BDNF gene hydroxymethylation in hippocampus related to neuroinflammation-induced depression-like behaviors in mice.

BACKGROUND: Neuroinflammation is a multifactorial condition related to glial cells and neurons activation, and it is implicated in CNS disorders including depression. BDNF is a crucial molecule that related to the pathology of depression, and it is the target of DNA methylation. DNA hydroxymethylation, an active demethylation process can convert 5-mC to 5-hmC by Tets catalyzation to regulate gene transcription. The regulatory function for BDNF gene in response to neuroinflammation remains poorly understood. METHODS: Neuroinflammation and depressive-like behaviors were induced by lipopolysaccharide (LPS) administration in mice. The microglial activation and cellular 5-hmC localization in the hippocampus were confirmed by immunostaining. The transcripts of Tets and BDNF were examined by qPCR method. The global 5-hmC levels and enrichment of 5-hmC in BDNF gene in the hippocampus were analyzed using dot bolt and hMeDIP-sequencing analysis. RESULTS: LPS administration induced a spectrum of depression-like behaviors (including behavioral despair and anhedonia) and increased expression of Iba-1, a marker for microglia activation, in hippocampus, demonstrating that LPS treatment cloud provide stable model of neuroinflammation with depressive-like behaviors as expected. Our results showed that Tet1, Tet2 and Tet3 mRNA expressions and consequent global 5-hmC levels were significantly decreased in the hippocampus of LPS group compared to saline group. We also demonstrated that 5-hmC fluorescence in the hippocampus located in excitatory neurons identified by CaMK II immunostaining. Furthermore, we demonstrated that the enrichment of 5-hmC in BDNF gene was decreased and corresponding BDNF mRNA was down-regulated in the hippocampus in LPS group compared to saline group. CONCLUSION: Neuroinflammation-triggered aberrant BDNF gene hydroxymethylation in the hippocampus is an important epigenetic element that relates with depression-like behaviors.

Inhibition by O-desmethyltramadol of glutamatergic excitatory transmission in adult rat spinal substantia gelatinosa neurons.

To reveal cellular mechanisms for antinociception produced by clinically used tramadol, we investigated the effect of its metabolite O-desmethyltramadol (M1) on glutamatergic excitatory transmission in spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons. The whole-cell patch-clamp technique was applied at a holding potential of -70 mV to SG neurons of an adult rat spinal cord slice with an attached dorsal root. Under the condition where a postsynaptic action of M1 was inhibited, M1 superfused for 2 min reduced the frequency of spontaneous excitatory postsynaptic current in a manner sensitive to a µ-opioid receptor antagonist CTAP; its amplitude and also a response of SG neurons to bath-applied AMPA were hardly affected. The presynaptic effect of M1 was different from that of noradrenaline or serotonin which was examined in the same neuron. M1 also reduced by almost the same extent the peak amplitudes of monosynaptic primary-afferent Aδ-fiber and C-fiber excitatory postsynaptic currents evoked by stimulating the dorsal root. These actions of M1 persisted for >10 min after its washout. These results indicate that M1 inhibits the quantal release of L-glutamate from nerve terminals by activating µ-opioid but not noradrenaline and serotonin receptors; this inhibition is comparable in extent between monosynaptic primary-afferent Aδ-fiber and C-fiber transmissions. Considering that the SG plays a pivotal role in regulating nociceptive transmission, the present findings could contribute to at least a part of the inhibitory action of tramadol on nociceptive transmission together with its hyperpolarizing effect as reported previously.

Presynaptic facilitation by tetracaine of glutamatergic spontaneous excitatory transmission in the rat spinal substantia gelatinosa - Involvement of TRPA1 channels.

The amide-type local anesthetic (LA) lidocaine activates transient receptor potential (TRP) ankyrin-1 (TRPA1) channels to facilitate spontaneous l-glutamate release onto spinal substantia gelatinosa (SG) neurons, which play a crucial role in regulating nociceptive transmission. In contrast, the ester-type LA procaine reduces the spontaneous release of l-glutamate in SG neurons. In order to determine whether TRPA1 activation by LAs is specific to amide-types, we examined the actions of tetracaine, another ester-type LA, and other amide-type LAs on glutamatergic spontaneous excitatory transmission in SG neurons by focusing on TRP activation. Whole-cell patch-clamp recordings were performed on SG neurons of adult rat spinal cord slices at a holding potential of -70mV. Bath-applied tetracaine increased spontaneous excitatory postsynaptic current (sEPSC) frequency in a concentration-dependent manner. Tetracaine activity was resistant to the voltage-gated Na+-channel blocker tetrodotoxin, the TRP vanilloid-1 antagonist capsazepine, and the TRP melastatin-8 antagonist BCTC, but was inhibited by the non-selective TRP antagonist ruthenium red and the TRPA1 antagonist HC-030031. With respect to amide-type LAs, prilocaine had a tendency to increase sEPSC frequency, while ropivacaine and levobupivacaine reduced the frequency. In conclusion, tetracaine facilitated spontaneous l-glutamate release from nerve terminals by activating TRPA1 channels in the SG, resulting in an increase in the excitability of SG neurons. TRPA1 activation was not specific to amide-type or ester-type LAs. The facilitatory action of LAs may be involved in pain occurring after recovery from spinal anesthesia.

TRPV1 agonist piperine but not olvanil enhances glutamatergic spontaneous excitatory transmission in rat spinal substantia gelatinosa neurons.

We examined the effects of TRPV1 agonists olvanil and piperine on glutamatergic spontaneous excitatory transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices with the whole-cell patch-clamp technique. Bath-applied olvanil did not affect the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC), and unchanged holding currents at -70 mV. On the other hand, superfusing piperine reversibly and concentration-dependently increased sEPSC frequency (half-maximal effective concentration: 52.3 µM) with a minimal increase in its amplitude. This sEPSC frequency increase was almost repetitive at an interval of more than 20 min. Piperine at a high concentration produced an inward current in some neurons. The facilitatory effect of piperine was blocked by TRPV1 antagonist capsazepine. It is concluded that piperine but not olvanil activates TRPV1 channels in the central terminals of primary-afferent neurons, resulting in an increase in the spontaneous release of l-glutamate onto SG neurons.

TRPA1 activation by lidocaine in nerve terminals results in glutamate release increase.

We examined the effects of local anesthetics lidocaine and procaine on glutamatergic spontaneous excitatory transmission in substantia gelatinosa (SG) neurons in adult rat spinal cord slices with whole-cell patch-clamp techniques. Bath-applied lidocaine (1-5 mM) dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) in SG neurons. Lidocaine activity was unaffected by the Na(+)-channel blocker, tetrodotoxin, and the TRPV1 antagonist, capsazepine, but was inhibited by the TRP antagonist, ruthenium red. In the same neuron, the TRPA1 agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. In contrast, procaine did not produce presynaptic enhancement. These results indicate that lidocaine activates TRPA1 in nerve terminals presynaptic to SG neurons to increase the spontaneous release of L-glutamate.

Muscarinic activity modulated by C-type natriuretic peptide in gastric smooth muscles of guinea-pig stomach.

Natriuretic peptides (NPs) are a cyclic guanosine monophosphate (cGMP) generation system like nitric oxide (NO) and play an inhibitory regulation in gastrointestinal motility but the effect of NPs on muscarinic activity is still unclear. This study was designed to investigate effect of C-type natriuretic peptide (CNP) on muscarinic control of gastric motility and its ion channel mechanism. The spontaneous contraction of gastric smooth muscle strip was recorded by using physiograph in guinea-pig. Membrane currents and potential were recorded by using whole-cell patch-clamp technique. CNP significantly inhibited muscarinic M receptor agonist carbachol (Cch)-induced contractions of gastric smooth muscle strips and dramatically hyperpolarized Cch-induced depolarization of membrane potential in gastric single smooth muscle cell. Muscarinic currents induced by both Cch and GTPgammaS, a G-protein agonist were significantly suppressed by CNP. 8-Br-cGMP mimicked the effect of CNP on Cch-induced muscarinic currents, and the peak holding current was decreased from -200.66+/-54.35 pA of control to -67.35+/-24.82 pA. LY83583, a guanylate cyclase nonspecific inhibitor, significantly weakened the inhibitory effect of CNP on muscarinic current while zaprinast, a cGMP sensitive phosphoesterase inhibitor, potentiated the inhibitory effect of CNP on muscarinic current. cGMP production was dramatically enhanced by CNP and this effect was suppressed by LY83583 in gastric smooth muscle. These results suggest that CNP modulates muscarinic activity via CNP-NPR-particulate guanylate cyclase (pGC)-cGMP pathway in guinea-pig.

Relationship between atrial natriuretic peptide-immunoreactive cells and microvessels in rat gastric mucosa.

AIM: To investigate the ultrastructural localization of atrial natriuretic peptide (ANP)-synthesizing cells and the relationship between ANP-synthesizing cells and microvessels in rat gastric mucosa. METHODS: Immunohistochemistry techniques and postembedding immunoelectron microscopy techniques were used to validate the findings regarding the expression of ANP-synthesizing cells and the ultrastructural localization of ANP-synthesizing cells in the gastric mucosa. Histochemistry techniques and the tannic acid-ferric chloride method (TA-Fe staining method) were used to reveal microvessel density and the distribution of ANP-synthesizing cells in different regions of the stomach. RESULTS: Cells expressing ANP were localized and ANP-synthesizing cells were identified as enterochromaffin (EC) cells in the gastric mucosa. ANP-synthesizing cells existed in different regions of the stomach. The percentage ANP-synthesizing cells in the mucosa was greatest in the fundus (46.7%+/-5.3%), intermediate in the antrum (40.1%+/-4.5%), and least in the body (21.6%+/-3.6%). There was a positive relationship between the percentage of ANP-synthesizing cells and the density of microvessels in the antral mucosa, but not in the fundus or body mucosa. CONCLUSION: ANP is synthesized by EC cells in rat gastric mucosa, and ANP-synthesizing cells are most dense in the gastric fundus. ANP may act not only as a regional autocrine and/or paracrine regulator, but also as an endocrine regulatory peptide in the gastrointestinal tract.

Role of arachidonic acid in hyposmotic membrane stretch-induced increase in calcium-activated potassium currents in gastric myocytes.

AIM: To study effects of arachidonic acid (AA) and its metabolites on the hyposmotic membrane stretch-induced increase in calcium-activated potassium currents (I(KCa)) in gastric myocytes. METHODS: Membrane currents were recorded by using a conventional whole cell patch-clamp technique in gastric myocytes isolated with collagenase. RESULTS: Hyposmotic membrane stretch and AA increased both I(K(Ca))) and spontaneous transient outward currents significantly. Exogenous AA could potentiate the hyposmotic membrane stretch-induced increase in I(K(Ca)). The hyposmotic membrane stretch-induced increase in I(K(Ca)) was significantly suppressed by dimethyleicosadienoic acid (100 micromol/L in pipette solution), an inhibitor of phospholipase A2. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly suppressed AA and hyposmotic membrane stretch-induced increases in I(K(Ca)). External calcium-free or gadolinium chloride, a blocker of stretch-activated channels, blocked the AA-induced increase in I(K(Ca)) significantly, but it was not blocked by nicardipine, an L-type calcium channel blocker. Ryanodine, a calcium-induced calcium release agonist, completely blocked the AA-induced increase in I(K(Ca)); however, heparin, a potent inhibitor of inositol triphosphate receptor, did not block the AA-induced increase in I(K(Ca)). CONCLUSION: Hyposmotic membrane stretch may activate phospholipase A2, which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca(2+) influx, which triggers Ca(2+)-induced Ca(2+) release and elicits activation of I(K(Ca)) in gastric antral circular myocytes of the guinea pig.