotaxic apparatus.Methods1. Food intake measurements: 22 adults Sprague-Dawley rats weighing 200-300g were divided into 3 groups: There were 10 in GABA group, 10 in ENK group and 2 in control group. The rats were individually housed in wire-mesh cages and maintained on a 12-hour light/ 12- hour dark cycle with Durine rat chow and water available ad libitum.All animals were allowed at least 5 days to accommodate the environment, and calculate food intake.2. SurgeryEach rat was pretreated with 2% pentobarbital sodium (45mg/kg.ip). The animal was mounted in a stereotaxic instrument with the use of a non-traumatic head holder. Bilateral stainless steel guide was aimed stereotaxically at the rNST region using the following coordinates: 12.80mm posteriorto the bregma suture, 1.2 mm lateral to either side of the sagittal suture and 6.0 mm from the top of the skull. Each rat was treated with Penicillin (20u/kg.i.m.) for 4 days. All animals were allowed at least 1 week to recover from stereotaxic surgery before behavioral testing began.GABA and ENK were dissolved in buffered physiological saline microinjections were administered bilaterally into the NST in 0.2 ul volumes over 30s. Physiological saline was initially used as a control injection for these experiments.3. Food intake measurements after surgeryCumulative intakes were assessed 4, 8 and 12h following the microinjection.4. HistologyAt the end of each experiment,males were deeply anesthetized with sodium pentobarbital (80mg/kg.i.p.) and perfused transcardially with 4% paraformadehyde in 0.1 M sodium phosphate-buffered saline (PBS,PH7.4).the brainstem were separated from the whole brain, post-fixed in the same fresh fixative for 1-2 days. The lower brainstem was cut into 20um thick frontal sections with a freezing microtome, and the sections were used in HE staining.Result1. Histological examination showed that the cannulae placements of all animals participating in the protocols were found to be within the region of the rNST.2. One rat of group GABA appeared postoperative infection, and was disused. GABA significantly decreased food intake.3. Rats of group ENK significantly decreased food intake.ConclusionFood intake was significantly decreased by local microinjection of GABA and ENK, because taste-responsive neurons of the NST were inhibited by GABA and ENK.Part 2 A morphological studies of conection between ENK immunoreactive terminals and GABA immunoreactive neurons in the rostral portion of the nucleus tractus solitarious of the ratObjectiveTo observe the connections between enkephalin immunoreactive (ENK-ir) terminals andγ-aminobutyric acid immunoreactive (GABA-ir) neurons in the rostral portion of the nucleus tractus solitarious (rNTS) of the rat.Methods1. Materials and methods:10 adult Sprague-Dawley rats weighing 200-300g were used. All rats were performed under anesthesia by intraperitoned injection of sodium pentobarbital (80mg/kg,i.p.), and perfused transcardially with 500ml of 0.1 M phosphate butter (PB, PH 7.4) containing 4% (W/V) paraformaldehyde and 75%(V/V)–saturated picric acid. After the perfueion, the brains were removed, post-fixed in the same fresh fixative and placed in 0.1 M PB containing 30%(W/V) sucrose overnight at 4℃. The lower brainstems were cut into 20um thick frontal sections with a freezing microtome and cut into 50um thick frontal sections with a microslicer.2. Double-immunofluorescent labelingThe sections were used for immunofluorescence histochemical double-staining for ENK/GABA: Mouse anti ENK (1:500) and rabbit anti GABA (1:5000) in PBS over night; biotinylated sheep anti-moouse IgG (1:200) in PBS for 8 hours; a mixture of Texas Red-labled avidin and Fluorescein- labled donkey anti-ribbit IgG in PBS for 6 hours; the sections were mounted onto clean glass slides, air-dried and cover slipped with PBS containing anti-fading reagent. All sections were observed with a confocal laser-scaning microscope.3. Electron microscopic immunohistochemical double-stainingSections were placed in 0.05 M PB containing 25% sucrose and 10% glycerol for 30 mintues for cryoprotection,followed by freeze-thawing with liquid nitrogen.Subsequently, the sections were incubated with 0.05M Tris-Hcl buffered-saline containing 30% normal goat serum for 30 mintues, and then processed for immunohistochemical double-staining, the immunogold-silver method for GABA was combined with the ABC method for ENK. The sections were incubation with first anti bodies for 24 hours.The sections incubated over night at room temperature with a mixture of coat anti-rabbit IgG conjugated to gold particle and biotinylated sheep anti-mouse IgG.The sections were postfixation with glutaraldehyde, silver enhancement with Silver kit, incubation with ABC Elite Kit, visualization of GABA and ENK-immunoreactivity with DAB, osmification ,counterstaining with uranyl acetate, flat-embeddingin Epon812 after dehydration and mounting onto silicon-coated glass slides.Then The sections were prepared for electron microscopy.Results1. Under the laser scanning confocal microscope1.1 There were dense ENK-ir fibers and terminals in the rNTS, they presented red.1.2 Some GABA-ir neurons were observed in the rNTS, they presented green.1.3 Close connections between ENK-ir terminals and GABA-ir cell bodies were also detected.2. By using the electron microscopic technique2.1 ENK-ir reaction products were mainly localized on the surface of round clear vesicles and dense-cored vesicles in the axon terminals.2.2 GABA-ir reaction products were mainly localized on the surface of rough endoplasmic reticulum and ribosome in dendrites.2.3 Synaptic connections between DAB staining ENK- immunoposi- tive terminals and nanogold labeling GABA- immunopositive or immunonegative dendrites or terminal in the rNTS:2.3.1 Symmetric (62%) and asymmetric synaptic (38%) connections were observed between ENK-immunopositive terminals and GABA-immunopositive cell bodies (26%) and dendrites (74%).2.3.2 Symmetric (73%) and asymmetric synaptic (27%) connections were observed between ENK-immunopositive terminals and GABA -imunonegative cell bodies (18%) and dendrites (82%).2.3.3 Slight symmetric synaptic connections were observed between ENK-ir terminals and GABA-imunonegative terminals, ENK-immunopositive terminals were presynaptic or postsynaptic components.ConclusionThe ENK-ir terminals might take part in the transmission and regulation of the taste information in the rNTS through inhibiting, exciting the GABAergic neurons or inhibit directly the activity of neurons within the rNTS.Part 3 An experimental study on GABA / MOR co-existed neurons and the connections between ENK immunoreactive terminals and MOR immunoreactive neurons in the rostral portion of the nucleus tractus solitarious of the ratObjectiveTo observe whetherγ-aminobutyric aicd (GABA) andμopioid receptor (MOR) co-exist in neurons of the rostral segment of the nucleus tractus solitarious (rNTS) in the rat, and the connections between Enkephalin immunoreactive (ENK-ir) terminals and MOR immunoreactive (MOR-ir) neurons.Methods1. Materials and methodsAll rats were performed under anesthesia by intraperitoned injection of sodium pentobarbital (80mg/kg, i.p.), and transcardially perfused.It was same with the above-mentioned method.2. Double-immunofluorescent labelingThe sections were used for immunofluorescence histochemical double-staining for MOR/GABA: It was same with the above-mentioned method. Guinea pig anti MOR (1:500) and rabbit anti GABA (1:5000) replace virgin first anti bodies. Cy3-labled donkey anti- guinea pig IgG(1:500) And Fluorescein- labled sheep anti-ribbit IgG(1:500) replace virgin second anti bodies.The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: It was same with the above-mentioned method. Mouse anti ENK (1:500) and Guinea pig anti MOR (1:500) replaced virgin first anti bodies. Biotinylated sheep anti-mouse IgG (1:200), Texas Red-labled avidin (1:200), Fluorescein-labled donkey anti-guinea pig IgG (1:500) replaced virgin second anti bodies.3. Electron microscopic immunohistochemical double-stainingThe sections were used for Electron microscopic immunohisto-chemical double-stainingfor MOR/GABA: It was same with the above-mentioned method. Guinea pig anti MOR (1:500) and rabbit anti GABA (1:5000) replace virgin first anti bodies. Biotinylated sheep anti-rabbit IgG and gold particle-labled coat anti-guinea pig IgG replaced virgin second anti bodies.The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: It was same with the above-mentioned method. Mouse anti ENK (1:500) and Guinea pig anti MOR (1:500) replaced virgin first anti bodies. Biotinylated sheep anti-mouse IgG and gold particle-labled coat anti-guinea pig IgG eplaced virgin second anti bodies.Results1. Under laser scanning confocal microscope1.1 The sections were used for immunofluorescence histochemical double-staining for MOR/GABA: Some GABA-ir neurons (red) and MOR-ir neurons (green) were observed in the rNTS; GABA/MOR co-existed neurons were also observed, and they were small-sized neurons.1.2 The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: There were dense ENK-ir fibers and terminals in the rNTS, they presented red; Close connections between ENK-ir terminals and MOR-ir cell bodies or dendrites were also detected.2. By using the electron microscopic technique2.1 The sections were used for Electron microscopic immunohisto- chemical double-stainingfor MOR/GABA : GABA and MOR co-exist in cell body and dendrites, GABA-ir reaction products were mainly localized on the surface of rough endoplasmic reticulum and ribosome in dendrites, MOR-ir reaction products were mainly localized on the membrane of dendrites and mitochondria, and GABA and MOR co-existed neurons formed symmetric synaptic(mainly) and asymmetric synaptic connections with the imunonegative terminals. 2.2 The sections were used for immunofluorescence histochemical double-staining for MOR/ENK: ENK-ir reaction products were mainly localized on the surface of round clear vesicles and dense-cored vesicles in the axon terminals; ENK-ir terminals formed symmetric synaptic (mainly) and asymmetric synaptic connections with MOR-ir cell bodies and dendrites; Slight MOR-ir reaction products were localized in the axon terminal, ENK-ir and MOR-ir reaction products co-existed,and formed symmetric synaptic connections with the imunonegative dendrite.ConclusionThese results indicate that ENK might have regulatory effects on GABAergic neurons in rNTS by bonding with MOR.