Low transition rate from normo- and low microalbuminuria to proteinuria in Japanese type 2 diabetic individuals: the Japan Diabetes Complications Study (JDCS).

AIMS/HYPOTHESIS: The aim of the study was to determine the transition rate and factors associated with the progression of normo- and low microalbuminuria to diabetic nephropathy (overt proteinuria). METHODS: For 8 years we prospectively observed 1,558 Japanese patients with type 2 diabetes mellitus whose basal urinary albumin:creatinine ratio (UACR) had been measured as <17.0 mg/mmol at entry. The incidence of nephropathy (UACR >33.9 mg/mmol) was determined by measuring UACR twice a year. RESULTS: Progression to nephropathy occurred in 74 patients. The annual transition rate was 0.67%, and was substantially higher for the low-microalbuminuric group than for the normoalbuminuric group (1.85% and 0.23%, respectively; hazard ratio for the low-microalbuminuric group 8.45, p < 0.01). The hazard ratio for an HbA(1c) of 7-9% or ≥9% was 2.72 (p < 0.01) or 5.81 (p < 0.01) relative to HbA(1c) <7.0%, respectively. In comparison with individuals with a systolic blood pressure (SBP) of <120 mmHg, the hazard ratios for patients with an SBP of 120-140 mmHg or ≥140 mmHg were 2.31 (p = 0.06) and 3.54 (p < 0.01), respectively. Smoking also affected progression to proteinuria (hazard ratio 1.99, p < 0.01). In contrast, 30.3% of the low-microalbuminuric group returned to normoalbuminuria (i.e. were in remission). CONCLUSIONS/INTERPRETATION: These results suggest that if patients with type 2 diabetes mellitus are receiving treatment from diabetologists for hyperglycaemia and hypertension when they are in the early stages of nephropathy (i.e. normo- or low microalbuminuria), their rate of transition to proteinuria is considerably lowered, and that differentiating patients with low microalbuminuria from those with high microalbuminuria might be clinically useful. TRIAL REGISTRATION: UMIN Clinical Trials Registry C000000222.

Accuracy of mandible-independent maxillary repositioning using pre-bent locking plates: a pilot study.

The double splint method is considered the gold standard for maxillary repositioning, but the procedure is lengthy and prone to error. Recent splintless methods have shown high repositioning accuracy; however, high costs and technical demands make them inaccessible to many patients. Therefore, a new cost-effective method of mandible-independent maxillary repositioning using pre-bent locking plates is proposed. Plates are bent on maxillary models in the planned position prior to surgery. The locations of the plate holes are replicated during surgery using osteotomy guides made from thermoplastic resin sheets. Pre-bent plates are subsequently fitted onto the maxilla, and plate holes are properly set to reposition the maxilla. The purpose of this study was to evaluate the accuracy of this method for maxillary repositioning and the reproducibility of the plate holes. Fifteen orthognathic surgery patients were evaluated retrospectively by superimposing preoperative simulations over their postoperative computed tomography models. The median deviations in maxillary repositioning and plate hole positioning between the preoperative plan and postoperative results were 0.43mm (range 0-1.55mm) and 0.33mm (range 0-1.86mm), respectively. There was no significant correlation between these deviations, suggesting that the method presented here allows highly accurate and reliable mandible-independent maxillary repositioning.

Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice.

The present study investigated whether the endogenous pro-inflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha)]-dependent expression of cyclooxygenase-2 (COX-2) mRNA within the spinal cord could be involved in the development of chronic inflammatory pain-like behaviors in mice. We demonstrated that the expression of COX-2 mRNA on the ipsilateral side of the spinal cord was significantly increased 6 h and 3 days after intraplantar injection of complete Freund's adjuvant (CFA), compared with the expression in saline-treated mice. In addition, the chronic pain-like behaviors following CFA injection were markedly suppressed by repeated intrathecal (i.t.) pre-treatment with the COX-2 inhibitor etodolac, but not with the COX-1 inhibitor mofezolac. The cytosolic level of the activated form of nuclear factor-kappa B (NF-kappaB), which is a major contributor to the induction of COX-2, on the ipsilateral side of the mouse spinal cord was also increased compared with that in the saline-treated mice. The key finding in the present study was that a single i.t. injection with either IL-1beta or TNF-alpha induced a marked increase in spinal COX-2 mRNA and persistent thermal hyperalgesia in mice. Furthermore, CFA-induced hypersensitivity to inflammatory pain was significantly reduced by repeated i.t. pre-injection of the recombinant Fc chimera of IL-1 receptor I or soluble TNF receptor I, which sequesters endogenous IL-1beta or TNF-alpha, respectively. In contrast, the expression of spinal COX-2 mRNA in CFA-treated mice was similar to that in saline-treated mice at 7 days after CFA injection. The present findings strongly indicate the early intrathecal use of the COX-2 inhibitor for the relief of chronic inflammatory pain. Furthermore, together with the result in a previous study that pro-inflammatory cytokines lead to stimulation of a NF-kappaB-dependent transcriptional pathway, these findings suggest that a spinal cytokine/NF-kappaB/COX-2 pathway may play an important role in the development, but not maintenance, of chronic pain following peripheral tissue inflammation.

Effects of oral administration of ciclosporin A on skin carcinogenesis: a study using the two-stage carcinogenesis protocol in mice.

BACKGROUND: Development of malignant skin neoplasms in patients receiving cyclosporin A (CsA) has been reported. The relationship between the pathogenesis of skin carcinogenesis and the dose of CsA is still unclear. AIM: To clarify the effect of oral administration of CsA, especially its dose, on skin carcinogenesis. METHODS: Hr-1 hairless mice were assigned to the following four groups: (i) control group (n = 8), given vehicle intragastrically six times/week and acetone applied to the skin of the back; (ii) chemical-alone (n = 11), given vehicle intragastrically + application of 7,12-dimethylbenz[a]anthracene (DMBA) once week and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) twice week to the back; (iii) CsA-alone group (n = 8), given CsA intragastrically (10 mg/kg) six times/week and vehicle applied to the back twice week; and (iv) CsA + chemical group (n = 8), given 10 mg/kg CsA intragastrically + topical DMBA and TPA. The number of papules > 3 mm in diameter that had developed on the back after 15 weeks was counted. The mean epidermal thickness and number of dermal infiltrates were determined. The same experiments were performed using CsA at doses of 5 and 20 mg/kg. RESULTS: Oral administration of either 10 or 20 mg/kg CsA significantly enhanced the formation of papillomas by DMBA and TPA, but no enhancement was observed when 5 mg/kg CsA was administered. The mean epidermal thickness and number of dermal infiltrates were significantly greater in the CsA + chemical group than in the chemical-alone group. CONCLUSION: These data suggest that oral administration of CsA in excess of a certain dose can accelerate tumour development in mice.

Localization of palatal area in human somatosensory cortex.

To determine the 'hard palate representing' area in the primary somatosensory cortex, we recorded somatosensory-evoked magnetic fields from the cortex in ten healthy volunteers, using magnetoencephalography. Following electrical stimulation of 3 sites on the hard palate (the first and third transverse palatine ridges, and the greater palatine foramen), magnetic responses showed peak latencies of 15, 65, and 125 ms. Equivalent current dipoles for early magnetic responses were found along the posterior wall of the inferior part of the central sulcus. These dipoles were localized anterior-inferiorly, compared with locations for the hand area in the cortex. However, there were no significant differences in three-dimensional locations among the 3 selected regions for hard palate stimulation. These results demonstrated the precise location of palatal representation in the primary somatosensory cortex, the actual area being small.

Computational fluid dynamics study of the pharyngeal airway space before and after mandibular setback surgery in patients with mandibular prognathism.

The purpose of this study was to investigate the relationship between the pressure drop in the pharyngeal airway space (ΔPPAS) and the minimum cross-sectional area (minCSA) of the pharyngeal airway before and after mandibular setback surgery using computational fluid dynamics, in order to prevent iatrogenic obstructive sleep apnoea. Eleven patients with mandibular prognathism underwent bilateral sagittal split osteotomy for mandibular setback. Three-dimensional models of the upper airway were reconstructed from preoperative and postoperative computed tomography images, and simulations were performed using computational fluid dynamics. ΔPPAS and the minCSA of the pharyngeal airway were calculated, and the relationship between them was evaluated by non-linear regression analysis. In all cases, the minCSA was found at the level of the velopharynx. After surgery, ΔPPAS increased significantly and the minCSA decreased significantly. The non-linear regression equation expressing the relationship between these variables was ΔPPAS=3.73×minCSA-2.06. When the minCSA was <1cm2, ΔPPAS increased greatly. The results of this study suggest that surgeons should consider bimaxillary orthognathic surgery rather than mandibular setback surgery to prevent the development of iatrogenic obstructive sleep apnoea when correcting a skeletal class III malocclusion.

mu-Opioid receptor internalization-dependent and -independent mechanisms of the development of tolerance to mu-opioid receptor agonists: Comparison between etorphine and morphine.

A growing body of evidences suggests that receptor desensitization is implicated in the development of tolerance to opioids, which is generally regulated by protein kinases and receptor trafficking proteins. In the present study, we demonstrated that repeated s.c. treatment with etorphine, but not morphine, produced a significant increase in protein levels of G protein-coupled receptor kinase 2, dynamin II, beta-arrestin 2 and phosphorylated-conventional protein kinase C in membranes of the mouse spinal cord, suggesting that the etorphine-induced mu-opioid receptor desensitization may result from G protein-coupled receptor kinase 2/dynaminII/beta-arrestin2-dependent phosphorylation of mu-opioid receptors. Unlike etorphine, morphine failed to change the levels of these trafficking proteins. Furthermore, we found that the level of glial fibrillary acidic protein in the mouse spinal cord was clearly increased by chronic in vivo and in vitro treatment with morphine, whereas no such effect was noted by etorphine. In the behavioral study, intraperitoneal pretreatment with the glial-modulating agent propentofylline suppressed the development of tolerance to morphine-induced antinociception. In addition, intrathecal injection of astrocytes and astrocyte-conditioned medium mixture, which were obtained from cultured astrocytes of the newborn mouse spinal cord, aggravated the development of tolerance to morphine. In contrast, these agents failed to affect the development of tolerance induced by etorphine. These findings provide direct evidence for the distinct mechanisms between etorphine and morphine on the development of tolerance to spinal antinociception. These findings raise the possibility that the increased astroglia response produced by chronic morphine could be associated with the lack of mu-opioid receptor internalization.

Correlation between reduction of syndecan-1 expression and clinico-pathological parameters in squamous cell carcinoma of tongue.

We examined expression of syndecan-1 in squamous cell carcinoma (SCC) of tongue using immunohistochemistry. Forty-three cases of SCC arising in lateral border of tongue were investigated. From the immunohistochemical staining pattern, the cases were divided into two groups based on expression of syndecan-1 at the supra-peripheral cells of the tumor nest: Group A, completely or mainly positive; Group B, sporadically positive or negative. Most poorly differentiated SCC cases were classified into Group B (81.8%). The number of Group B cases in T1-2 was different from that in T3-4. The number of cases where syndecan-1 expression was reduced was much greater in T3-4, and represented the majority of Group B (86.7%). More than 80% of Grade 4D cases were in Group B (83.3%) based on the Yamamoto-Kohama criteria. These results indicate that reduction of syndecan-1 correlates to histological grade, tumor size and mode of invasion in tongue SCC.

Localization of aldose reductase in the human eye.

The presence of the enzyme aldose reductase is increasingly being linked to diabetic complications. The distribution of this enzyme in human cornea, lens, retina, and optic nerve has been studied using specific antibodies against purified human placental aldose reductase raised in both rabbit and goat. The antisera from both animals gave equal, specific reactions. In frozen sections of ocular tissues, significant aldose reductase localization was reproducibly demonstrated in the endothelium and epithelium of the cornea and in the basal cell layers of the conjunctiva. In the lens, staining was observed in the epithelium and superficial lens fibers. In retinal sections, the presence of aldose reductase was demonstrated in the Mueller's cells, especially near the inner limiting membrane. It was also found in some ganglion and cone cells. In the optic nerve, positive staining was observed in the axon. All other cells of the tissues examined revealed only weak, nonspecific staining.

Is ACE gene polymorphism a useful marker for diabetic albuminuria in Japanese NIDDM patients?

OBJECTIVE: We studied the relationship between an insertion/deletion (I/D) polymorphism in the ACE gene and albuminuria/proteinuria in Japanese NIDDM patients. RESEARCH DESIGN AND METHODS: A total of 142 Japanese NIDDM patients (89 men, 53 women) with a known diabetes duration of 14 +/- 5 (mean +/- SD) years and an age of 56 +/- 6 years were divided into three groups according to the stage of nephropathy: 41 patients with normoalbuminuria, 47 patients with microalbuminuria, and 54 with overt proteinuria. The three groups were similar in age, diabetes duration, and recent HbAic level. RESULTS: The distribution of DD, ID, and II genotypes of the ACE gene did not differ among the three groups (10, 46, and 44% in the normoalbuminuric patients; 13, 53, and 34% in the microalbuminuric patients; and 15, 46, and 39% in the proteinuric patients, respectively). Meanwhile, the frequency of the D allele in the proteinuric male patients was slightly higher than in the normoalbuminuric male patients (45 vs. 27%, chi 2 = 3.9, P < 0.05), while the D allele frequency was nonsignificantly lower in the proteinuric female patients than in the normoalbuminuric female patients. CONCLUSION: These results did not support the hypothesis that the genotype of the ACE gene would be a clinically useful genetic marker for predicting the development of nephropathy in Japanese NIDDM patients. However, the role of D allele of ACE gene in the progression of nephropathy in male patients remains to be seen.

Mastoparan-stimulated prolactin secretion in rat pituitary GH3 cells involves activation of Gq/11 proteins.

Mastoparan has been reported to induce a wide variety of cellular actions by activating GTP-binding proteins (G proteins) in various cells. Here, we demonstrate that mastoparan is able to stimulate the secretion of PRL from rat anterior pituitary tumor GH3 cells in dose- and time-dependent manners. Mastoparan had no effect on the accumulation of intracellular cAMP; however, it induced a rapid increase in the intracellular Ca2+ concentration in GH3 cells. Extracellular Ca2+ was required for mastoparan-induced PRL secretion, which was inhibited by nifedipine, an L-type Ca2+ channel blocker. Incubation of mastoparan with myo-[3H]inositol-labeled GH3 cells also resulted in the increased formation of inositol phosphates (InsPs) compared with control cells. Neomycin sulfate and U73122, both phospholipase C inhibitors, suppressed mastoparan-induced PRL secretion. Guanosine 5'-1beta-thioldiphosphate (GDPbetaS) encapsulated in GH3 cells by reversible electropermeabilization suppressed the response to mastoparan. However, pretreatment with pertussis toxin had no effect on the stimulation of PRL secretion by mastoparan, and both Mas7 (a highly active analogue of mastoparan) and Mas17 (an inactive analogue) enhanced the secretion of PRL to a similar level to that of mastoparan-induced GH3 cells. In contrast, the substance P-related peptide GPant-2A, a Gq antagonist, inhibited mastoparan-induced PRL release, whereas GPant-2, a G(i/o) antagonist, did not in electropermeabilized GH3 cells. Moreover, a specific G(q/11) antibody against the carboxyl terminus of the G(q/11) alpha-subunit blocked the stimulatory effect of mastoparan on secretion and mastoparan-stimulated InsPs production in digitonin-permeabilized GH3 cells. These results indicate that mastoparan induces the Ca2+-regulated secretion of PRL from GH3 cells by activating G(q/11) and the phospholipase C pathway.

The stimulatory and inhibitory guanine nucleotide-binding proteins of adenylate cyclase in erythrocytes from patients with pseudohypoparathyroidism type I.

Both the inhibitory and stimulatory guanine nucleotide-binding proteins of the adenylate cyclase complex were measured in erythrocyte membranes from patients with pseudohypoparathyroidism (PHP). The inhibitory guanine nucleotide-binding protein (Ni) of adenylate cyclase was measured by incorporation of [32P]ADP-ribose from [32P]NAD into the 39K subunit of Ni catalyzed by pertussis toxin. The ADP-ribosyltransferase activity of the toxin was expressed through incubation with dithiothreitol and erythrocyte membranes. Erythrocytes from 12 patients with PHP type I (PHP-I) had Ni values similar to those of 9 normal subjects and 2 patients with pseudopseudohypoparathyroidism. In 6 PHP-I patients, decreased activity of the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase, as determined by reconstitution of adenylate cyclase in the Ns-deficient membranes of cyc-S49 cells, corresponded with the reduced degree of ADP-ribosylation of the 42K subunit of Ns catalyzed by cholera toxin. These data suggest that the defect of Ns results in reduced stimulation of adenylate cyclase in some PHP-I patients, and that enhanced inhibition of the enzyme due to an increase in the 39K subunit of Ni does not account for the biochemical lesion in PHP-I patients.

Epidermal growth factor receptors in plasma membranes of normal and diseased human thyroid glands.

We studied the characteristics of epidermal growth factor (EGF) receptors in plasma membrane fractions derived from normal and diseased human thyroid tissues. The mean maximal specific binding of EGF to membrane fractions of normal thyroid tissue (n = 25) was 1.46 +/- 0.47 (+/- SD) fmol/mg protein. The maximal specific binding was higher than the upper limit of the normal range (2.40) in 12 of the 39 (31%) differentiated carcinomas, 2 of the 3 (67%) undifferentiated carcinomas, and 1 squamous cell thyroid carcinoma. In contrast, the maximal specific binding in samples derived from adenomas (1.13 +/- 0.91), adenomatous goiters (0.92 +/- 0.56), and hyperplastic (Graves') thyroids (1.57 +/- 0.61) was not different from that in normal thyroid tissue. Scatchard plot analysis revealed that all thyroid membrane fractions had two classes of specific receptors for EGF. The mean association constant for the high affinity EGF receptors in normal thyroid tissue was 7.9 +/- 2.9 (+/- SD) X 10(9) mol/L-1, and the capacity was 22.9 +/- 7.0 fmol/mg protein. The capacity of the high affinity receptors was higher (P less than 0.05) in differentiated carcinoma (37.2 +/- 25.5) and undifferentiated carcinoma (32.7 +/- 11.6) than in normal thyroid tissue. In one squamous cell carcinoma, the capacities for the two classes of binding sites were about 15-fold greater than in normal thyroid tissue. In contrast, the association constants of the high affinity receptors from carcinomas (differentiated, 6.9 +/- 2.8; undifferentiated, 11.8 +/- 4.1; squamous cell, 8.2) were similar to that of normal thyroid tissue. In the thyroid tissues from eight patients with Graves' disease the capacity of the high affinity binding sites (37.5 +/- 12.3 fmol/mg protein) was higher than that in normal tissue, but the affinity (4.4 +/- 1.6 X 10(9) mol/L-1) was less, and the maximal specific binding was similar in the two types of tissue. These results suggest that a significant increase in the number of high affinity EGF receptors may play a role in the pathogenesis of human thyroid carcinoma.

Ultrastructural characterization of pharyngeal and esophageal motoneurons in the nucleus ambiguus of the rat.

The viscerotopic organization of the upper alimentary tract has been established in the nucleus ambiguus, but there is little information about the morphology of the individual neurons innervating the pharynx and esophagus. We studied the ultrastructure of pharyngeal (PH), cervical esophageal (CE), and subdiaphragmatic esophageal (SDE) motoneurons labeled by retrogradely transported wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) in the compact formation of the nucleus ambiguus. WGA-HRP was injected into the lower pharynx, or the cervical and subdiaphragmatic esophagus of male rats. The retrogradely labeled PH neurons in the rostral portion of the compact formation were large (26.1 x 50.1 microns, 906.7 microns2), polygonal, and contained well-developed cell organelles with a round nucleus. Subsurface cisterns connected with rough endoplastic reticulum were often present near the postsynaptic membrane. Both CE and SDE neurons in the compact formation were medium-sized, round or oval, and contained well-developed cell organelles, although the SDE neuron was significantly larger than the CE neuron (24.9 x 33.6 microns, 593.0 microns2 in the SDE neuron, and 19.5 x 30.2 microns, 440.3 microns2 in the CE neuron). The average number of axosomatic terminals in a sectional plane was largest in PH neurons (29.0), smaller in CE neurons (7.9), and smallest in SDE neurons (4.2). The number of axosomatic terminals containing round vesicles (Gray's type I) was almost equal to that of terminals containing pleomorphic vesicles (Gray's type II) in PH and CE neurons, but there were few Gray's type II axosomatic terminals in SDE neurons. Desmosome-like junctions at somato-somatic or somato-dendritic apposition were often present in the area surrounding SDE neurons. There were also small unlabeled neurons (9.5 x 18.1 microns, 131.8 microns2) in the compact formation of the nucleus ambiguus. The small neurons contained poorly developed cell organelles and an irregular shaped nucleus with invaginated nuclear membrane, and had no Nissl bodies. These results indicate that PH neurons have the characteristics of somatic motoneurons, and that CE and SDE neurons are similar to visceral motoneurons.

Direct synaptic projections to esophageal motoneurons in the nucleus ambiguus from the nucleus of the solitary tract of the rat.

Neurons of the nucleus of the solitary tract (NTS) serve as interneurons in swallowing. We investigated the synaptology of the terminals of these neurons and whether they project directly to the esophageal motoneurons in the compact formation of the nucleus ambiguus (AmC). Following wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) injection into the NTS, many anterogradely labeled axodendritic terminals were found in the neuropil of the AmC. The majority of labeled axodendritic terminals (89%) contained round vesicles and made asymmetric synaptic contacts (Gray's type I), but a few (11%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II). More than half of the labeled terminals contacted intermediate dendrites (1-2 microm diameter). There were no retrogradely labeled medium-sized motoneurons, but there were many retrogradely labeled small neurons having anterogradely labeled axosomatic terminals. A combined retrograde and anterograde transport technique was developed to verify the direct projection from the NTS to the esophageal motoneurons. After the esophageal motoneurons were retrogradely labeled by cholera toxin subunit B conjugated HRP, the injection of WGA-HRP into the NTS permitted ultrastructural recognition of anterogradely labeled axosomatic terminals contacting directly labeled esophageal motoneurons. Serial sections showed that less than 20% of the axosomatic terminals were labeled in the esophageal motoneurons. They were mostly Gray's type I, but a few were Gray's type II. In the small neurons, more than 30% of axosomatic terminals were labeled, which were exclusively Gray's type I. These results indicate that NTS neurons project directly not only to the esophageal motoneurons, but also to the small neurons which have bidirectional connections with the NTS.

Synaptology of the direct projections from the nucleus of the solitary tract to pharyngeal motoneurons in the nucleus ambiguus of the rat.

During the pharyngeal phase of the swallowing reflex, the nucleus of the solitary tract (NTS) receives peripheral inputs from the pharynx by means of the glossopharyngeal ganglion and is the location of premotor neurons for the pharyngeal (PH) motoneurons. The semicompact formation of the nucleus ambiguus (AmS) is composed of small and medium-sized neurons that do not project to the pharynx, and large PH motoneurons. We investigated whether the neurons in the NTS projected directly to the PH motoneurons or to the other kinds of neurons in the AmS by using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) injections into the pharyngeal muscles of male Sprague-Dawley rats, many nerve terminals anterogradely labeled with WGA-HRP were found to contact PH motoneurons retrogradely labeled with CT-HRP. Most of the labeled axodendritic terminals (63%) contained pleomorphic vesicles with symmetric synaptic contacts (Gray's type II), and the remaining ones contained round vesicles with asymmetric synaptic contacts (Gray's type I). About 14% of the axosomatic terminals on PH motoneuron in a sectional plane were anterogradely labeled, and about 70% of the labeled axosomatic terminals were Gray's type II. Observations of serial ultrathin sections revealed that both the small and the medium-sized neurons received only a few labeled axosomatic terminals that were exclusively Gray's type I. These results indicate that the NTS neurons may send mainly inhibitory as well as a few excitatory inputs directly to the PH motoneurons in the AmS.

GABA(A) receptor-mediated inhibition in the nucleus ambiguus motoneuron.

GABA-sensitive ambiguus motoneurons were investigated by microiontophoretic application of GABAergic drugs including bicuculline and muscimol in alpha-chloralose- and urethane-anaesthetized rats. Ambiguus motoneurons were activated by recurrent laryngeal nerve stimulation through a small cuff electrode and identified as laryngeal motoneurons when they met the conventional criteria for antidromic activation. GABA(A) antagonist, bicuculline, and its agonist, muscimol, were iontophoretized on ambiguus motoneurons through a three-multibarrel electrode glued to the recording microelectrode. One-hundred and nineteen out of 155 neurons sampled from the loose formation and its vicinity were found to be respiratory neurons, most of which were inspiratory neurons. A small proportion (32 neurons) was classified as laryngeal motoneurons according to the criteria. A majority of laryngeal motoneurons was found to be GABA sensitive. Namely, application of GABA and its antagonist and agonist affected the antidromic spikes in a dose-dependent manner; GABA and muscimol usually decreased the amplitude and slowed the slope in the spike, whereas bicuculline, reversed these inhibitory effects. The dose-dependent relationships were limited exclusively to the measurements analysed in the negative-going phase but not in the positive-going phase in the antidromic spike. GABA and muscimol decreased but bicuculline increased the ratios to control in these measurements. The effects of distributions of the histograms shifting towards the opposite direction were statistically significant. The line of evidence suggests strongly that a majority of laryngeal motoneurons located in the nucleus ambiguus presumably possesses GABA(A) receptors on their postsynaptic membrane. These GABA-sensitive laryngeal motoneurons may receive inputs either from inhibitory interneurons subserving the reciprocal inhibition in the reflexive integration or from inhibitory respiratory interneurons which control the synchronized glottic movements during vocalization and respiration.

Ambiguous respiratory neurons are modulated by GABA(A) receptor-mediated inhibition.

A group of respiratory neurons in the rostral nucleus ambiguus complex is known to generate the inspiratory and expiratory drives which enable spontaneous respiration to be sustained. Since previous studies indicated that mutual synaptic inhibition is required to produce oscillations between inspiratory and expiratory neurons, it may implicate GABAergic synaptic transmission between each group of neurons. In this study we tried to determine whether most ambiguous respiratory neurons are influenced by GABA(A) receptor-mediated inhibition. Eighty-eight respiratory interneurons showing rhythmic activity in synchrony with the spontaneous respiration were recorded in urethane-chloralose anesthetized Wistar rats. Multibarrel iontophoretic application of GABA(A) antagonist bicuculline produced a remarkable facilitation in maximum burst discharge rate, whereas the agonist muscimol reversed this effect completely. Simultaneous application of GABA and bicuculline increased the discharge rate more than in any single application or in the simultaneous application of GABA and muscimol. These results were statistically significant. These findings suggest strongly that GABA(A) receptors in the ambiguous respiratory neurons may have an inhibitory role in the synaptic transmission for maintaining the respiratory oscillation in the nucleus ambiguus.

Involvement of protein kinase Cgamma isoform in morphine-induced reinforcing effects.

The present study was designed to investigate the role of protein kinase C (PKC) isoform in the morphine-induced reinforcing effect in mice. An intracerebroventricular injection of calphostin C, a specific PKC inhibitor, produced a dose-dependent reduction in the morphine-induced place preference. The protein level of PKCgamma was significantly up-regulated in membrane preparations of the limbic forebrain obtained from the morphine-conditioned mice compared to that from the saline-conditioned mice. However, the protein levels of PKCalpha, betaI, betaII and epsilon were not affected in the same preparation. By contrast, there were no changes in the protein level of all five PKC isoforms in the lower midbrain. Furthermore, we investigated the rewarding properties of morphine in mice lacking PKCgamma gene. A significant place preference was observed following treatment with morphine in wild-type mice, whereas such an effect of morphine was not found in PKCgamma knockout mice. These findings suggest that activated PKCgamma in the limbic forebrain following the treatment with morphine may be critical for the development and/or maintenance of reinforcing effects induced by morphine in mice.

Implication of brain-derived neurotrophic factor in the release of dopamine and dopamine-related behaviors induced by methamphetamine.

It is widely recognized that methamphetamine enhances the release of dopamine at dopaminergic neuron terminals of the mesolimbic system, which induces dopamine-related behaviors. Brain-derived neurotrophic factor (BDNF), a neurotrophin, binds to and activates its specific receptor tyrosine kinase, TrkB. BDNF has been shown to influence the release of dopamine in the mesolimbic dopamine system. The present study was designed to investigate roles of BDNF and TrkB in the expression of methamphetamine-induced dopamine release in the nucleus accumbens and dopamine-related behaviors induced by methamphetamine in rats. Methamphetamine (1 mg/kg, s.c.) produced a substantial increase in the extracellular levels of dopamine and induced a progressive augmentation of dopamine-related behaviors such as rearing and sniffing. In contrast, both the stimulation of dopamine release and induction of dopamine-related behaviors by methamphetamine were significantly suppressed by pretreatment with intra-nucleus accumbens injection of either BDNF (2.0 microl/rat, 1:1000, 1:300 and 1:100) or TrkB (2.0 microl/rat, 1:1000 and 1:100) antibody. Furthermore, the basal level of dopamine in the nucleus accumbens was not affected by treatment with both BDNF and TrkB antibodies. These findings provide further evidence that BDNF/TrkB pathway is implicated in the methamphetamine-induced release of dopamine and the induction of dopamine-related behaviors.