Discrimination analysis of excitatory and inhibitory neurons using Raman spectroscopy.

We have succeeded in discriminating between intact excitatory and inhibitory neuronal cells with Raman analysis. Excitatory and inhibitory neurons have several differences in their electric activities, but it can be difficult to determine their types based only on visual appearances. As Raman spectroscopy does not require any staining or labeling, its use in live neuronal cells is possible. In the present study, we used primary neurons obtained from rat cerebral cortexes, which we cultured on a glial feeder layered culturing dish for 15 days. The Raman spectra of the intact neurons on the dish were obtained; the neurons were then immunostained and their types determined. Partial least squares regression-discriminant analysis (PLSR-DA) was employed for classification of the excitatory and inhibitory neurons. The results demonstrated a high feasibility for use of Raman spectroscopy for discrimination analysis of inhibitory and excitatory neurons in a nondestructive manner.

Raman endoscopy for monitoring the anticancer drug treatment of colorectal tumors in live mice.

A miniaturized Raman endoscope (mRE) system was employed to study the effects of anticancer treatment on colorectal tumors in a live murine model. The endoscope is narrow enough to observe the inside of the mouse colon under anesthesia. It has a channel for a ball lens mounted on a hollow fiber Raman probe (BHRP) to measure any targeted point under the visual control of the endoscope. Colorectal cancer tissue was observed to study the alterations of the tissue in response to anticancer drug treatment. Three anticancer drugs, 5-fluorouracil (5-FU), cisplatin (CDDP), and docetaxel, were employed. Although no alteration was recognized in the endoscopic visual observations at 2 weeks after the drug treatment, the Raman spectra obtained in the live mouse colon indicated that molecular changes of lipids and proteins were observed. This study demonstrates that in situ Raman analysis is highly sensitive for detecting the effects of anticancer drugs.

Differential contribution of Kv4-containing channels to A-type, voltage-gated potassium currents in somatic and visceral dorsal root ganglion neurons.

Little is known about electrophysiological differences of A-type transient K(+) (KA) currents in nociceptive afferent neurons that innervate somatic and visceral tissues. Staining with isolectin B4 (IB4)-FITC classifies L6-S1 dorsal root ganglion (DRG) neurons into three populations with distinct staining intensities: negative to weak, moderate, and intense fluorescence signals. All IB4 intensely stained cells are negative for a fluorescent dye, Fast Blue (FB), injected into the bladder wall, whereas a fraction of somatic neurons labeled by FB, injected to the external urethral dermis, is intensely stained with IB4. In whole-cell, patch-clamp recordings, phrixotoxin 2 (PaTx2), a voltage-gated K(+) (Kv)4 channel blocker, exhibits voltage-independent inhibition of the KA current in IB4 intensely stained cells but not the one in bladder-innervating cells. The toxin also shows voltage-independent inhibition of heterologously expressed Kv4.1 current, whereas its inhibition of Kv4.2 and Kv4.3 currents is voltage dependent. The swapping of four amino acids at the carboxyl portion of the S3 region between Kv4.1 and Kv4.2 transfers this characteristic. RT-PCRs detected Kv4.1 and the long isoform of Kv4.3 mRNAs without significant Kv4.2 mRNA in L6-S1 DRGs. Kv4.1 and Kv4.3 mRNA levels were higher in laser-captured, IB4-stained neurons than in bladder afferent neurons. These results indicate that PaTx2 acts differently on channels in the Kv4 family and that Kv4.1 and possibly Kv4.3 subunits functionally participate in the formation of KA channels in a subpopulation of somatic C-fiber neurons but not in visceral C-fiber neurons innervating the bladder.

Development of in-situ Raman diagnosis technique of eosinophil esophagitis.

The spectra of the live tissue with blood flow measured with 785 nm-excitation light showed a very weak signal due to hemoglobin (Hb). It suggested the possibility to detect eosinophil accumulation in the tissue with the 785 nm-excitation light. The excitation wavelength of 633 nm induced strong fluorescence of sapphire glass that is a material of the ball lens of BHRP (Ball lens top hollow optical fiber Raman probe). On the other hand, the previous study suggested that eosinophil including eosinophil peroxidase (EPO) that showed a strong resonance Raman effect with 633 nm-excitation light. The purpose of the present study is to collect basic information and to evaluate the viability of Raman spectroscopic analysis for the detection of eosinophil accumulation in the live esophagus. BHRP with a sapphire ball lens with 500 µm diameter was applied for measurement of live esophagus tissue of a mouse. In this study, Raman spectra of eosinophil were measured with 633 and 785 nm-excitation. The Raman spectra of eosinophil showed a strong contribution of EPO, suggested that a heme chromophore in EPO had pre-resonance enhancement via Q band with the 785 nm-excitation light. Principal component analysis (PCA) is applied for the analysis of Raman spectra of eosinophil, erythrocyte and other granulocytes. Eosinophil was successfully discriminated from other blood cells in the PCA score plots built for the datasets of the spectra measured with 633 and 785 nm-excitation wavelengths. Consequently, our study demonstrates that Raman spectroscopy with 785 nm-excitation had high viability for in situ analysis of eosinophilic esophagitis (EoE).

Comparison of effects of a selective 5-HT reuptake inhibitor versus a 5-HT4 receptor agonist on in vivo neurogenesis at the rectal anastomosis in rats.

It was recently reported that activation of enteric neural 5-HT(4) receptors (SR4) promotes reconstruction of enteric neural circuit injury in distal gut of guinea pigs and that this reconstruction involves neural stem cells. We aimed to explore a novel approach using a selective serotonin reuptake inhibitor (SSRI), which increases endogenous 5-HT, to repair enteric nerve fiber injury in the rat distal gut. Enteric nerve fiber injury was performed by rectal transection and subsequent end-to-end one-layer anastomosis. The SSRI fluvoxamine maleate (100 µmol/l) was applied locally at the anastomotic site to compare with the 5-HT(4) agonist mosapride citrate (100 µmol/l) (applied for patent) applied locally and orally. Unlike mosapride, fluvoxamine failed to promote the regeneration of the nerve fiber tract across the anastomosis. Furthermore, fluvoxamine did not generate anti-distal-less homeobox 2 (DLX2)- and anti-SR4-positive cells (neural stem cells) and/or anti-neurofilament (NF)-positive cells (neural cells) in newly formed granulation tissue at the anastomosis, whereas these cell types were observed in mosapride-treated preparations. In contrast to its effects in guinea pigs, mosapride generated 5-bromo-2'-deoxyuridine (BrdU)-positive neural cells in ganglia sites 3 mm oral and anal from the anastomosis 2 wk after nerve fiber injury. All actions of mosapride were observed after local and or oral applications. These findings indicate that local SSRI treatment does not induce in vivo nerve fiber tract growth across the anastomosis in the rat distal gut. Mosapride induces nerve fiber tract growth across the anastomosis, mediated through enteric neural stem cells possibly from neural crest-derived stem cells or mesenchymal stem cells in the bone marrow.

In vitro enhanced differentiation of neural networks in ES gut-like organ from mouse ES cells by a 5-HT4-receptor activation.

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a "gut", from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified, but enteric neural networks were not identified. In the present study, we succeeded in forming dense enteric neural networks by a 5-HT(4)-receptor (SR4) agonist, mosapride citrate (1-10 µM) added only during EB formation. Addition of an SR4-antagonist, GR113808 (10 µM) abolished the SR4-agonist-induced formation of enteric neural networks. The SR4-agonist (1 µM) up-regulated the expression of mRNA of SR4 and the SR4-antagonist abolished this upregulation. 5-HT per se exerted similar effects to those of SR4-agonist, though less potent. These results suggest SR4-agonist differentiated enteric neural networks, mediated via activation of SR4 in the ES gut.

Coherent Anti-Stokes Raman Scattering Spectroscopy Using a Double-Wavelength-Emission Electronically Tuned Ti:Sapphire Laser.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy is a powerful tool for Raman imaging technology. In contrast, conventional spontaneous Raman spectroscopy is often used for biological analysis with multivariate analysis. This study develops a new type of CARS instrument with a double-wavelength-emission, background-free, electronically tuned Ti:sapphire laser (DW-ETL). DW-ETL generates two laser pulses with different wavelengths simultaneously within its single resonator. The pulse wavelength and buildup time are regulated by acousto-optical tunable filter in the resonator. The present DW-ETL CARS system is free from any mechanical movement to measure a CARS spectrum by controlling each laser pulse of the emission throughout the fingerprint region. Consequently, it is theoretically able to provide stable CARS spectra to apply multivariate analysis in biological applications. The present study demonstrates that the DW-ETL CARS system provides spectra of biomedical samples in the full finger-print region, and the stability and controllability of the system are evaluated.

Cardioprotective effects of a novel calpain inhibitor SNJ-1945 for reperfusion injury after cardioplegic cardiac arrest.

We have previously indicated that calpain inhibitor-1 prevents the heart from ischemia- reperfusion injury associated with the impairment of total Ca(2+) handling by inhibiting the proteolysis of alpha-fodrin. However, this inhibitor is insoluble with water and inappropriate for clinical application. The aim of the present study was to investigate the protective effect of a newly developed calpain inhibitor, SNJ-1945 (SNJ), with good aqueous solubility on left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts. SNJ (150 microM) was added to KCl (30 meq) cardioplegia (CP). Mean end-systolic pressure at midrange LV volume (ESP(mLVV)) and systolic pressure-volume area (PVA) at mLVV (PVA(mLVV); a total mechanical energy per beat) were hardly changed after CP plus SNJ arrest-reperfusion (post-CP + SNJ), whereas ESP(mLVV) and PVA(mLVV) in post-CP group were significantly (P < 0.01) decreased. Mean myocardial oxygen consumption for the total Ca(2+) handling in excitation-contraction coupling did not significantly decrease in post-CP + SNJ group, whereas it was significantly (P < 0.01) decreased in post-CP group. The mean amounts of 145- and 150-kDa fragments of alpha-fodrin in the post-CP group were significantly larger than those in normal and post-CP + SNJ groups. In contrast, the mean amounts of L-type Ca(2+) channel and sarcoplasmic reticulum Ca(2+)-ATPase were not significantly different among normal, post-CP, and post-CP + SNJ groups. Our results indicate that soluble SNJ attenuates cardiac dysfunction due to CP arrest-reperfusion injury associated with the impairment of the total Ca(2+) handling in excitation-contraction coupling by inhibiting the proteolysis of alpha-fodrin.

Inhibition of cytochrome c release by 10-N-nonyl acridine orange, a cardiolipin-specific dye, during myocardial ischemia-reperfusion in the rat.

The release of cytochrome c from the mitochondria to the cytosol is a critical step for downstream caspase-mediated apoptotic signal transduction in ischemia-reperfusion (I/R)-induced myocardial tissue injury. 10-N-nonyl acridine orange (NAO), a cardiolipin-specific dye, has been shown to inhibit Bid-mediated cytochrome c release from isolated mitochondria in vitro; however, the possible protective effects of NAO and the mechanisms underlying the protection from myocardial I/R-induced tissue injury in a rat model are unknown. Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion. All rats received either vehicle or NAO (100 microg/kg iv) 10 min before the occlusion. The infarct size in the heart at 24 h after reperfusion was significantly reduced in NAO-treated rats compared with vehicle-treated rats. NAO treatment significantly reduced the cytosolic cytochrome c contents and caspase-9 activity in the ischemic region but did not affect caspase-8 activity. Furthermore, NAO treatment markedly suppressed the translocation of truncated Bid, a proapoptotic Bcl-2 family member, to the mitochondrial fraction. NAO also suppressed the mitochondrial swelling and oxygen uptake stimulated by calcium overload. The results suggest that NAO possesses protective effects against myocardial I/R injury, which may be due to the suppression of cytochrome c release through blockade of truncated Bid translocation to mitochondria and inhibition of the opening of mitochondrial permeability transition pores.

A cardioprotective agent of a novel calpain inhibitor, SNJ-1945, exerts beta1 actions on left ventricular mechanical work and energetics.

We have previously shown that a newly developed calpain inhibitor, SNJ-1945 (SNJ), with good aqueous solubility prevents the heart from KCl arrest-reperfusion injury associated with the impairment of total Ca(2+) handling by inhibiting the proteolysis of alpha-fodrin as a cardioplegia. The aim of the present study was to investigate certain actions of this calpain inhibitor, SNJ, on left ventricular (LV) mechanical work and energetics in cross-circulated excised rat hearts undergoing blood perfusion with 40 microM SNJ. Mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at mLVV (a total mechanical energy/beat) were significantly increased by SNJ perfusion (P < 0.01). Mean myocardial oxygen consumption per beat (Vo(2)) intercepts (Vo(2) for the total Ca(2+) handling in excitation-contraction coupling and basal metabolism) of Vo(2)-PVA linear relations were significantly increased (P < 0.01) with unchanged mean slopes of Vo(2)-PVA linear relations. Pretreatment with the selective beta(1)-blocker landiolol (10 microM) blocked these effects of SNJ perfusion. There were no significant differences in mean basal metabolic oxygen consumption among normal, 40 microM SNJ, and 10 microM landiolol + 40 microM SNJ groups. Our results indicate that water-soluble SNJ exerted positive actions on mechanical work and energetics mediated via beta(1)-adrenergic receptors associated with the enhancement of total Ca(2+) handling in excitation-contraction coupling and with unchanged contractile efficiency. In clinical settings, this pharmacological action of SNJ is beneficial as an additive agent for cardioplegia.

Determinants of the epithelial-muscular axis on embryonic stem cell-derived gut-like structures.

Dome-like structures with epithelial-muscular layers resembling the gut have been derived from mouse embryonic stem (ES) cells. These domes have been reported to show spontaneous contractions and are called ES gut. In the present study, we examined the epithelial-muscular axis of these domes by detecting differentiation markers. A normal epithelial-muscular axis was exhibited in the domes with spontaneous motility, whereas the domes without spontaneous motility showed either an inverted or obscure axis. To investigate the factors affecting the epithelial-muscular axis, we examined the expression of hedgehog signaling factors in the domes. Expression of hedgehog family factors was detected in the epithelial components of the domes with motility, whereas this expression was inverted or obscure in the domes without motility. Out of the 25 domes, 10 of the 10 motility (+) domes showed a normal epithelial-muscular axis, whereas 14 of the 15 motility (-) domes lacked a normal epithelial-muscular axis. This implies that activin A upregulated the expression of sonic hedgehog and intestinal alkaline phosphatase in the embryoid bodies. These findings suggest that the motility of the ES gut depends on the domes' epithelial-muscular axis.

Effects of angiotensin type I receptor blockade on the cardiac Raf/MEK/ERK cascade activated via adrenergic receptors.

A close interaction between adrenergic nerves and angiotensin systems has been documented. The present study was designed to investigate the mechanisms of angiotensin-receptor blocker (ARB) suppression of beta-adrenergic receptor stimulation-induced cardiac hypertrophy. Chronic isoproterenol (ISO)-induced cardiac hypertrophy was inhibited in wild-type mice and AT1aR(-/-) mice treated with the ARB Candesartan (CV11974). Acute ISO-induced increase in phosphorylation levels of ERK MAPK was completely inhibited and increases in phosphorylation levels of p38 and JNK MAPKs were partially suppressed in both types of mice. Analysis of the activity of the small GTPase-regulating protein Raf indicated that the mechanisms by which ARB inhibits the Raf/MEK/ERK pathway under beta-adrenergic receptor stimulation basically depended on changes in the binding activities of Ras (stimulatory to Raf cascade) and Rap-1 (inhibitory to Raf cascade). Binding activities of Ras and Rap-1 in the heart were markedly augmented by ISO, whereas ARB suppressed only Ras, but not Rap-1, binding activity. Raf immunoprecipitation results confirmed that ISO-induced increases in its association with total and phosphorylated forms of MEK were completely normalized by ARB. These results might provide a molecular basis for the beneficial effects of AT1-receptor antagonists on cardiac remodeling and functions in patients with sympatho-excitatory heart failure.

Left ventricular function of isoproterenol-induced hypertrophied rat hearts perfused with blood: mechanical work and energetics.

We investigated left ventricular (LV) mechanical work and energetics in the cross-circulated (blood-perfused) isoproterenol [Iso 1.2 mg x kg(-1).day(-1) for 3 days (Iso3) or 7 days (Iso7)]-induced hypertrophied rat heart preparation under isovolumic contraction-relaxation. We evaluated pressure-time curves per beat, end-systolic pressure-volume and end-diastolic pressure-volume relations, and myocardial O(2) consumption per beat (Vo(2))-systolic pressure-volume area (PVA; a total mechanical energy per beat) linear relations at 240 beats/min, because Iso-induced hypertrophied hearts failed to completely relax at 300 beats/min. The LV relaxation rate at 240 beats/min in Iso-induced hypertrophied hearts was significantly slower than that in control hearts [saline 24 microl/day for 3 and 7 days (Sa)] with unchanged contraction rate. The Vo(2)-intercepts (composed of basal metabolism and Ca(2+) cycling energy consumption in excitation-contraction coupling) of Vo(2)-PVA linear relations were unchanged associated with their unchanged slopes in Sa, Iso3, and Iso7 groups. The oxygen costs of LV contractility were also unchanged in all three groups. The amounts of expression of sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban (PLB), phosphorylated-Ser(16) PLB, phospholemman, and Na(+)-K(+)-ATPase are significantly decreased in Iso3 and Iso7 groups, although the amount of expression of NCX1 is unchanged in all three groups. Furthermore, the marked collagen production (types I and III) was observed in Iso3 and Iso7 groups. These results suggested the possibility that lowering the heart rate was beneficial to improve mechanical work and energetics in isoproterenol-induced hypertrophied rat hearts, although LV relaxation rate was slower than in normal hearts.

Maternal immune activation in mice delays myelination and axonal development in the hippocampus of the offspring.

Epidemiological data suggest a relationship between maternal infection and a high incidence of schizophrenia in offspring. An animal model based on this hypothesis was made by injecting double-stranded RNA, polyinosinic-polycytidylic acid (poly-I:C), into early pregnant mice, and their offspring were examined for biochemical and histological abnormalities. Mouse brains were examined with special reference to oligodendrocytes, which have been implicated in several neurodevelopmental disorders. We detected a significant decrease of myelin basic protein (MBP) mRNA and protein at early postnatal periods in poly-I:C mice. MBP immunocytochemistry and electron microscopy revealed that the hippocampus of juvenile poly-I:C mice was less myelinated than in PBS mice, with no significant loss of oligodendrocytes. In addition, axonal diameters were significantly smaller in juvenile poly-I:C mice than in control mice. These abnormalities reverted to normal levels when the animals reached the adult stage. These findings suggest that retarded myelination and axonal abnormalities in early postnatal stages caused by maternal immune activation could be related to schizophrenia-related behaviors in adulthood.

Genetic fate mapping of Olig2 progenitors in the injured adult cerebral cortex reveals preferential differentiation into astrocytes.

Olig2 is a basic helix-loop-helix (bHLH) transcription factor essential for development of motoneurons and oligodendrocytes. It is known that Olig2(+) cells persist in the central nervous system (CNS) from embryonic to adult stages and that the number of Olig2(+) progenitors increases in the injured adult CNS. Recent studies have demonstrated an inhibitory action of Olig2 on neurogenesis in adult CNS, but the fate of Olig2(+) cells in the injured state remains largely unknown. To trace directly the fate of Olig2 cells in the adult cerebral cortex after injury, we employed the CreER/loxP system to target the olig2 locus. In this genetic tracing study, green fluorescent protein (GFP) reporter-positive cells labeled after cryoinjury coexpressed glial fibrillary acidic protein (GFAP), an astrocytic marker. Electron microscopy also showed that GFP(+) cells have the ultrastructural characteristics of astrocytes. Furthermore, GFP(+) cells labeled before injury, most of which had been NG2 cells, also produced bushy astrocytes. Here we show direct evidence that Olig2(+) cells preferentially differentiate into astrocytes, which strongly express GFAP, in response to injury in the adult cerebral cortex. These results suggest that reactive astrocytes, known to be the main contributors to glial scars, originate, at least in part, from Olig2(+) cells.

Knockdown of the L3/Lhx8 gene suppresses cholinergic differentiation of murine embryonic stem cell-derived spheres.

L3/Lhx8, a member of the Lim-homeobox gene family, is selectively and specifically expressed in the murine embryonic medial ganglionic eminence (MGE). Our previous study demonstrated that L3/Lhx8-deficient mice specifically lack cholinergic neurons in the basal forebrain. In this manuscript, we report the in vitro effects of reduced L3/Lhx8 gene expression on cholinergic differentiation in murine embryonic stem (ES) cell-derived spheres without dissociation. The knockdown of L3/Lhx8 gene expression dramatically decreased the cholinergic phenotype of spheres without altering other known phenotypes (TuJ1, GABA and GFAP). These results strongly suggest that L3/Lhx8 is a key factor for cholinergic differentiation of murine ES cell-derived spheres and is involved in basal forebrain development.

Effects of intrathecal injection of a hyperpolarization-activated channel (Ih) inhibitor ZD7288 on bladder function in urethane-anesthetized rats.

AIMS: The role of I(h) channels at the spinal level in the control of bladder function was examined in urethane anesthetized female rats. METHODS: Bladder activity was recorded via a transurethral catheter under isovolumetric conditions or via an intravesical catheter during continuous infusion cystometry. ZD7288, a selective I(h) channel inhibitor, was administered intrathecally at L6-S1 segmental levels of the spinal cord. RESULTS: Under isovolumetric conditions, intrathecal 0.3 microg of ZD7288 was inactive, whereas 1 and 3 microg abolished bladder activity for 5.6 +/- 1.8 and 20.7 +/- 3.9 min, respectively. Three micrograms also tended to decrease the amplitude of the contractions when they reappeared. During continuous cystometry, an intrathecal injection of ZD7288 at 0.3-3 microg dose-dependently increased the intercontraction intervals by 7.5%, 33.1%, and 57.5% from pre-drug values. A higher dose of ZD7288 (3 microg) tended to increase the pressure threshold for inducing micturition by 16.6% and the maximum voiding pressure by 11.1%. On the other hand, cardiovascular parameters such as heart rate and mean blood pressure were not affected by 0.3-3 microg of intrathecal ZD7288. CONCLUSIONS: These results indicate that I(h) channels play an important role in the control of micturition. Because I(h) channel inhibition in the lumbosacral spinal cord reduced the frequency of the micturition reflex without significantly affecting the amplitude of reflex bladder contraction, I(h) channels might preferentially be involved in afferent processing in the spinal cord to control the micturition reflex.

Development of Quantitative Analysis Techniques for Saccharification Reactions Using Raman Spectroscopy.

A technique for the analysis of saccharification reactions by a specific enzyme was developed on the basis of Raman spectroscopy using multivariate analysis. It is a microvolume, quantitative, and in situ technique, which can be used for studying saccharification processes in plant tissues. Prediction models for quantitative analysis of maltose, glucose, and starch were built with partial least squares regression (PLSR) analysis to monitor the saccharification process caused by α-amylase. We examined the reliability of the prediction models built using seven test samples. The spectral regions used to build the models were optimized for each sugar and were selected in such a manner that they did not overlap with strong protein and lipid bands that generally exist in plant tissues. The models were validated by monitoring the composition of reduced sugars and starch in a reactor and by comparing the results with those obtained by a conventional method. The results of Raman analysis and the conventional method showed good agreement for the reaction with α-amylase; however, it is not perfect for reactions with a different enzyme, especially ß-amylase. The results suggest that the present Raman technique is reliable and useful for sugar analysis. However, the prediction model built for a specific enzyme is valid only for that enzyme.

Early detection of virus infection in live human cells using Raman spectroscopy.

Virus infection of a human cell was determined only 3 h after invagination. We used viral vector Ad-CMV-control (AdC), which lacks the E1 gene coding for early polypeptide 1 (E1). AdC can replicate in human embryonic kidney 293 (HEK293) cells into which the E1 gene has been transfected. According to partial least-square regression discriminant analysis, it was assumed that two kinds of reaction take place in the cell during viral invasion. The first response of the cell was determined 3 h after the virus invasion, and the second one was determined ∼9 h later. The first one seems to be due to compositional changes in DNA. Analysis of large-scale datasets strongly indicated that the second reaction can be attributed to a reduction in protein concentration or uptake of phenylalanine into the nucleus.

Characterization of hyperpolarization-activated current (Ih) in dorsal root ganglion neurons innervating rat urinary bladder.

Afferent pathways innervating the urinary bladder consist of myelinated Adelta-fibers and unmyelinated C-fibers. Normal voiding is dependent on mechanoceptive Adelta-fiber bladder afferents that respond to bladder distention. However, the mechanisms for controlling the excitability of Adelta-fiber bladder afferents are not fully understood. We therefore used whole cell patch-clamp techniques to investigate the properties of hyperpolarization-activated, cyclic nucleotide-gated (HCN) currents (I(h)) in dorsal root ganglion (DRG) neurons innervating the urinary bladder of rats. The neurons were identified by axonal tracing with a fluorescent dye, Fast Blue, injected into the bladder wall. Hyperpolarizing voltage step pulses from -40 to -130 mV produced voltage- and time-dependent inward I(h) currents in bladder afferent neurons. The amplitude and current density of I(h) at a holding potential of -130 mV was significantly larger in medium-sized bladder afferent neurons (diameter: 37.8 +/- 0.3 microm), a small portion (19%) of which were sensitive to capsaicin (1 microM), than in uniformly capsaicin-sensitive small-sized (27.6 +/- 0.5 microm) bladder neurons. In medium-sized bladder neurons, a selective HCN channel inhibitor, ZD7288, dose-dependently inhibited I(h) currents. ZD7288 (10 microM) also increased the time constant of the slow depolarization phase of spike after-hyperpolarization from 91.8 to 233.0 ms. These results indicate that I(h) currents are predominantly expressed in medium-sized bladder afferent neurons innervating the bladder and that inhibition of I(h) currents delayed recovery from the spike after-hyperpolarization. Thus, it is assumed that I(h) currents could control excitability of mechanoceptive Adelta-fiber bladder afferent neurons, which are usually capsaicin-insensitive and larger in size than capsaicin-sensitive C-fiber bladder afferent neurons.