Rapid Qualitative and Quantitative Analysis of Caffeine and Sodium Benzoate in Annaca by Infrared Spectroscopy.

ABSTRACT: Objective To establish an infrared spectroscopic method for the rapid qualitative and quantitative analysis of caffeine and sodium benzoate in Annaka samples. Methods Qualitative and quantitative modeling samples were prepared by mixing high-purity caffeine and sodium benzoate. The characteristic absorption peaks of caffeine and sodium benzoate in Annaka samples were determined by analyzing the infrared spectra of the mixed samples. The quantitative model of infrared spectra was established by partial least squares （PLS）. Results By analyzing the infrared spectra of 17 mixed samples of caffeine and sodium benzoate （the purity of caffeine ranges from 10% to 80%）, the characteristic absorption peaks for caffeine were determined to be 1 698, 1 650, 1 237, 972, 743, and 609 cm-1. The characteristic absorption peaks for sodium benzoate were 1 596, 1 548, 1 406, 845, 708 and 679 cm-1. When the detection of all characteristic absorption peaks was the positive identification criteria, the positive detection rate of caffeine and sodium benzoate in 48 seized Annaka samples was 100%. The linear range of PLS quantitative model for caffeine was 10%-80%, the coefficient of determination （ R2） was 99.9%, the root mean square error of cross validation （RMSECV） was 0.68%, and the root mean square error of prediction （RMSEP） was 0.91%; the linear range of PLS quantitative model for sodium benzoate was 20%-90%, the R2 was 99.9%, the RMSECV was 0.91% and the RMSEP was 1.11%. The results of paired sample t test showed that the differences between the results of high performance liquid chromatography method and infrared spectroscopy method had no statistical significance. The established infrared quantitative method was used to analyze 48 seized Annaka samples, the purity of caffeine was 27.6%-63.1%, and that of sodium benzoate was 36.9%-72.3%. Conclusion The rapid qualitative and quantitative analysis of caffeine and sodium benzoate in Annaka samples by infrared spectroscopy method could improve identification efficiency and reduce determination cost.

Modulation of synchronous sympathetic firing behaviors by endogenous GABA(A) and glycine receptor-mediated activities in the neonatal rat spinal cord in vitro.

Delivering effective commands in the nervous systems require a temporal integration of neural activities such as synchronous firing. Although sympathetic nerve discharges are characterized by synchronous firing, its temporal structures and how it is modulated are largely unknown. This study used a collagenase-dissociated splanchnic sympathetic nerve-thoracic spinal cord preparation of neonatal rats in vitro as an experimental model. Several single-fiber activities were recorded simultaneously and verified by rigorous computational algorithms. Among 3763 fiber pairs that had spontaneous fiber activities, 382 fiber pairs had firing positively correlated. Their temporal relationship was quantitatively evaluated by cross-correlogram. On average, correlated firing in a fiber pair occurred in scales of ∼40ms lasting for ∼11ms. The relative frequency distribution curves of correlogram parametrical values pertinent to the temporal features were best described by trimodal Gaussians, suggesting a correlated firing originated from three or less sources. Applications of bicuculline or gabazine (noncompetitive or competitive GABA(A) receptor antagonist) and/or strychnine (noncompetitive glycine receptor antagonist) increased, decreased, or did not change individual fiber activities. Antagonist-induced enhancement and attenuation of correlated firing were demonstrated by a respective increase and decrease of the peak probability of the cross-correlograms. Heterogeneity in antagonistic responses suggests that the inhibitory neurotransmission mediated by GABA(A) and glycine receptors is not essential for but can serve as a neural substrate to modulate synchronous firing behaviors. Plausible neural mechanisms were proposed to explain the temporal structures of correlated firing between sympathetic fibers.

Reduction of bladder activity on FDG PET/CT scan in patients with urinary bladder carcinoma. A prospective study with a patient-friendly protocol.

UNLABELLED: Oral hydration with large volume of fluid, high dose of diuretics and maximum bladder distension are frequently required to achieve an adequate urinary FDG clearance in an FDG PET/CT protocol for bladder cancer or pelvic tumour evaluation. Although most patients tolerated these procedures for eliminating urinary FDG activity, these procedures may be still unpleasant. AIM: Is there a more patient-friendly protocol which is less burdensome and yet provides a satisfactory FDG clearance in urine? In this study, we established a patient-friendly FDG PET/CT protocol without compromising urinary FDG clearance. PATIENTS, METHODS: 23 patients with biopsy-proven urinary bladder cancers were recruited to evaluate the effectiveness of this patient-friendly protocol on reducing urinary bladder FDG activity. The patient-friendly protocol includes encouraging patients to take a tolerable amount of fluid, delaying the administration of low dose diuretic, shortening the urine holding time and using delayed imaging. RESULTS: All of the patients tolerated this patient-friendly procedure well. In addition, the patient-friendly protocol was effective in reducing FDG activity in the urine. One hundred percent of primary bladder cancer were visualized on the FDG PET/CT images using this patient-friendly protocol. CONCLUSIONS: This patient-friendly FDG PET/CT protocol is less intrusive yet effective in reducing urinary FDG activity.

Kidneys from standard-criteria donors with different severities of terminal acute kidney injury.

OBJECTIVES: High terminal serum creatinine level in a deceased donor has been reported as the second most frequent cause of refusal for kidney transplantation. A growing body of evidence has shown a comparable outcome of kidney transplantation from deceased donors with acute kidney injury (AKI). However, the influence of the severity of AKI on graft outcomes remains to be elucidated. METHODS: In this retrospective cohort study, 84 consecutive kidney transplants from 57 standard-criteria donors were classified into 4 groups by RIFLE (Risk, Injury, Failure, Loss of function, and End-stage renal disease) classification according to donor AKI severity before kidney procurement. The donor and recipient characteristics and graft outcomes were compared. RESULTS: Of 84 kidney transplants, 56, 11, 10, and 7 recipients were in the Non-AKI, Risk, Injury, and Failure groups. The mean terminal creatinine was 1.1, 1.6, 2.3, and 4.4 mg/dL in these 4 groups. However, the graft outcomes, including primary nonfunction rate, delayed graft function rate, acute rejection rate, renal function, graft survival and overall survival over the first 5 years had no statistical difference. A trend toward increasing delayed graft function rate as the severity of AKI increased was observed (Non-AKI, Risk, Injury, and Failure: 26.8%, 36.4%, 60.0%, and 57.1%, P = .099). CONCLUSIONS: Our study demonstrates that AKI before procurement does not cause adverse long-term graft outcomes. Standard-criteria donors with AKI are suitable for kidney transplantation, even with a high severity of AKI.

Long-term outcomes of living kidney donors over the past 28 years in a single center in Taiwan.

BACKGROUND: The chronic shortage of kidneys for transplantation has increased the number of living donations, but demand remains high, which has created a long waiting list of end-stage kidney disease patients. Donors with decreased renal mass may suffer a higher risk of developing proteinuria, hypertension (HTN), and chronic renal disease (CKD) during long-term follow-up. METHODS: We retrospectively retrieved medical data of living kidney donors at our hospital over the past 28 years. RESULTS: There were 45 male and 60 female donors with a mean donation age of 46.34 ± 12.47 years (range = 20-70y). The mean follow-up duration was 4.67 ± 4.78 years. The serum creatinine (Cr) at donation was 0.93 ± 0.22 mg/dL, while the latest Cr was 1.26 ± 0.45 mg/dL (P < .001). The mean age at follow-up was 50.95 ± 14.57 years. At last follow-up, eight subjects (7.6%) displayed HTN requiring treatment, 10 (9.5%), proteinuria and 55.4%, an estimated glomerular filtration rate (eGFR) of less than 60 mL/min, including one with diabetic nephropathy at 10 years after donation who required long-term hemodialysis. Although gender did not correlate with occurrence of HTN, proteinuria, and CKD, the occurrence of CKD was associated with age at donation (P < .001, odds ratio [OR] = 1.076), and age at follow-up (P < .001, OR = 1.071). HTN donors were older (P = .036, OR = 1.057) with longer follow-up durations (P = .007, OR = 1.166) and had higher Cr values at donation (P = .044, OR = 94.4). Donors with proteinuria were not related to gender, follow-up duration, initial Cr, warm ischemic time, or duration of admission. eGFR was indeed worse after donation (P = .002). CONCLUSIONS: Our results indicated a significant proportion of living donors may develop CKD upon long-term follow-up. The factors affecting donor risk of CKD were baseline renal function, older age, and duration after kidney donation.

In vivo monitoring of quantum dots in the extracellular space using push-pull perfusion sampling, online in-tube solid phase extraction, and inductively coupled plasma mass spectrometry.

To monitor the dynamic changes of extracellular quantum dots (QDs) in vivo in the livers of anesthetized rats, we developed an automatic online analytical system comprising push-pull perfusion (PPP) sampling, the established in-tube solid phase extraction (SPE) procedure, and inductively coupled plasma mass spectrometry (ICPMS). The method takes advantage of the retention of QDs onto the interior surface of a polytetrafluoroethylene (PTFE) tube as a means of extracting the QDs from complicated push-pull perfusates. For the injected QDs present in the liver extracellular fluid (ECF) at low picomolar levels, a temporal resolution of 10 min was required to collect sufficient amounts of QDs to meet the sensitivity requirements of the ICPMS system. To the best of our knowledge, this study is the first to exploit the PPP technique for the collection of QDs from living animals and PTFE tubing as a SPE adsorbent for the online extraction of QDs and the removal of biological matrix prior to ICPMS analysis of cadmium-containing inorganic nanocrystal. We confirmed the analytical reliability of this method from measurements of the spike recoveries of saline samples; in addition, we demonstrated the systems' applicability through in vivo monitoring of the time-dependent concentration profile of liver extracellular QDs in living rats after intravenous administration.

Online in-tube solid-phase extraction using a nonfunctionalized PVC tube coupled with ICPMS for in vivo monitoring of trace metals in rat brain microdialysates.

We have developed a simple, automatic, online in-tube solid-phase extraction (SPE) method for the preconcentration of trace elements from saline samples. The method takes advantage of the adsorption of trace metal ions onto the interior of a nonfunctionalized PVC tube as a means of separation from the matrix salt. The adsorption of transition metal ions is presumably dominated by Lewis acid/base interactions, which facilitate the formation of metal-PVC complexes. For simultaneous determination of multiple trace metals in the extracellular fluid from the brains of anesthetized rats, we developed an online analytical system comprising microdialysis sampling, the established in-tube extraction procedure, and ICPMS. In the extraction step, the efficiency was optimal when the pH of the sample was adjusted to 8.0 using phosphate buffer. After extraction onto the interior of PVC tube, the adsorbed analytes were eluted with 0.5% HNO 3 prior to online ICPMS measurement. For those elements present in the extracellular fluid at less than nanogram-per-milliliter concentrations (i.e., Cu, Zn, and Mn), a temporal resolution of 12 min was required to collect enough microdialysate to meet the sensitivity requirements of the ICPMS instrument. Noteworthily, because washing and postconditioning the interior of PVC tube are not necessary, a relatively unsophisticated and clean procedure was attained and extremely low detection limits in the range of 5.0 to 510 ng L (-1) were thus obtained for the analysis of Mn, Cu, Zn, Pb, Cd, Ni, and Co in microdialysate samples of 8 microL by ICPMS. To the best of our knowledge, this study is the first to exploit PVC peristaltic tubing as an SPE adsorbent in the laboratory-on-valve mode for online sample treatment and trace metal preconcentration prior to ICPMS measurement. We confirmed the analytical reliability of this method through the analysis of the certified reference material SLEW-3 (estuarine water) and 2670a (human urine) and demonstrated its applicability through simultaneous in vivo monitoring of the basal concentrations and concentration profiles of multiple metal ions in the brain extracellular fluid of a living rat.

Biophysical and histological determinants underlying natural firing behaviors of splanchnic sympathetic preganglionic neurons in neonatal rats.

Isolated thoracic spinal cords of neonatal rats spontaneously generate splanchnic sympathetic nerve discharge (SND) with a quasiperiodic rhythm approximately 1-Hz. Using in vitro nerve-cord preparations that retained T6-T12 spinal segments, we investigated whether the natural firing behavior of sympathetic preganglionic neurons (SPNs) encoded the SND rhythm and what were the main biophysical and histological determinants of SPN firing. Under extracellular recording conditions, electrical stimulation of splanchnic nerves elicited antidromic responses in 212 SPNs. Among them, 92 SPNs were quiescent; 120 active SPNs had an average firing rate of 0.72+/-0.04 Hz, which was close to the quasiperiodic rhythm of SND. SPNs with rhythmic burst firing were rare. Probability plots of interspike intervals were constructed to extract mathematical features underlying SPN firing. Most active SPNs (88%) had a firing well described by unimodal Gaussian, suggesting a predominantly tonic pattern with normal variations. Biophysical properties of 112 SPNs were measured under whole-cell recording conditions. The charging time constant, tau, is positively correlated with the average firing rate. Histological properties were examined in 45 SPNs with intracellular diffusion of Lucifer Yellow or biocytin. SPNs with pyramidal somata and multipolar dendrites tend to be spontaneously active. In contrast, those with bipolar somata and fewer dendritic branches were quiescent in firing. These observations suggest that activity levels of SPNs are correlated with their capacity for temporal and spatial summation of synaptic inputs. How the seemingly tonic firing of individual SPNs is integrated into whole-nerve SND with quasiperiodic rhythms is discussed.

Comparison of effects of propofol and midazolam at sedative concentrations on sympathetic tone generation in the isolated spinal cord of neonatal rats.

BACKGROUND: Propofol and midazolam are common sedatives for critically ill patients. Little is known about the effects of propofol and midazolam on central sympathetic activity when drug concentrations in extracellular milieu are under precise control. Previous work using an in vitro neonatal rat splanchnic nerve-spinal cord preparation has demonstrated that tonic sympathetic activity is generated spontaneously in the thoracic spinal cord. The aim of this study was to investigate the concentration effects of propofol and midazolam on spinally generated sympathetic activity. METHODS: Using an in vitro neonatal rat splanchnic nerve-spinal cord preparation that allows the precise control of drug concentrations, the central sympathetic effects elicited by the application of propofol (10-640 microM) and midazolam (10-640 microM) were compared. RESULTS: There was a prompt decrease in sympathetic activity on application of propofol or midazolam in a concentration-dependent manner. A significant decrease in sympathetic activity was observed on application of propofol at 80-640 microM; however, the application of propofol at 10-40 microM caused only a slight alteration in activity. The sympathetic activity was not altered significantly by 10 microM of midazolam, but the application of midazolam at 20-640 microM caused a significant decrease in activity. Thus, in these experimental conditions, the minimum concentration of propofol causing a significant decrease in sympathetic activity was 80 microM and that of midazolam was 20 microM. CONCLUSIONS: The current findings suggest that the administration of 9-19 microM of propofol or 0.7-0.9 microM of midazolam, the clinically relevant concentrations for sedation, does not alter central sympathetic outflow at the spinal cord level. However, propofol at a concentration of 86 microM, which could be achieved by a single-bolus loading dose to induce sedation, depresses central sympathetic activity.

On-line HPLC-UV/Nano-TiO2-ICPMS system for the determination of inorganic selenium species.

We have developed an UV/nano-TiO2 vapor generation (VG) device that when coupled between a chromatographic column and an ICP mass spectrometer provides a simple and sensitive hyphenated method for the determination of Se(IV) and Se(VI) without the need to use conventional chemical VG techniques. Because our proposed VG device allows both Se(IV) and Se(VI) species in the column effluent to be converted on-line into volatile Se products, which are then measured directly by the ICPMS, the safety risks and the probability of contamination arising from the use of additional chemicals are both low. To achieve the maximum signal intensity, we optimized a number of the operating parameters of the UV/nano-TiO2 VG device, including the acidity, the amounts of TiO2 and formic acid, and the length of the reaction coil, with respect to their effects on the reduction efficiency of the analyte species. This hyphenated method achieves excellent detection limits-0.06 and 0.03 ng mL(-1) for Se(IV) and Se(VI), respectively-because of the high efficiencies of the conversions of Se(IV) and Se(VI) to their respective volatile products and the lower blank level achieved, relative to those of other traditional systems. In addition, because the conversion efficiency of the analyte selenium species reached its maximum level within 36 s of irradiation, the working time (approximately 12 min) was limited primarily by time required for the chromatographic separation. A series of validation experiments-analysis of the 1643e Standard Reference Material and natural water samples-indicated that our proposed methods can be applied satisfactorily to the determination of inorganic selenium species in water samples.

Combination regimen in the treatment of chronic prostatitis.

The aim of the study was to report our experiences in the treatment of chronic prostatitis using combination regimen including ciprofloxacin, doxazosin, allopurinol and biofeedback perineal massage. From May 2003 to April 2004, 7 patients with NIH Category II-chronic bacterial prostatitis and 7 patients with NIH Category IIIA-inflammatory chronic pelvic pain syndrome were treated. The NIH-Chronic Prostatitis Symptom Index (NIH-CPSI) was scored by the patient before and after the treatment, 6 months later. In Category II patients, the bacterial eradication rate was 71% after ciprofloxacin treatment during a follow-up of 6 months. The beneficial response rate to allopurinol, doxazosin and biofeedback perineal massage was 50%, 42% and 85%, respectively. In NIH Category IIIA patients, the individual beneficial response rate to ciprofloxacin, allopurinol, doxazosin and biofeedback perineal massage was 57%, 100%, 71% and 100%, respectively. Comparing pre-treatment and post-treatment results of the combination regimen, there was a statistically significant improvement in the 3 domains of pain score, urinary symptoms and quality of life impact of the NIH-CPSI. Combination regimen including ciprofloxacin, doxazosin allopurinol and biofeedback perineal massage in the treatment of chronic prostatitis is a safe and effective modality in our limited experience.

Mechanisms underlying the cardioinhibitory and pressor responses elicited from the medullary neurons in the gigantocellular tegmental field of cats.

A stimulation of the gigantocellular tegmental field (FTG) in the medulla oblongata often increases systemic arterial blood pressure (SAP) and decreases heart rate (HR). We investigated if the cardioinhibitory/depressor areas, including the nucleus ambiguus (NA), the dorsal motor nucleus of vagus (DMV) and the caudal ventrolateral medulla (CVLM), underlied the functional expression of FTG neurons in regulating cardiovascular responses. In 73 chloralose-urethane anesthetized cats, the HR, SAP and vertebral nerve activity (VNA) were recorded. Neurons in the FTG, NA, DMV and CVLM were stimulated by microinjection of sodium glutamate (25 mM Glu, 70 nl). To study if the NA, DMV, and CVLM relayed the cardioinhibitory messages from the FTG, 24 mM kainic acid (KA, 100 nl) was used as an excitotoxic agent to lesion neurons in the NA, DMV or CVLM. We found that the cardioinhibition induced by FTG stimulation was significantly reduced by KA lesioning of the ipsilateral NA or DMV. Subsequently, a bilateral KA lesion of NA or DMV abolished the cardioinhibitory responses of FTG. Compared to the consequence of KA lesion of the DMV, only a smaller bradycardia was induced by FTG stimulation after KA lesion of the NA. The pressor response induced by Glu stimulation of the FTG was reduced by the KA lesion of the CVLM. Such an effect was dominant ipsilaterally. Our findings suggested that both NA and DMV mediated the cardioinhibitory responses of FTG. The pressor message from the FTG neurons might be partly working via a disinhibitory mechanism through the depressor neurons located in the CVLM.

The nNOS/cGMP signal transducing system is involved in the cardiovascular responses induced by activation of NMDA receptors in the rostral ventrolateral medulla of cats.

Nitric oxide (NO) is synthesized from L-arginine by NO synthase (NOS). NO stimulates the soluble form of guanylyl cyclase (sGC) and induces accumulation of cyclic guanosine monophosphate (cGMP). The purpose of this study was to examine whether the cardiovascular responses induced by N-methyl-D-aspartate (NMDA) in the rostral ventrolateral medulla (RVLM) depend on the actions of NOS and sGC. In anesthetized cats, the extracellular NO level was measured by in vivo voltammetry using a nafion/porphyrine/o-phenylenediamine-coated carbon-fiber electrode. Microinjection of NMDA into the RVLM produced hypertension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 7-nitroindazole, a neuronal NO synthase (nNOS) inhibitor, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a sGC inhibitor. These findings suggest that NO is involved in the NMDA-induced cardiovascular responses in the RVLM.

Intraspinal amino acid neurotransmitter activities are involved in the generation of rhythmic sympathetic nerve discharge in newborn rat spinal cord.

Endogenous neurotransmitter activities underlying the sympathetic nerve discharge (SND) generated by newborn rat spinal cord in vitro were investigated using glutamatergic, glycinergic, and GABAergic antagonists. Under control conditions, the SND power spectrum had two major frequency components: synchronous bursting SND (bSND) with power dominant at < 0.1 Hz and quasiperiodic SND (qSND) oscillating at 1-2 Hz. Using high Mg2+ solution (12-24 mM) to block Ca2+-dependent synaptic transmission reversibly abolished SND. An interruption of glutamatergic neurotransmission by CNQX (non-NMDA receptor blocker) or L-AP4 (reducing the synaptic release of glutamate) failed to affect qSND, but consistently reduced bSND. Application of kynurenate, a broad-spectrum ionotropic glutamate receptor blocker, only caused an unstable SND but did not reduce SND. In contrast, strychnine (Stry, glycine receptor antagonist) consistently reduced qSND in a dose-dependent manner. Bicuculline (Bic, GABA(A) receptor antagonist) induced a synchronous bSND of irregular rhythm, which could be further regularized by adding Stry. Bic-induced bSND was reversibly abolished by CNQX or L-AP4. In conclusion, intraspinal glycinergic, GABAergic, and glutamatergic activities are involved in the generation of the spinal cord-derived SND in newborn rats. Intraspinal GABAergic interneurons may tonically inhibit the glutamatergic bursting neurons that generate a synchronous bSND. Activities of these glutamatergic bursting neurons may also be modulated by intraspinal glycinergic interneurons.

Activation of noradrenergic mechanism attenuates glutamate-induced vasopressor responses in the pons and medulla of cats in vivo.

1. Using anesthetized cats, the authors examined the noradrenergic modulation of the glutamate induced pressor and depressor responses in various brainstem areas, including pontine gigantocellular tegmental field (FTG), dorsomedial medulla (DM), rostral ventrolateral medulla (RVLM), and caudal ventrolateral medulla (CVLM). 2. Unilateral microinjection of L-glutamate (Glu, 3 nmol in 30 nL saline) into FTG, DM and RVLM produced an increase in systemic arterial pressure (SAP) and a decrease in heart rate (HR), while into CVLM produced decreases of SAP and HR. 3. Application of norepinephrine (NE) into the pressor areas (0.05 to 5 nmol) did not alter the resting SAP and HR, but significantly attenuated the Glu-induced pressor response with an order of potency: FTG > DM > RVLM. In the depressor CVLM, NE alone produced a dose-dependent decrease of resting SAP and HR, but did not affect the Glu-induced depressor responses. 4. The involvement of different adrenoceptor subtypes was further investigated by application of selective adrenoceptor agonists including phenylephrine (alpha1), clonidine (alpha2), and isoproterenol (beta). Responses to these agonists are similar to those elicited by NE, except that only alpha-adrenoceptor agonists could antagonize the Glu-induced pressor responses of the RVLM. 5. Our observations indicate that NE not only inhibits the pressor mechanisms in various brainstem areas but also elicits a direct depressor response in CVLM. These findings also suggest that NE acts more likely a neurotransmitter, rather than a modulator, in the CVLM.

Rhythmic sympathetic nerve discharges in an in vitro neonatal rat brain stem-spinal cord preparation.

To understand the origination of sympathetic nerve discharge (SND), I developed an in vitro brain stem-spinal cord preparation from neonatal rats. Ascorbic acid (3 mM) was added into the bath solution to increase the viability of preparations. At 24 degrees C, rhythmic SND (recorded from the splanchnic nerve) was consistently observed, but it became quiescent at <16 degrees C. Respiratory-related SND (rSND) was discernible and was well correlated with C(4) root activity. Power spectral analysis of SND revealed a dominant 2-Hz oscillation. In most preparations (86%), such oscillation was persistent, whereas it only slightly reduced its magnitude after isolation from the brain stem. The removal of neural structures rostral to the superior cerebellar artery (equivalent to the level of facial nuclei) reduced rSND, increased tonic SND, but did not affect the temporal coupling between SND and C(4) root activity. Our data suggest a prominent contribution of SND from the neural mechanisms confined within the neonatal rat spinal cord. This ascorbic acid-enhanced in vitro preparation is a very useful model to study neural mechanisms underlying sympathorespiratory integration.

Correlation of vasomotor- and respiratory-controlling mechanisms around the caudal ventrolateral medulla in cats.

We examined the involvement of caudal ventrolateral medulla (CVLM) in respiratory control. Microinjection of glutamate (Glu) into CVLM decreased systemic arterial blood pressure (SAP) and altered phrenic nerve activities (PNA). Among 143 depressor sites, 55% (78/143) increased respiratory frequency (Rf), while 72% altered PNA amplitude (36% increased and 36% decreased). A small but significant positive correlation was observed between the magnitudes of depressor responses and inhibition of PNA amplitude (r = 0.1718, n = 143), indicating a substantial cross-talk between depressor and PNA inhibitory neurons. Furthermore, microinjections of acetylcholine (ACh) mimicked the Glu-induced depressor responses. However, ACh did not alter Rf, but still reduced PNA amplitude. Our findings suggest that Rf-regulating and depressor neurons are two separate neuronal populations, coexisting in CVLM. The PNA inhibitory and depressor neurons, in contrast, could have stronger correlation.

A single minute lesion around the ventral respiratory group in medulla produces fatal apnea in cats.

In 35 adult cats anesthetized with intraperitoneal chloralose and urethane, the ventrolateral medulla was explored by microinjection of kainic acid (KA, 24 mM, 200 nl) with metal electrode-tubing or glass micropipette to determine regions which elicit persistent apnea. Persistent apnea is defined as: (1) In spontaneously breathing cats, termination of respiration over 3 min with a decrease of the mean systemic arterial pressure (MSAP) to 25 mm Hg. (2) In animals under artificial ventilation and paralyzed by gallamine, cessation of bilateral phrenic nerve (PNA) activities over 25 min. The apnea producing area was located dorsal to the rostral pole of the lateral reticular nucleus, ventromedial to the ambiguous nucleus and immediately caudal to the retrofacial nucleus. Functionally, this region includes the rostral part of the ventral respiratory group (rVRG) encompassing the pre-BOtzinger area. We define this region as the VRG apnea producing area (VRG-Apa). Fatal apneusis was observed under following conditions: (1) Persistent apnea was produced after a single KA microinjection in one side of the VRG-Apa (5 animals). Microinjection of sodium glutamate (0.25 M, 70-200 nl) in the same area produced only brief apnea, while microinjection of kynurenic acid (0.1 M, 200 nl) showed little effect on the respiration but slightly increased the SAP. (2) Positioning an electrode nearby but not in the VRG-Apa with or without KA injection did not produce apnea. But when a second electrode insertion to the opposite VRG-Apa immediately produced persistent apnea even without KA injection (6 animals). (3) Midsagittal division of the medulla 0-5 mm rostral to the obex produced persistent silence of PNA on both sides in artificial ventilated animals (7 animals), while similar division 0-5 mm caudal to the obex (4 animals) produced a brief but reversible quiescence of PNA. In conclusion, findings of the present study support the existence of a restricted region of VRG-Apa. VRG-Apa on both sides are closely connected, and integrity of both VRG-Apa is essential for normal respiration.

GABAergic inhibition of neonatal rat phrenic motoneurons.

Phrenic motoneurons (PMNs) receive intermittently glutaminergic inspiratory drives and GABAergic inhibition in adult mammals. Since gamma-amino-butyric acid (GABA) might act as an excitatory amino acid in early stages of development, we here investigated if GABA(A) receptors inhibit PMNs in neonates. Using in vitro neonatal rat brainstem-spinal cord preparation, local application of GABA and muscimol (a GABA(A) receptor agonist) to the vicinity of PMNs consistently reduced the inspiratory activity of C4 ventral roots. Under whole-cell patch-clamp conditions and in the presence of 0.5 microM TTX to block synaptic transmission, muscimol (10 microM) decreased whole-cell input resistance, and surprisingly, when PMNs were voltage-clamped at their resting membrane potential, muscimol induced depolarizing-inward, rather than hyperpolarizing-outward membrane current. Our findings indicate that GABA(A) receptors mediate a depolarizing blockade of the glutaminergic excitatory inputs to neonatal rat PMNs.