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BackgroundIncreased secretion of oxytocin and arginine vasopressin (AVP) from hypothalamic magnocellular neurosecretory cells (MNCs) is a key physiological response to lactation. In the current study, we sought to test the hypothesis that the GABAA receptor-mediated inhibition of MNCs is altered in lactating rats.ResultsGramicidin-perforated recordings in the rat supraoptic nucleus (SON) slices revealed that the reversal potential of GABAA receptor-mediated response (EGABA) of MNCs was significantly depolarized in the lactating rats as compared to virgin animals. The depolarizing EGABA shift was much larger in rats in third, than first, lactation such that GABA exerted an excitatory, instead of inhibitory, effect in most of the MNCs of these multiparous rats. Immunohistochemical analyses confirmed that GABAergic excitation was found in both AVP and oxytocin neurons within the MNC population. Pharmacological experiments indicated that the up-regulation of the Cl(-) importer Na(+)-K(+)-2Cl(-) cotransporter isotype 1 and the down-regulation of the Cl(-) extruder K(+)-Cl(-) cotransporter isotype 2 were responsible for the depolarizing shift of EGABA and the resultant emergence of GABAergic excitation in the MNCs of the multiparous rats.ConclusionWe conclude that, in primiparous rats, the GABAergic inhibition of MNCs is weakened during the period of lactation while, in multiparous females, GABA becomes excitatory in a majority of the cells. This reproductive experience-dependent alteration of GABAergic transmission may help to increase the secretion of oxytocin and AVP during the period of lactation.

Abstract Background Neuropathic pain is a chronic and intractable symptom associated with nerve injury. The periaqueductal gray (PAG) is important in the endogenous pain control system and is the main site of the opioidergic analgesia. To investigate whether neuropathic pain affects the endogenous pain control system, we examined the effect of neuropathic pain induced by sacral nerve transection on presynaptic GABA release, the kinetics of postsynaptic GABA-activated Cl- currents, and the modulatory effect of μ-opioid receptor (MOR) activation in mechanically isolated PAG neurons with functioning synaptic boutons. Results In normal rats, MOR activation inhibited the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) to 81.3% of the control without any alteration in their amplitude. In neuropathic rats, the inhibition of mIPSC frequency by MOR activation was 82.4%. The frequency of GABAergic mIPSCs in neuropathic rats was 151.8% of normal rats without any difference in the mIPSC amplitude. Analysis of mIPSC kinetics showed that the fast decay time constant and synaptic charge transfer of mIPSCs in neuropathic rats were 76.0% and 73.2% of normal rats, respectively. Conclusions These results indicate that although the inhibitory effect of MOR activation on presynaptic GABA release is similar in both neuropathic and normal rats, neuropathic pain may inhibit endogenous analgesia in the PAG through an increase in presynaptic GABA release.

Microglia are activated by inflammatory and pathophysiological stimuli in neurodegenerative diseases, and activated microglia induce neuronal damage by releasing cytotoxic factors like nitric oxide (NO). Activated microglia synthesize a significant amount of vitamin D3 in the rat brain, and vitamin D3 has an inhibitory effect on activated microglia. To investigate the possible role of vitamin D3 as a negative regulator of activated microglia, we examined the effect of 25-hydroxyvitamin D3 on NO production of lipopolysaccharide (LPS)-stimulated microglia. Treatment with LPS increased the production of NO in primary cultured and BV2 microglial cells. Treatment with 25-hydroxyvitamin D3 inhibited the generation of NO in LPS-activated primary microglia and BV2 cells. In addition to NO production, expression of 1-α-hydroxylase and the vitamin D receptor (VDR) was also upregulated in LPS-stimulated primary and BV2 microglia. When BV2 cells were transfected with 1-α-hydroxylase siRNA or VDR siRNA, the inhibitory effect of 25-hydroxyvitamin D3 on activated BV2 cells was suppressed. 25-Hydroxyvitamin D3 also inhibited the increased phosphorylation of p38 seen in LPS-activated BV2 cells, and this inhibition was blocked by VDR siRNA. The present study shows that 25-hydroxyvitamin D3 inhibits NO production in LPS-activated microglia through the mediation of LPS-induced 1-α-hydroxylase. This study also shows that the inhibitory effect of 25-hydroxyvitamin D3 on NO production might be exerted by inhibiting LPS-induced phosphorylation of p38 through the mediation of VDR signaling. These results suggest that vitamin D3 might have an important role in the negative regulation of microglial activation.

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA(A) receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E(GABA)) in MNCs. This E(GABA) shift was associated with increased expression of Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA(A) receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl(-) gradients.

Anemarrhena asphodeloides is widely used in traditional Chinese medicine, and is known to have anti-diabetic and diuretic effects. In this study, we evaluated the anti-inflammatory effects of anemarsaponin B (ASB), a steroidal saponin isolated from the rhizomes of A. asphodeloides (Liliaceae), in LPS-stimulated RAW 264.7 macrophage cell line. ASB significantly and dose-dependently decreased the protein and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). ASB also reduced the expressions and productions of pro-inflammatory cytokines, including those of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Electrophoretic mobility shift assay (EMSA) and reporter gene assays revealed that ASB attenuated the LPS-induced DNA binding and transcriptional activity of nuclear factor-kappa B (NF-kappaB). In addition, it was found that pretreatment with ASB significantly inhibited the nuclear translocation of the p65 subunit of NF-kappaB by blocking the phosphorylation of inhibitory kappa B-alpha (IkappaB-alpha). On the other hand, ASB inhibited the phosphorylation of MAP kinase kinases 3/6 (MKK3/6) and mixed lineage kinase 3 (MLK3), which are both involved in the p38 pathway. Taken together, these results suggest that anti-inflammatory effect of ASB in LPS-treated RAW 264.7 macrophages is associated with the inhibition of NF-kappaB transcriptional activity, possibly via the p38 MAP kinase pathway.

The medial preoptic area (MPOA) of the hypothalamus is critically involved in the regulation of male sexual behavior and has been implicated in several homeostatic processes. Serotonin (5-hydroxytryptamine, 5-HT) inhibits sexual behavior via effects in the MPOA, where there are high densities of 5-HT(1A) and 5-HT(1B) receptor subtypes. We used whole-cell recordings under voltage-clamp conditions to investigate the serotonergic modulation of gamma-aminobutyric acid (GABA)ergic and glutamatergic synaptic transmission in mechanically dissociated rat MPOA neurons with native presynaptic nerve endings. Spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the MPOA were completely blocked by bicuculline. Serotonin reversibly reduced the GABAergic mIPSC frequency without affecting the mean current amplitude. Serotonergic inhibition of mIPSC frequency was mimicked by (+/-)-8-hydroxy-2-dipropylaminotetralin hydrobromide, a specific 5-HT(1A) receptor agonist, and blocked by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide, a specific 5-HT(1A) receptor antagonist. 6-Cyano-7-nitroquinoxaline-2,3-dione completely blocked spontaneous glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in the MPOA. Serotonin reversibly decreased the glutamatergic mEPSC frequency without affecting the mean current amplitude. Serotonergic inhibition of mEPSC frequency was mimicked by CGS 12066B, a specific 5-HT(1B) receptor agonist, and blocked by SB 216641, a specific 5-HT(1B) receptor antagonist. Stimulation of adenylyl cyclase with forskolin increased the frequencies of GABAergic mIPSCs and glutamatergic mEPSCs, and blocked the inhibitory effects of 5-HT. H-89, a selective protein kinase A (PKA) inhibitor, decreased the frequencies of GABAergic mIPSCs and glutamatergic mEPSCs, and blocked their reduction by 5-HT. These findings suggest that 5-HT reduces the frequency of GABAergic mIPSCs and glutamatergic mEPSCs through 5-HT(1A) and 5-HT(1B) receptor-mediated inhibition, respectively, of the PKA-dependent pathway in the presynaptic nerve terminals of MPOA neurons.