Cellular localization of angiotensin type 1 receptor and angiotensinogen mRNAs in the subfornical organ of the rat brain

The cellular localization of angiotensin type 1 receptor (AT 1) and angiotensinogen mRNA expression in the subfornical organ (SFO) of the rat brain has been studied by means of non-radioactive in situ hybridization combined with immunocytochemistry for glial fibrillary acidic protein (GFAP) and Neutral red staining. The AT 1 receptor mRNA expression is shown to be within putative nerve cells without any association with the glial fibrillary acidic protein (GFAP)-immunoreactive (IR) cells. In contrast the angiotensinogen cRNA expression is associated predominantly with GFAP-IR cells. The results demonstrate that a neuronal AT 1 receptor mediates the actions of circulating angiotensin II on the SFO and that the angiotensinogen mRNA is predominantly expressed in the SFO astroglial cells.     

Cell subpopulations of nicotine-sensitive subfornical organ neurons in rat

The subfornical organ (SFO), which is related to drinking and cardiovascular regulation, is activated by central application of nicotine (NIC) and angiotensin II (ANG). However, NIC-induced drinking is much smaller than ANG-induced one although approximately 60% of SFO neurons are affected by both NIC and ANG. Therefore, some specific subpopulations of SFO neurons for NIC or ANG may be related to such different drinking responses. To clarify subpopulations of NIC-sensitive neurons, electrophysiological properties of SFO neurons with the application of NIC was investigated at whole-cell patch-clamp recordings. Based on our developed electrophysiological criteria of the recovery kinetics of tetraethylammonium-resistant transient outward K(+) currents, two sub-types (F- and S-type neurons) were distinguished. Twenty-nine dissociated SFO neurons were examined to determine whether they showed NIC-induced inward currents. Most F-type neurons (n=19/21) showed NIC sensitivity, but most S-type neurons (n=7/8) did not. Our previous study had demonstrated that half of the F-type and all of the S-type units showed ANG sensitivity. These suggests that almost all of the NIC-sensitive SFO neurons were electrophysiologically classified as the F-type, but not S-type, and this differs in part from angiotensin sensitivity. The different subpopulations for chemical sensitivities in the SFO may be related to different drinking responses.     

c-Fos protein expression in the rat subfornical organ following osmotic stimulation.

To examine the role of the subfornical organ (SFO) in the osmotic activation of hypothalamic neurons, the responses of the SFO to osmotic stimulation were evaluated by using c-Fos protein immunohistochemistry. Numerous c-Fos-immunoreactive nuclei were found in the SFO of rats injected i.p. with hypertonic saline solution as early as 30 min after stimulation, and the effect lasted up to 3 h. Only a few c-Fos-positive cells were detected in the SFO of rats injected with isotonic saline. However, electrolytic lesions of the SFO did not prevent the osmotic activation of the hypothalamic paraventricular and supraoptic nuclei. These data suggest that the SFO and the hypothalamic magnocellular nuclei are simultaneously but separately activated by osmotic stress.     

糖尿病大鼠穹窿下器室管膜细胞的形态学变化

目的:观察糖尿病大鼠不同时期穹窿下器的室管膜细胞的形态学变化.方法:雄性Wistar大鼠,每只大鼠给予链脲佐菌素60 mg/kg,腹腔一次性注射,建立Ⅰ型糖尿病大鼠模型.造模成功后分别于2、4及8周,扫描电镜和透射电镜观察穹窿下器不同时期室管膜细胞的形态学变化.结果:扫描电镜观察,对照组细胞膨隆,表面光滑,有清晰的微绒毛.2周组细胞扁平、塌陷,细胞表面可见到圆形小破孔.4周组室管膜细胞表面凹凸不平,出现皱褶,细胞膜可见大小不一的多个小破孔,并可见细胞膜缺失、细胞器裸露的细胞.8周组室管膜细胞表面皱褶明显,可见大量的球形分泌颗粒.透射电镜观察显示,对照组穹窿下器室管膜细胞脑室面完整,有少量的微绒毛和乳头状微突起,模型组室管膜面可见大量分泌颗粒.结论:糖尿病可导致大鼠穹窿下器室管膜细胞破损及分泌大量的分泌颗粒.     

SOX2/Nestin阳性细胞在糖尿病大鼠穹窿下器中的表达

目的:观察神经干细胞特异性标记物性别决定基因高迁移率组蛋白(SOX2)、巢蛋白(nestin)在不同时期糖尿病大鼠穹窿下器(SFO)中的表达情况并探讨其意义。方法:雄性Wistar大鼠给予链脲佐菌素(STZ,60mg/kg,腹腔一次性注射)建立Ⅰ型糖尿病大鼠模型。用免疫组织化学染色方法观察SOX2、Nestin阳性表达细胞在正常大鼠及2、4、8、12周糖尿病大鼠SFO的表达情况。结果:模型组各个时间点的SOX2和Nestin的阳性表达细胞数量及平均光密度均高于正常对照组,且均在第4周表达达到高峰,而后逐渐降低,差异有统计学意义(P0.05)。结论:在不同病程糖尿病大鼠模型中,SOX2及Nestin在SFO的阳性表达一过性增强,表明糖尿病时可能会短暂性提高SFO神经干细胞的增殖功能。     

Activation of metabotropic glutamate receptors inhibits GABAergic transmission in the rat subfornical organ

Glutamate is known to increase neuronal excitability in the subfornical organ, a circumventricular organ devoid of the blood-brain barrier. To understand the synaptic mechanism of neuronal excitation by glutamate in this nucleus, we examined the effects of glutamate on GABAergic spontaneous inhibitory postsynaptic currents recorded from subfornical organ neurons in the rat brain slice. The baseline frequency, amplitude and decay time-constant of such spontaneous synaptic currents were 5.60 Hz, 119 pA and 17.3 ms, respectively. Glutamate (10-1000 microM) selectively inhibited the frequency of spontaneous GABAergic inhibitory postsynaptic currents (half-maximal effective concentration=47 microM) with little effects on their amplitudes and decay time constants. The inhibitory effect of glutamate on the frequency of spontaneous GABAergic postsynaptic currents was not blocked by tetrodotoxin (1 microM), or by the antagonists of ionotropic glutamate receptors. In contrast, such inhibitory effect of glutamate was mimicked by general or group II selective metabotropic glutamate receptor agonists such as DCGIV (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (half-maximal effective concentration=112 nM), but not by the agonists for group I or group III metabotropic glutamate receptors. Under current clamp mode, glutamate reduced the frequencies of spontaneous inhibitory postsynaptic potentials and action potentials in subfornical organ neurons. Our data indicate that glutamate decreases the frequency of spontaneous inhibitory postsynaptic currents by acting on the group II metabotropic glutamate receptors on axonal terminals in the subfornical organ. From these results we suggest that the glutamate-induced modulation of tonic GABAergic inhibitory synaptic activity can influence the excitability of subfornical organ neurons.     

Region-specific projections from the subfornical organ to the paraventricular hypothalamic nucleus in the rat

Neurons in the subfornical organ (SFO) project to the paraventricular hypothalamic nucleus (PVN) and there, in response to osmolar and blood pressure changes, regulate vasopressin neurons in the magnocellular part (mPVN) or neurons in the parvocellular part (pPVN) projecting to the cardiovascular center. The SFO is functionally classified in two parts, the dorsolateral peripheral (pSFO) and ventromedial core parts (cSFO). We investigated the possibility that neurons in each part of the SFO project region-specifically to each part of the PVN, using anterograde and retrograde tracing methods. Following injection of an anterograde tracer, biotinylated dextran amine (BDX) in the SFO, the respective numbers of BDX-uptake neurons in the pSFO and cSFO were counted and the ratio of the former to the latter was obtained. In addition, the respective areas occupied by BDX-labeled axons per unit area of the mPVN and pPVN were measured and the ratio of the former to the latter was obtained. Similarly, following injection of the retrograde tracer in the PVN, the respective areas occupied by tracer per unit area of the mPVN and pPVN were measured and the ratio of the former to the latter was obtained. The respective numbers of retrogradely labeled neurons in the pSFO and cSFO were also counted and the ratio of the former to the latter was obtained. It became clear by statistical analyses that there are strong positive correlations between the ratio of BDX-uptake neuron number in the SFO and the ratio of BDX-axon area in the PVN in anterograde experiment (correlation coefficient: 0.787) and between the ratio of retrograde neuron number in the SFO and the ratio of tracer area in the PVN in retrograde experiment (correlation coefficient: 0.929). The result suggests that the SFO projects region-specifically to the PVN, the pSFO to the mPVN and the cSFO to the pPVN.     

The subfornical organ and the integration of multiple factors in thirst

Rats with lesions centered on the ventral stalk of the subfornical organ (SFO) were used to characterize the participation of this structure in the control of drinking. It is concluded that the SFO does indeed play some minor role in the mediation of drinking following intraventricular injections of angiotensin. Further, it is shown that lesions of the SFO, but not lesions of the adjacent septal-hippocampal tissue, attenuate osmotic thirst elicited by two doses of hypertonic saline. Diminished drinking responses following water deprivation, and normal feeding responses following food deprivation, underscore the importance of the SFO for drinking behavior in general, and an expanded role for the SFO in fluid regulation is suggested. However, some incidental observations suggest that the SFO is less than an equal partner with structures in the AV3V region in the overall control of water balance.     

Atrial natriuretic peptide in the subfornical organ reduces drinking induced by angiotensin or in response to water deprivation

Three experiments tested whether the subfornical organ (SFO) could be a site of action for the antidipsogenic effects of atrial natriuretic peptide (ANP) in rats. Pretreatment with 100, 230, or 500 pmol ANP in the SFO reduced drinking induced by 10 pmol angiotensin II in the SFO. Drinking in response to water deprivation was reduced by ANP in rats having cannulas in or near the SFO, but not in rats having cannulas distant from the SFO or in the ventricles. Finally, ANP had no effect on eating or drinking after food deprivation, suggesting that the rats in the other experiments were not acutely incapacitated. The SFO may mediate the central effects of ANP on drinking induced by angiotensin or in response to water deprivation and could play a similar role in the central effects of ANP on salt appetite, diuresis, vasopressin secretion, and blood pressure.     

Angiotensin-converting enzyme is present in the subfornical organ and other circumventricular organs of the rat

Angiotensin-converting enzyme (ACE, kininase II, EC 3.4.15.1) activity was measured by a radiochemical assay in isolated circumventricular organs of the rat. ACE activity was unevenly distributed, with a 100-fold difference between the lowest (subcommisural) and the highest (subfornical organ) activities. Our results suggest that angiotensin II could be locally formed in circumventricular organs and especially in the subfornical organ. The high angiotensin-converting enzyme activity in the subfornical organ could indicate a physiological role of endogenous angiotensin II in this structure.     

Morphological plasticity of cultured astrocytes derived from the subfornical organ of the adult rat

The morphological plasticity of astrocytes from the subfornical organ of the adult rat has been examined using an explant culture preparation. Astrocytes migrate out of the explant and form a monolayer of amorphous, non-stellate cells. This non-stellate form was maintained when cultures were incubated in a HEPES buffered salt solution (HBSS) for 50 minutes. The fraction of cells that was stellate in these cultures was significantly increased when cultures were incubated in HBSS supplemented with forskolin (5 M; but not 1,9-dideoxyforskolin) or with nitroprusside (10–100M)indicating that elevation of intracellular cAMP or cGMP mediates stellation. The stellation responses induced by forskolin and by nitroprusside were blocked by inclusion of serum (0.5%) or of LY83,583 (10M), an inhibitor of soluble guanylate cyclase, in the incubation medium. The relevance of the data to neuroglial plasticity in the subfornical organ in vivo is discussed.     

Diversity of the muscarinic and nicotinic responses of subfornical organ neurons in rat slice preparations

Recently we found that subfornical organ (SFO) neurons were activated through nicotinic receptors as well as muscarinc. In this study, the preferential responses of single SFO neurons to both muscarine and nicotine were examined by using rat slice preparations, and current and voltage clamp recordings. When the amplitudes of the depolarizations and inward currents by muscarine and nicotine in single SFO neurons were compared, some neurons showed a higher sensitivity to muscarine than to nicotine while others showed vice versa. These data indicate that acetylcholine activates SFO neurons preferentially through either muscarinic or nicotinic receptors and suggest a diversity of cholinergic responses in the SFO.     

Intrinsic osmosensitivity of subfornical organ neurons

The constancy of plasma osmolality demands that salt and water concentration within the extracellular fluid be constantly monitored and regulated within a few percentage points. The circumventricular organs in general, and the subfornical organ in particular, have long been proposed to be the site of the osmosensitivity. Isolated subfornical organ neurons of male rats were studied using the whole-cell patch-clamp technique and both action potential frequency and whole cell currents were measured as bath osmolality was changed, from 240 to 330mOsm, by altering the amount of mannitol and maintaining the concentrations of electrolytes constant. Out of 64 cells, 66% responded to changes in bath osmolality in a predictable manner, exhibiting a hyperpolarization and decrease in spike frequency in hypo-osmotic solutions and a depolarization and increase in action potential frequency during hyperosmotic exposure. Cells (34%) defined as non-responders exhibited no significant modulation during identical changes in extracellular osmolality. The responses to changing extracellular osmolality were dose dependent; the activity of subfornical organ neurons was significantly modulated by changes in extracellular osmolality of less than 10mOsm. By regression analysis, this osmosensitivity was approximately 0. 1Hz/mOsm change throughout a +/-10mOsm range and was maintained throughout the range of osmolalities studied (270-330mOsm). The mechanism underlying this osmosensitivity remains unclear, although the non-selective cation conductance and the volume-activated chloride conductance do not seem to be involved.This intrinsic osmosensitivity of subfornical organ within the normal physiological range supports the view that this circumventricular structure plays a role in normal osmoregulation.     

Involvement of the septum in the regulation of paraventricular vasopressin neurons by the subfornical organ in the rat

Extracellular recordings were obtained from 58 phasically active neurosecretory neurons in the hypothalamic paraventricular nucleus (PVN) of urethane-anesthetized male rats. Of these PVN neurons, 39 exhibited an increase and 11 displayed a reduction in ongoing activity following electrical stimulation of the subfornical organ (SFO), while the remaining neurons were unresponsive. Microinjection of the local anesthetic lidocaine into the medial septum reversibly abolished the SFO stimulus-evoked reduction in 7 out of 9 PVN neurons tested, whereas similar injection was without effect on the stimulus-evoked increase in 18 out of 20 PVN neurons tested. These results suggest that the SFO efferents through the medial septum to the PVN exert a predominantly inhibitory influence on the excitability of putative vasopressin (VP)-secreting neurons in the PVN.     

Neurons in the nucleus of the solitary tract with ascending projections to the subfornical organ in the rat

Thirty-one neurons in the region of the nucleus of the solitary tract (NTS) were antidromically activated by electrical stimulation of the subfornical organ (SFO) in male rats under urethane anesthesia. The activity of these identified neurons was tested for a response to activation of peripheral baroreceptors, achieved by rising arterial blood pressure with an intravenous administration of the alpha-agonist metaraminol. Of the neurons tested, 17 displayed an increase and 6 exhibited a reduction in neuronal firing that accompanied a 40- to 60-mmHg elevation in mean arterial pressure, while 8 were unresponsive. The results suggest that neurons projecting to the SFO in the region of the NTS may be important for carrying peripheral baroreceptor information to the SFO.