Pacemaker properties of respiratory neurons of the ventrolateral medullary regions in early postnatal rats

Spike activity of respiratory neurons of the ventrolateral medullary regions was studied under conditions of blocking of synaptic transmission. The experiments were carried out on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of newborn (1st day of life) and 4- to 5-day-old rats. Part of the pre-inspiratory and (to a somewhat lesser extent) expiratory neurons of newborn rats appeared most resistive to superfusion of preparations with a low-Ca²⁺ (0.2 mM) and Mg²⁺-rich (5.0 mM) solution. Spike activity in some neurons of these groups was preserved up to 40 and 25 min, respectively, after mass inspiratory discharges in then. phrenicus had disappeared. Similar neurons in 4- to 5-day-old SIMSP were less resistive. Inspiratory neurons in animals of both age groups demonstrated no pacemaker properties. Coagulation of the regions where pre-inspiratory neurons are localized (the retrofacial zone) did not evoke irreversible blockade of respiratory rhythm in all SIMSP of 4- to-5-day-old rats and in most SIMSP of newborn animals. At the same time, coagulation of the zone where inspiratory neurons are concentrated (the pre-Bötzinger complex) resulted in the blockade of respiratory rhythm in all SIMSP, with no exceptions.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 273–284, November–December, 1996.     

Participation of the opioid- and serotonergic systems of the ventrolateral medullary regions in the control of respiratory activity in early postnatal rats

The effects of blockers of opioid and serotonin receptors (naloxone and methisergide, respectively) on the respiratory activity recorded from then. phrenicus of semi-isolated medullo-spinal preparations (SIMSP) were studied in newborn (1-day-old) and 4- to 5-day-old rats. The preparations were superfusedin situ, and inspiratory discharges (ID) were recorded before and after transverse sections of the ventrolateral medullary regions (VLMR) at different levels. Naloxone evoked an increase in the ID frequency in then. phrenicus both at the initial configuration of preparations and after successive transections of the VLMR between theM andS chemosensitive zones and between theS andL zones. The relative intensity of this effect was significantly higher in newborn rats than in 4- to 5-day-old ones. In contrast, methisergide made the ID frequency lower at all configurations of the SIMSP, and this effect was more intensive in 4- to 5-day-old animals. In the course of progressive separations of more rostral VLMR zones, the effects of naloxone and methisergide applications on the ID frequency became weaker. This allows us to suppose that in newborn rats the rostral VLMR portions provide more intensive opioidergic inhibitory control influences on the activity of respiratory networks than those in 4- to 5-day-old animals. At the same time, tonic activating influences from serotonergic VLMR neurons on the mechanisms generating respiratory rhythm are more intensive in 4- to 5-day-old animals than those in newborn ones. Thus, it can be supposed that the levels of maturation of the opioid- and serotonergic neurotransmitter systems, which provide tonic control of respiratory activity generated in the VLMR, are different in newborn and 4- to 5-day-old animals. The problems of how the opioid- and serotonergic mechanisms controlling respiratory rhythm generation are formed in the course of early stages of ontogenetic development are discussed.Neirofiziologiya/Neurophysiology, Vol. 28, No. 1, pp. 62–73, January–February, 1996.     

Localization of the medullary respiratory neurons in rats by microelectrode recording.

Neuronal activity has been recorded extracellularly from the medulla of anesthetized rats. Units whose discharge frequency varied in phase with respiratory airflow were located bilaterally between 1.5 and 2 mm lateral to midline, extending from 1 mm caudal to 1.5 mm rostral to the obex, in the ventral two-thirds of the medulla. Expiratory units predominated and were intermingled with inspiratory units. Ten different patterns of discharge were distinguished, varying from a short burst at the beginning of expiration to a resting discharge which increased in frequency during either inspiration or expiration. Evidence was also obtained that fiber tracts from other areas of the brain cross midline just caudally to the obex and pass to the respiratory centers on which they apparently exert and excitatory action.     

Topico-anatomical relations among the structures of the caudal medullary region in early postnatal rats

Anatomical and topographical organization of the structures of the caudal medullary region was studied in 3- and 21-day-old rats. A system of stereotaxic coordinates was proposed, and brain maps for the animals of these age groups were plotted. Topographic characteristics were given for the nuclei forming the medullary respiratory center: the lateral reticular nucleus,n. ambiguus, and the nuclei of the solitary tract, vagus and hypoglossal cranial nerves. These structures differ from each other in their cytoarchitectonics and in the changes in number during their neurons' postnatal development. The differences are suggested to result from the differences in morphological maturation of the neurons at early ontogenic stages.Neirofiziologiya/Neurophysiology, Vol. 26, No. 4, pp. 243–250, July–August, 1994.     

肾缺血增强大鼠延髓腹外侧头端区神经元电话动和Fos蛋白表达

在67只切断两侧缓冲神经的麻醉Sprague-Dawley大鼠,应用细胞外记录的电生理方法和免疫组织化学技术,分别观察肾缺血对延髓腹外侧头端区巨细胞旁外侧核神经元自发放电活动和Fos蛋白表达的影响.所得结果如下(1)左肾动脉阻断后,28个单位的放电频率由11.40±1.08增至21.1±1.74spikes/s(P<0.001),血压和心率无明显变化(P>0.05);(2)在17个放电单位中,应用腺苷受体拮抗剂8-苯茶碱(8-phenyltheophylline,10mg/kg)可明显抑制肾缺血的兴奋效应(P<0.05);(3)肾缺血后,延髓腹外侧头端区的Fos蛋白样免疫反应神经元显著增加(P<0.01);(4)预先应用8-苯茶碱可明显减弱肾缺血所激活的Fos蛋白表达反应(P<0.05).以上结果提示肾缺血增强延髓腹外侧头端区神经元的放电活动和Fos蛋白表达,而此作用可能与肾脏缺血所产生的腺苷激活肾内感受器有关.     

辣椒素对大鼠延髓腹外侧头端区神经元电活动的影响

在35只切断两侧缓冲神经的麻醉大鼠,应用细胞外记录的电生理学方法,观察颈总动脉注射辣椒素(capsaicin)对延髓腹外侧头端区(RVLM)巨细胞旁外侧核(PGL)自发电活动的影响。所得结果如下:(1)颈动脉注射辣椒素(10μmol,01ml),MAP由1074±013升至1256±021kPa(P<0001);HR由374±4增至395±5bpm(P<0001);30个PGL神经元自发放电单位的放电频率由126±07增至209±11spikes/s(P<0001)。(2)在10个放电单位,应用辣椒素受体阻断剂钌红(rutheniumred;200mmol,01ml)后,明显抑制辣椒素的上述效应。以上结果提示,辣椒素可能通过激活RVLM神经元上的辣椒素受体,进而兴奋PGL神经元     

内皮素通过最后区易化大鼠延髓腹外侧头端区神经元活动

在３５只切断双侧缓冲神经、用氨基甲酸乙酯－α氯醛糖混合麻醉的Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠,应用细胞外记录的电生理学方法,由ＲＭ－６０００型多道生理记录仪和ＷＳ－６８２Ｇ热阵记录器（频响范围０～２．８ｋＨｚ）同步记录血压、心率和单位神经元放电,观察颈动脉注射内皮素对８７个延髓腹是头端区（ＲＶＬＭ）自发放电神经元活动的影响,所得结果如下;（１）颈动脉注射ＥＴ－１（０．３ｎｍｏｌ／ｋｇ）时３６个单位     

Roles of NMDA receptors and nitric oxide in responses of the medullary respiratory generator of early postnatal rats to hypoxia and acidosis

The dynamics of changes in the frequency of the respiratory activity recorded from the n. phrenicus under conditions of 3-min-long applications of 5 μM N-methyl-D-aspartate (NMDA), an anoxic gas mixture-saturated saline, or an acidified (pH 7.0) solution were studied in the experiments on superfusedin situ semi-isolated medullo-spinal preparations (SIMSP) of 3- to 4-day-old rats. Test applications were performed on the intact SIMSP or on those preliminarily influenced by the following substances: a non-competitive NMDA receptor blocker, ketamine (10 μM); an inhibitor of NO synthase, methyl ester of N^G-nitro-L-arginine (MENA, l0 μM); hemoglobin, which binds NO (Hb, 0.3 μM); an NO donor, sodium nitroprusside (SNP, 10 μM); or/and a competitive blocker of non-NMDA receptors, CNQX (1.0 μM). Application of NMDA increased the frequency of the respiratory discharges, and the effect was blocked by MENA, Hb, and SNP. Addition of Hb to the SNP-containing solution neutralized the effect of the latter. In hypoxia, ketamine blocked an increase in the respiratory frequency within the initial 90-sec segment of the test and decreased the rhythm suppression within the second test half. MENA increased the respiration discharge frequency throughout the test. CNQX exerted no Influence on the frequency in the initial period and decreased its suppression within the second test half. Preliminary ketamine and MENA applications made smaller the increment of the discharge frequency at application of the solution with pH 7.0; the MENA effect was stronger. In addition, using a histochemical technique, we studied spatial distribution of the neurons containing an NO synthase marker, NADPH-diaphorase (NADPH-d), in frontal sections of the medulla of 4-day-old rats. NADPH-d-positive cells were observed within the limits of the dorsal and ventral respiratory neuronal groups (DRG and VRG, respectively). Their density was the highest in the rostral VRG part (in the region of the lateral paragigantocellular nucleus). Our results show that in early postnatal rats NMDA receptors and endogenous NO are actively involved in the control of respiratory rhythm generated by SIMSP under hypoxic and acidotic conditions. The results of morphohistochemical study can be considered a neuroanatomical support for the active NO role in the control of medullary respiratory rhythm in the early postnatal period.     

颈动脉注射17β -雌二醇对去缓冲神经大鼠延髓腹外侧头端区神经元放电活动的影响

本研究旨在观察17β-雌二醇(E2)对雄性大鼠延髓腹外侧头端区(RVLM)神经元自发放电活动的影响.在切断双侧缓冲神经的麻醉雄性Sprague-Dawley大鼠上,同步记录血压、心率和RVLM神经元的自发放电活动.颈动脉内注射E2 (10 ng/kg),30个RVLM神经元自发放电单位中有25个单位的放电频率由14.46±0.47降至9.73±0.33 spikes/s (P<0.05),与此同时血压和心率无明显改变.E2的抑制效应在1 min内起效,持续时间长于5 min.雌激素受体拮抗剂tamoxifen (5 mg/kg)不能阻断E2 的抑制效应.预先给予一氧化氮(NO)合酶阻断剂L-NAME (2.7 μg/kg)能明显阻断E2的抑制效应.应用NO供体SIN-1 (0.5 μg/kg)可增强E2的抑制效应.以上结果提示,E2可通过非基因组效应激活RVLM神经元的NOS而引发NO释放,进而抑制其自发放电活动.     

NADPH-Diaphorase-containing neurons in the medullary structures involved in generation of the respiratory activity in neonatal rats

Using a histochemical technique, we examined distribution of the neurons containing a marker of nitric oxide synthase (NOS), NADPH-diaphorase (NADPH-d), on frontal slices of the medulla and upper cervical spinal segments of 4-day-old rats. It was demonstrated that NADPH-d-positive cells are present within the dorsal and ventral medullary respiratory groups. The highest density of the labeled middle-size multipolar neurons (27.9±2.6 cells per 0.1 mm² of the slice) was observed in the rostral part of the ventral respiratory group, within the reticular lateral paragigantocellular nucleus. Similar NADPH-d-positive neurons were also observed in other reticular formation structures: rostroventrolateral reticular, gigantocellular, and ventral medullary nuclei, and in the ventral part of the paramedial nucleus. There were no labeled neurons in the lateral reticular nucleus. Single small and medium-size labeled neurons were found at all rostro-caudal levels of thenucl. ambiguous (nuclei retrofacialis, ambiguous, andretroam-biguous). Groups of NADPH-d-positive neurons were also revealed within the dorsal respiratory group, along the whole length of thenucl. tractus solitarii (mostly in its ventrolateral parts). Single labeled neurons were also observed in thenucl. n. hypoglossi, and their groups were observed in the dorsal motor part of thenucl. n. vagus. Involvement of the structures containing NADPH-d-positive neurons in the processes related to generation of the respiratory activity is discussed. Our neuroanatomical experiments prove that in early postnatal mammals NO is actively involved in generation and regulation of the medullary respiratory rhythm. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 128–136, March–April, 2000.     

Voltage-activated potassium currents in the somatic membrane of sensory neurons of early postnatal rats

Transmembrane ion currents were studied in the somatic membrane of freshly isolated neurons from the spinal ganglia of early postnatal (younger than 15-day-old) rats. According to their dissimilar voltage dependence and different sensitivity to external application of tetraethylammonium (TEA) and 4-aminopyridine (4-AP), three types of outward potassium currents were identified. Fast-inactivating K⁺ current was activated at the most negative values of the membrane potential and showed the highest sensitivity to external application of 4-AP. The threshold for activation of slow-inactivating K⁺ current was within a −40 ... −30 mV range. Non-inactivating delay-rectified current showed the highest sensitivity to TEA. All three types of K⁺ currents could be found in all studied neurons of animals of three age groups: 1, 5 to 6, and 14 to 15 postnatal days. The mean density of fast-inactivating K⁺ current significantly increased during the first two weeks of postnatal ontogenesis. Within the studied period, the mode of a normal (Gaussian) distribution of fast K⁺ current shifted toward higher current density values. The mean density of slow-inactivating K⁺ current also increased with the age. Yet, the mean density of non-inactivating delay-rectified K⁺ current significantly dropped during the first five days of the postnatal development and remained stable during the following time interval.     

Enhanced catabolism to acetaldehyde in rostral ventrolateral medullary neurons accounts for the pressor effect of ethanol in spontaneously hypertensive rats

We have previously shown that ethanol microinjection into the rostral ventrolateral medulla (RVLM) elicits sympathoexcitation and hypertension in conscious spontaneously hypertensive rats (SHRs) but not in Wistar-Kyoto (WKY) rats. In this study, evidence was sought to implicate the oxidative breakdown of ethanol in this strain-dependent hypertensive action of ethanol. Biochemical experiments revealed significantly higher catalase activity and similar aldehyde dehydrogenase (ALDH) activity in the RVLM of SHRs compared with WKY rats. We also investigated the influence of pharmacological inhibition of catalase (3-aminotriazole) or ALDH (cyanamide) on the cardiovascular effects of intra-RVLM ethanol or its metabolic product acetaldehyde in conscious rats. Compared with vehicle, ethanol (10 μg/rat) elicited a significant increase in blood pressure in SHRs that lasted for the 60-min observation period but had no effect on blood pressure in WKY rats. The first oxidation product, acetaldehyde, played a critical role in ethanol-evoked hypertension because 1) catalase inhibition (3-aminotriazole treatment) virtually abolished the ethanol-evoked pressor response in SHRs, 2) intra-RVLM acetaldehyde (2 μg/rat) reproduced the strain-dependent hypertensive effect of intra-RVLM ethanol, and 3) ALDH inhibition (cyanamide treatment) uncovered a pressor response to intra-RVLM acetaldehyde in WKY rats similar to the response observed in SHRs. These findings support the hypothesis that local production of acetaldehyde, due to enhanced catalase activity, in the RVLM mediates the ethanol-evoked pressor response in SHRs.     

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 μm² ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 μm² ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.     

Effects of angiotensin II on the spontaneous activity of rostral ventrolateral medullary cardiovascular neurons and blood pressure in spontaneously hypertensive rats

The interactive role of rostral ventrolateral medulla (RVL) cardiovascular neurons and brain angiotensin II (Ang II) in regulating the arterial blood pressure was examined by recording simultaneously the spontaneous activity of these spinal projecting neurons and the arterial blood pressure in the pentobarbital-anesthetized spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar Kyoto rat (WKY). It was found that Ang II elicited dose-dependent excitatory responses in a subpopulation of RVL cardiovascular neurons, followed by a subsequent increase in blood pressure. These effects of Ang II were significantly greater in SHR than in WKY. The effects were attenuated or abolished by co-administration of Ang II antagonist, [Sar¹, Ile⁸]-Ang II. Pre-administration of [Sar¹, Ile⁸]-Ang II to RVL using bilateral microinjection attenuated the blood pressure effects of intracerebroventricularly administered Ang II by as much as 70%. These results indicated that spinal projecting RVL cardiovascular neurons are important in mediating the pressor action of Ang II. The enhanced sensitivity and responsiveness of RVL cardiovascular neurons to Ang II may be pertinent to the genesis of hypertension in adult SHR.     

Breathing of awake goats during prolonged dysfunction of caudal M ventrolateral medullary neurons

Forster, H. V., L. G. Pan, T. F. Lowry, T. Feroah, W. M. Gershan, A. A. Whaley, M. M. Forster, and B. Sprtel. Breathing ofawake goats during prolonged dysfunction of caudal M ventrolateral medullary neurons. J. Appl. Physiol.84(1): 129-140, 1998.Cooling the caudal M ventrolateralmedullary (VLM) surface for 30 s results in a sustained apnea inanesthetized goats but only a 30% decrease in breathing in awakegoats. The purpose of the present study was to determine, in the awakestate, the effect of prolonged (minutes, hours) caudal M neuronaldysfunction on eupneic breathing andCO₂ sensitivity. Dysfunction wascreated by ejecting excitatory amino acid receptor antagonists or aneurotoxin on the VLM surface through guide tubes chronically implantedbilaterally on a 10- to 12-mm²portion of the caudal M VLM surface of 12 goats. Unilateral and bilateral ejections (1 µl) of selective antagonists forN-methyl-D-aspartic acid ornon-N-methyl-D-asparticacid receptors had no significant effect on eupneic breathing orCO₂ sensitivity. Unilateralejection of a nonselective excitatory amino acid receptor antagonistgenerally had no effect on eupneic breathing orCO₂ sensitivity. However, bilateral ejection of this antagonist resulted in a significant 2-Torrhypoventilation during eupnea and a significant reduction inCO₂ sensitivity to 60 ± 9% ofcontrol. Unilateral ejection of the neurotoxin kainic acid initiallystimulated breathing; however, breathing then returned to near controlwith no incidence of apnea. After the kainic acid ejection,CO₂ sensitivity was reducedsignificantly to 60 ± 7% of control. We conclude that in the awakestate a prolonged dysfunction of caudal M VLM neurons results incompensation by other mechanisms (e.g., carotid chemoreceptors, wakefulness) to maintain near-normal eupneic breathing, butcompensation is more limited for maintainingCO₂ sensitivity.

     

Differing activities of medullary respiratory neurons in eupnea and gasping

Our purpose was to compare further eupneic ventilatory activity with that of gasping. Decerebrate, paralyzed, and ventilated cats were used; the vagi were sectioned within the thorax caudal to the laryngeal branches. Activities of the phrenic nerve and medullary respiratory neurons were recorded. Antidromic invasion was used to define bulbospinal, laryngeal, or not antidromically activated units. The ventilatory pattern was reversibly altered to gasping by exposure to 1% carbon monoxide in air. In eupnea, activities of inspiratory neurons commenced at various times during inspiration, and for most the discharge frequency gradually increased. In gasping, the peak discharge frequency of inspiratory neurons was unaltered. However, all commenced activities at the start of the phrenic burst and reached peak discharge almost immediately. The discharge frequencies of all groups of expiratory neurons fell in gasping, with many neurons ceasing activity entirely. These data are consistent with the hypothesis that brain stem mechanisms controlling eupnea and gasping differ fundamentally.     

Effect of neonatal androgenization on the septal neurons of the brain in female rats in early postnatal ontogeny

Newborn female rats were androgenized, and the reaction of neurons of brain septum on excessive quantity of exogenous androgens, introduced during so-called "crucial" period of formation of centers of gonadotropic regulation of sexual cycles, has been studied in 3, 5, 7, 10, 20, 30, and 60 days old animals. Morphometry of brain septum cell nuclei revealed that most neuron nuclei shrink after androgenization. Monoamine content was significantly increased in septum nuclei of experimental animals. Neonatal androgenization led to the increased capacity of septal complex neurons to bind 3H-estradiol and to the decreased 3H-testosterone binding. The data obtained suggest that the brain septum neurons of female rats depend on sex steroids, particularly during "crucial" period of development.