Kainic acid on the rostral ventrolateral medulla inhibits phrenic output and CO2 sensitivity

We used the neurotoxin, kainic acid, which is known to stimulate neuronal cell bodies as opposed to axons of passage by binding to specific amino acid receptors to determine whether cells with such receptors have access to the ventrolateral medullary surface and are involved in central ventilatory chemosensitivity. Pledgets with 4.7 mM kainic acid were placed bilaterally on the rostral, intermediate, or caudal ventilatory chemosensitive areas for 1-2 min in chloralose-urethan-anesthetized, paralyzed, vagotomized, glomectomized, and servo-ventilated cats. Application of kainic acid on the caudal or intermediate areas produced no consistent significant effects on eucapnic phrenic output or on the slope or maximum value of the phrenic nerve response to increased end-tidal PCO2. Rostral area kainic acid produced immediate augmentation and then diminution of blood pressure and phrenic output. Apnea developed in six of nine cats by 40 min. In all five cats in which it could be tested, the slope of the CO2 response was clearly decreased. Of [3H]kainic acid applied to the rostral area, 88.4% was shown to be within 2 mm of the ventral surface. Comparison of surface application sites of this and other studies suggests that an area overlapping the border of the original rostral and intermediate areas allows access to neurons involved in the chemoreception process, which may also provide tonic facilitatory input to cardiorespiratory systems.     

DIDS decreases CSF HCO3- and increases breathing in response to CO2 in awake rabbits

An inhibitor of the HCO3-/Cl- exchange carrier protein, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or vehicle was infused in mock cerebrospinal fluid (CSF) via the cisterna magna in conscious rabbits at 10 mumol/l for 40 min at 10 microliter/min. Neither treatment had any effect over 2-5 h on the non-CO2-stimulated CSF ion values or blood gases. With CO2 stimulation such that arterial PCO2 (PaCO2) was increased 25 Torr over 3 h, DIDS treatment significantly decreased the stoichiometrically opposite changes in CSF [HCO3-] and [Cl-] that normally accompany hypercapnia and reflect ionic mechanisms of CSF pH regulation. Expressed as delta CSF [HCO3-]/delta PaCO2, DIDS treatment decreased the CSF ionic response by 35%. In a separate paired study design DIDS administration via the same protocol had no effect on resting ventilation but significantly increased the ventilation and tidal volume responses to a 28-Torr increase in PaCO2. Expressed as change in minute ventilation divided by delta PaCO2, DIDS treatment produced a 39.6% increase. The results support the concept of a DIDS-inhibitable anion exchange carrier being involved in CSF pH regulation in hypercapnia and suggest a DIDS-related effect on the ventilatory response to CO2.     

Fluorescence location of RVLM kainate microinjections that alter the control of breathing

Kainic acid (4.7 mM) applied to the rostral ventrolateral medulla (RVLM) surface decreases phrenic output, CO2 sensitivity, and blood pressure in chloralose-urethan-anesthetized, vagotomized, paralyzed, glomectomized, servoventilated cats. In this study using the same preparation, bilateral 50- to 100-nl kainate injections just below the RVLM surface better localized these responses topographically. The physiological responses to unilateral 10-nl kainate injections were then correlated with anatomic location determined by fluorescent microbeads (0.5 micron diam). Many sites were associated with no effect, a few rostral and caudal sites with increased phrenic activity, and cluster of sites with decreased phrenic activity often to apnea, decreased CO2 sensitivity, and decreased responses to carotid sinus nerve stimulation. Blood pressure was unaffected. These sites, within 400 microns of the surface, were ventral to the facial nucleus, ventrolateral to the nucleus paragigantocellularis lateralis, caudal to the superior olive, and rostral to the retrofacial nucleus. They appeared to be within the recently described retrotrapezoid nucleus, which contains cells with respiratory-related activity and projections to the dorsal and ventral respiratory groups. Cells within this site appear able to provide tonic input to respiration and to affect peripheral and central chemoreception.     

RTN TRH causes prolonged respiratory stimulation

Cream, Carlos L., Aihua Li, and Eugene E. Nattie. RTNTRH causes prolonged respiratory stimulation. J. Appl.Physiol. 83(3): 792-799, 1997.We injectedthyrotropin-releasing hormone (TRH; 10 nl; 0.25, 0.5, 1.0, or 10 mM),its inactive free acid form (TRHOH; 1 mM), or a metabolite with lowTRH-receptor binding affinity, histidine-proline diketopiperazine (cHP;1 mM), into the retrotrapezoid nucleus of anesthetized rats. Injectionlocation was verified by anatomic analysis. Lower doses (0.25-0.5mM) significantly increased both the product of integrated phrenicamplitude and frequency(Phr · f) and f for 20-30min compared with artificial cerebrospinal fluid control injections. Higher doses (1.0-10 mM) produced greater and long-lastingstimulation of Phr · f,Phr, and f and of blood pressure. Thisstimulation reached values 150% of baseline and durations of 270 minafter a single injection. TRHOH (1 mM ) or cHP (1 mM) had no effect onPhr but increased f, as did 1 mM TRH. We concludethat TRH has a very powerful stimulatory effect in the retrotrapezoidnucleus region on Phr · f, withthe Phr response seemingly specific for TRHreceptors. Similar responses of f to TRHOH and cHP suggest it may benonspecific.

     

Brain water and electrolytes in response to respiratory acidosis and brain edema in the mutant mouse,Jimpy

Compared to littermate controls, unstressed Jimpy mice have higher brain water, sodium, potassium and chloride contents and lower carbonic anhydrase activity. When stressed by CO₂ to produce a respiratory acidosis or by injection of distilled water to produce brain edema, the Jimpy mouse brain has water and ionic responses essentially like those in controls.     

Brain stem lesion size determined by DEAD red or conjugation of neurotoxin to fluorescent beads

Neurotoxinmicroinjected into the retrotrapezoid nucleus of anesthetized ratsdecreases phrenic activity and eliminates the response toCO₂. In unanesthetized rats, suchtreatment has no effect on awake, resting breathing and decreasesCO₂ sensitivity by 40% (M. Akilesh, M. Kamper, A. Li, and E. E. Nattie. J. Appl. Physiol. 82: 469-479, 1997). One important factorin explaining these disparate results is the actual size of theanatomic lesion. In the present study, we injected ibotenic acid intothe retrotrapezoid nucleus of anesthetized rats and evaluated lesionsize by using two new approaches: 1)DEAD red, a fluorescent probe that enters impaired cells through leakymembranes and binds to nucleic acids, and2) conjugation of toxin tofluorescent beads. With the use of DEAD red, the region containinglabeled dying cells was 313 ± 104 nl(n = 4), six times larger than theinitial injected volume, and the physiological effects on phrenicamplitude, the CO₂ response, andblood pressure began within minutes and were substantial. Withconjugated toxin, in theory, neuronal damage would be limited to theregion of detectable fluorescence (49 ± 10 nl;n = 4). Effects on phrenicamplitude, CO₂ sensitivity, andblood pressure were absent until ~2 h postinjection. Controlexperiments, with 2 h of in vitro incubation of theneurotoxin-microbead conjugate and injection of the supernatant aftercentrifugation, showed similar results that suggest release ofconjugated neurotoxin. We conclude that DEAD red provides a usefulmeans to monitor neuronal impairment in acute studies in vivo.Conjugation of neurotoxin to microbeads may be less reliable in this regard.

     

Acoustic plethysmography measures breathing in unrestrained neonatal mice.

Measurement of breathing volumes in neonatal mice is of growing importance in order to characterize the influence of development and genetic modifications on respiratory control to evaluate hypotheses concerned with human infant deficits that may affect sudden infant death syndrome, for example. Current techniques require undesirable physical constraints or incur possible artifacts specific to very small animals. We have examined the utility of a recently proposed approach using an acoustic resonance procedure that does not require undue physical constraint beyond placement in the acoustic plethysmograph. We show here that this approach can be applied to baby mice 5 days after birth and that it can be accurately calibrated. In addition, this approach should be useful to study unrestrained neonatal mice under conditions where body temperature approaches environmental temperature and barometric plethysmography cannot be used.     

CO2 dialysis in nucleus tractus solitarius region of rat increases ventilation in sleep and wakefulness.

To evaluate the function of widely distributed central chemoreceptors during sleep and wakefulness in the rat, we focally stimulate single chemoreceptor sites during naturally occurring sleep-wake cycles by microdialysis of artificial cerebrospinal fluid equilibrated with 25% CO2. In retrotrapezoid nucleus, this increased ventilation (tidal volume) by 24% only in wakefulness (Li A, Randall M, and Nattie E. J Appl Physiol 87: 910-919, 1999). In caudal medullary raphé, it increased ventilation (frequency) by 15-20% only in sleep (Nattie EE and Li A. J Appl Physiol 90: 1247-1257, 2001). Here, in nucleus tractus solitarius (NTS), focal acidification significantly increased ventilation by 11% in sleep and 7% in wakefulness rostrally (n = 5) and by 16% in sleep and 28% in wakefulness caudally (n = 5). The sleep-wake cycle was unaltered. Dialysis with 5% CO2 had no effect. Dialysis with 50% CO2 caudally did not further stimulate ventilation but did disrupt sleep. Central chemoreceptors in the NTS affect breathing in both sleep and wakefulness. The threshold for arousal in caudal NTS is greater than that for the stimulation of breathing.