Ventilatory effects of glial dysfunction in a rat brain stem chemoreceptor region

Glia are thoughtto be important in brain extracellular fluid ion and pH regulation, buttheir role in brain stem sites that sense pH and stimulate breathing isunknown. Using a diffusion pipette, we administered the glial toxin,fluorocitrate (FC; 1 mM) into one such brain stem region, theretrotrapezoid nucleus (RTN) for 45-60 min. This dose and timeperiod were chosen so that the effects of FC would be largelyreversible. Within minutes, tissue pH decreased, and respiratory outputincreased. Both recovered almost completely after cessation of FCadministration. The response to systemicCO₂ stimulation was unaffected byFC treatment compared with that following control diffusion. Anatomicanalysis showed, at the center of FC administration, some small (meandiameter = 5.1 µm) cells that stained for DEAD Red, a marker foraltered cell membrane permeability, and some fragmented glia (glialfibrillary acidic protein immunohistochemistry). The average RTN tissuevolume that contained such DEAD Red-positive cells was 271 nl, ~23%of the volume of one RTN region. Reversible disruption of glia in theRTN, a region known to contain central chemoreception, results in anacidic local pH and in stimulation of respiratory output.

     

NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat

N-methyl-D-aspartate(NMDA) glutamate receptors mediate critical components ofcardiorespiratory control in anesthetized animals. The role of NMDAreceptors in the ventilatory responses to peripheral and centralchemoreceptor stimulation was investigated in conscious, freelybehaving rats. Minute ventilation(E)responses to 10% O₂, 5%CO₂, and increasing intravenousdoses of sodium cyanide were measured in intact rats before and afterintravenous administration of the NMDA receptor antagonist MK-801 (3 mg/kg). After MK-801, eupcapnic tidal volume(VT) decreased while frequencyincreased, resulting in a modest reduction inE.Inspiratory time (TI) decreased, whereas expiratory time remained unchanged. TheE responsesto hypercapnia were qualitatively similar in control and MK-801conditions, with slight reductions in respiratory drive (VT/TI)after MK-801. In contrast, responses to hypoxia were markedly attenuated after MK-801 and were primarily due to reduced frequency changes, whereas VT wasunaffected. Sodium cyanide doses associated with significantEincreases were 5 and 50 µg/kg before and after MK-801,respectively. Thus 1-log shift to the right of individual dose-responsecurves occurred with MK-801. Selective carotid body denervation reducedE duringhypoxia by 70%, and residual hypoxic ventilatory responses wereabolished after MK-801. These findings suggest that, in conscious rats,carotid and other peripheral chemoreceptor-mediated hypoxic ventilatoryresponses are critically dependent on NMDA receptor activation and thatNMDA receptor mechanisms are only modestly involved during hypercapnia.

     

Hemodynamic effects of hypertonic saline in the conscious rat

The present study examines the role of vasopressin and the sympathetic nervous system on the hemodynamic effects of an infusion of hypertonic saline (NaCl 1.5 M) in conscious rats. The cardiovascular response to hypertonic saline was similar in both untreated and hexamethonium-pretreated rats. Mean arterial pressure increased by 15 mmHg as a consequence of the elevation of total peripheral resistance, while cardiac index was decreased. The administration of an antagonist to the pressor activity of vasopressin in rats with intact reflexes, partially decreased mean arterial pressure and total peripheral resistance and increased cardiac index toward basal values. In contrast, the hemodynamic response to hypertonic saline was totally reverted when the vasopressin antagonist was injected in the hexamethonium-pretreated rats. The results of the present study indicate that the hypertensive response induced by hypertonic saline in conscious rats is due to the vasoconstrictor effects of both vasopressin and the sympathetic nervous system.     

Urinary bladder function in conscious rat pups: a developmental study

Cystometric studies of bladder function in anesthetized neonatal rats have suggested specific changes in urodynamic parameters that coincide with the development of a mature bladder-to-bladder micturition reflex. Here, we used a conscious cystometry model that avoids the potentially confounding effects of anesthesia to characterize voiding patterns and urodynamic parameters during early postnatal development in healthy rat pups. Cystometry was performed on postnatal day (P)0, 3, 7, 14, and 21 rats with continuous intravesical instillation of NaCl via a bladder catheter. Micturition cycles were analyzed with respect to voiding pattern, nonvoiding contractions, infused volume, and basal, filling, threshold, and micturition pressures. Reproducible micturition patterns were obtained from all age groups. The time from stimulation to contraction was significantly longer (P ≤ 0.001) in ≤1-wk-old rats (～10 s) than that in older rats (～3 s). An interrupted voiding pattern was observed in ≤10-day-old subgroups. Micturition pressure progressively increased with age (from 21.77 ± 1.92 cmH(2)O at P0 to 35.47 ± 1.28 cmH(2)O at P21, P ≤ 0.001), as did bladder capacity. Nonvoiding contractions were prominent in the P3 age group (amplitude: 4.6 ± 1.3 cmH(2)O, frequency: ～4.0 events/100 s). At P7, the pattern of spontaneous contractions became altered, acquiring a volume-related character that persisted in a less prominent manner through P21. Bladder compliance increased with age, i.e., maturation. In conclusion, conscious cystometry in rat pups resulted in reproducible micturition cycles that yielded consistent data. Our results revealed immature voiding and prolonged micturition contractions during the first 10 neonatal days and provide evidence for age-related changes in urodynamic parameters.     

MicroPET imaging of noxious thermal stimuli in the conscious rat brain

Small animal positron emission tomography (microPET) has been utilized in the investigation of nociception. However, a possible drawback from previous studies is the reduced activation pattern due to the application of anesthesia. The purpose of the present study was to demonstrate a potential means of avoiding anesthesia during stimulation, as well as minimizing the confounding anesthetic effect. Sodium pentobarbital and ketamine were first evaluated to determine their effect on microPET images in the current study. [¹⁸F]-Fluorodeoxyglucose (¹⁸F-FDG) was an appropriate radiotracer to reveal activated regions in rat brains. Pentobarbital anesthesia significantly reduced ¹⁸F-FDG uptake in neural tissues, blurrier to lower contrast; therefore, ketamine was used to anesthetize animals during microPET. After the rats were anesthetized and secured in a laboratory-made stereotaxic frame, a simple, noninvasive stereotaxic technique was used to position their heads in the microPET scanner and to roughly conform the images in the stereotaxic atlas. For functional imaging, conscious rats were restrained in cages with minimal ambient noise; short repetitive thermal stimuli were applied to each rat's tail subsequently. The rats were adequately anesthetized with ketamine following 30 min of scanning without stimulation. An activation index (AI) was calculated from microPET data to quantify the local metabolic activity changes according to the normalized ¹⁸F-FDG dosage. The average AI indicated a side-to-side difference for all innocuous stimulations in the thalamus. However, such side-to-side difference was only observed for noxious heat and cold stimulations in primary somatosensory cortex (SI), secondary somatosensory cortex (SII), and agranular insular cortex (AIC). The present study demonstrated the feasibility of the microPET technique to image metabolic functions of the conscious rat brain, offering better rationale and protocol designs for future pain studies.     

Glia and glial polyamines. Role in brain function in health and disease

The roles of glia and polyamines (PA) in brain function and dysfunction are highlighted in this review. We emphasize that PA accumulation preferentially in glia, but not in neurons, is clearly evolutionarily determined; it is found throughout the brain, retina, peripheral nervous system, and in glial-neuronal co-cultures of multiple species, including man. This phenomenon raises key questions: (i) What are the mechanisms that underlie such uneven distribution, accumulation and release from glia? (ii) What are the consequences of PA fluxes within the brain on neuronal function? (iii) What are the roles of PAs in brain disorders and diseases? This review includes suggestions on the roles of PAs, such as putrescine (PT), spermidine (SPD), spermine (SPM) and their derivatives as novel glio-transmitters in brain since PA affect many neuronal and glial receptors, channels and transporters. Polyamines hitherto have been neglected, although it is evident that these molecules are key elements for normal brain function and their metabolic disorders, apparently, cause the development of many pathological syndromes and diseases. The study of endogenous PA allows one to put forward the basic principles of scientific research on glio-neuronal interactions and clinical therapies, which are based on the exclusivity of glial cells in terms of accumulation of PA and PA-dependent functions.     

Altered baroreflex function after tail suspension in the conscious rat

Experiments were performed on conscious chronically instrumented rats to determine the contribution of peripheral V2-vasopressinergic receptors in any alteration of baroreceptor reflex (BRR) sensitivity on release from 1 wk of 30 degrees head-down tilt resulting from tail suspension. Initial experiments determined changes in plasma volume (PV) occurring over this period by use of the Evans Blue dye dilution technique. PV was determined immediately before tail suspension and on day 7 of the stimulus. PV, erythrocyte volume, and total blood volume were all significantly diminished on day 7, whereas hematocrit was unchanged. Other rats were instrumented with pulsed Doppler flow probes on the ascending aorta for determination of cardiac output and with arterial and venous catheters 7-10 days before study. Immediately before tail suspension, control cardiac output, mean arterial blood pressure, and heart rate values were determined. In addition, BRR sensitivity was estimated both before and after intravenous administration of a V2-receptor antagonist by assessing the slope of the pulse interval-mean arterial blood pressure relationship in response to a series of pressor doses of phenylephrine. BRR sensitivity was determined on the last day of head-down tilt, 1 min after release from tail suspension, and 10 min after administration of a specific V2-vasopressinergic antagonist. BRR sensitivity tended to fall on day 7 of tail suspension compared with control and was significantly increased after release. However, BRR sensitivity was not altered by intravenous V2 antagonist administration either before tail suspension or after release.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro analysis of the effects of cholecystokinin on rat brain stem motoneurons

Using whole cell patch clamp in thin brain stem slices, we tested the effects of cholecystokinin (CCK) on identified gastric-projecting neurons of the rat dorsal motor nucleus of the vagus (DMV). Perfusion with the sulfated form of CCK octapeptide (CCK8s, 30 pM-300 nM, EC50 approximately 4 nM) induced a concentration-dependent inward current in 35 and 41% of corpus- and antrum/pylorus-projecting DMV neurons, respectively. Conversely, none of the fundus-projecting DMV neurons responded to perfusion with CCK8s. The CCK8s-induced inward current was accompanied by a 65 +/- 17% increase in membrane input resistance and reversed at 90 +/- 4 mV, indicating that the excitatory effects of CCK8s were mediated by the closure of a potassium conductance. Pretreatment with the synaptic blocker TTX (0.3-1 microM) reduced the CCK8s-induced current, suggesting that a portion of the CCK8s-induced current was mediated indirectly via an action on presynaptic neurons apposing the DMV membrane. Pretreatment with the selective CCK-A receptor antagonist lorglumide (0.3-3 microM) attenuated the CCK8s-induced inward current in a concentration-dependent manner, with a maximum inhibition of 69 +/- 12% obtained with 3 microM lorglumide. Conversely, pretreatment with the selective CCK-B antagonist triglumide did not attenuate the CCK8s-induced inward current; pretreatment with triglumide (3 microM) and lorglumide (1 microM) attenuated the CCK8s-induced current to the same extent as pretreatment with lorglumide alone. Immunohistochemical experiments showed that CCK-A receptors were localized on the membrane of 34, 65, and 60% of fundus-, corpus-, and antrum/pylorus-projecting DMV neurons, respectively. Our data indicate that CCK-A receptors are present on a subpopulation of gastric-projecting neurons and that their activation leads to excitation of the DMV membrane.     

Effect of pancreastatin on pancreatic endocrine function in the conscious rat

We have studied the effects of pancreastatin on insulin and glucagon secretions in vivo in the conscious rat. Rats were prepared with a gastric fistula and with both external jugular veins cannulated. We found that an i.v. infusion of pancreastatin (1 and 10 nmol/kg/h) inhibited the plasma insulin response and increased the plasma glucose response to the intragastric infusion of glucose in a dose-dependent manner. Furthermore, the infusion of pancreastatin increased the plasma glucagon response to the i.v. infusion of arginine in a dose-dependent manner, and it inhibited the plasma insulin response. However, such an infusion of pancreastatin had no effect on the basal plasma glucose level, nor did it have any effect on plasma insulin and glucagon concentrations. Thus, it is suggested that in the rat, the newly discovered pancreastatin is a regulator of islet cell function.     

The disappearance rate of intraventricular bradykinin in the brain of the conscious rat

To explore the possibility that cyclic nucleotides control green algal growth and division, $\text{Chlamydomonas}$ chemostat cultures were assayed for cyclic nucleotides. Substantial qualities of cAMP were found in cells and in extracellular millieu. Most cGMP molecules were extracellular. Slowing cell growth by slowing chemostat dilution caused reversible changes in cellular morphology and cyclic nucleotide levels. During slowed growth cAMP level increased dramatically; cGMP level decreased. New cells resulting from division were not released from original cell wall.     

Prostaglandin modulation of the vascular effects of vasopressin in the conscious rat

Experiments were performed on conscious, male Sprague-Dawley rats to determine whether cyclooxygenase inhibition affects the pressor response to exogenous vasopressin. The rise in arterial blood pressure was tested in response to 1.0, 2.5, 5.0, and 12.5 mU synthetic arginine vasopressin both before and following cyclooxygenase inhibition with either meclofenamate or the structurally dissimilar inhibitor ibuprofen. In addition, time control experiments were also performed where only the saline vehicle for the drugs was administered. In all animals tested, the increase in arterial pressure in response to the highest three concentrations of vasopressin was greater following cyclooxygenase inhibition than before, while the saline vehicle had no effect. The baroreceptor-mediated bradycardia accompanying the rise in blood pressure was variable, but unaffected by meclofenamate or ibuprofen. It is concluded that vasodilator prostaglandins are released in response to pressor levels of vasopressin, which act to modulate the pressor response of the peptide.     

The renin-angiotensin system in the rat brain. Immunocytochemical localization of angiotensinogen in glial cells and neurons

The distribution of angiotensinogen containing cells was determined in the brain of rats using immunocytochemistry. Specific angiotensinogen immunoreactivity is demonstrated both in glial cells and neurons throughout the brain, except the neocortical and cerebellar territories. Positive neurons are easily and invariably detected in female brains, and haphazardly in male brain (sex hormone dependent). Angiotensinogen immunoreactivity in male brain neurons can be induced by water deprivation or binephrectomy in some areas and particularly in paraventricular nuclei. Finally, the highest concentrations of positive neurons are found in the anterior and lateral hypothalamus, preoptic area, amygdala and some well known nuclei of the mesencephalon and the brainstem. Our results confirm the wide distribution of angiotensinogen mRNA in the brain reported recently by Lynch et al. (1987). Thus the demonstration of angiotensinogen in neurons and glial cells allows a greater understanding of the biochemical and physiological data in accordance with multiple brain renin angiotensin systems.     

Thyroidal regulation of different isoforms of NaKATPase in glial cells of developing rat brain.

The developmental profile of the different isoforms of NaKATPase have been investigated during the first three weeks of postnatal development using primary cultures of isolated glial cells derived from neonatal rat cerebra. Northern and Western blot analysis show that the expression of four isoforms (alpha1, alpha2, beta1 and beta2) in these cells increases progressively between 5 to 20 days of culture. Comparison of the mRNA levels of these isoforms in thyroid hormone deficient (TH def) and thyroid hormone supplemented (TH sup) cells cultured for 5-10 days, revealed for the first time that all four isoforms are sensitive to T3 in the glial cells. Furthermore immunocytochemical staining of these cells with isoform specific NaKATPase antibodies also showed that the localization of the different isoforms in the TH def cells were altered in comparison to that in the TH sup cells. These results establish glial cells as the target cells for the regulation of NaKATPase by TH in the developing brain.     

Stability of neuronal and glial marker enzymes in post-mortem rat brain

The enzymatic activities in post-mortem rat brain kept at 4°C and at 25°C were determined for a number of enzymes localized in specific cell types in the central nervous system. Choline acetyltransferase (CAT), glycerol-3-phosphate dehydrogenase (GPDH), glutamine synthetase (GS), lactate dehydrogenase (LDH) and 2,3-cyclic nucleotide phosphohydrolase (CNPase) were found to be very stable at both 4°C and 25°C with only slight, if any, losses of activity being seen even at periods as long as 72 hr. Glutamic acid decarboxylase (GAD) activity was less stable than that of the other enzymes. In brains kept at 4°C GAD activity was stable out to 24 hr after which it began to decline rapidly to 65% of control at 72 hr. In brains kept at 25°C, GAD activity was stable for 6–8 hr and then began to steadily decline to 58% of control at 24 hr and 29% of control at 72 hr. Assuming that these enzymes have similar stabilities in post-mortem human brain, the effect of post-mortem delay in processing tissues may be of lesser significance than other factors with regard to the measured enzyme activities in human brain samples.