The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis.

Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.

Synergism between cocaine and atropine at the caudal ventrolateral medulla of cats.

We have previously reported that the anticholinergic properties of cocaine may be important in cocaine induced apneusis. We have studied the effects of the cholinergic muscarinic antagonist atropine (ATR) on cocaine induced apneusis at the caudal chemosensitive areas of the ventrolateral medulla oblongata (CVLM). Experiments were performed in urethane anesthetized and tracheotomized cats with the CVLM surgically exposed. Topical application of ATR (44 mM ) to the CVLM produced significant decrements in minute ventilation (V(E)) and mean arterial blood pressure (MABP) (P<0.05) but the effects on tidal volume (V(T)), respiratory frequency (f) and heart rate (HR) were not significant. Administration of cocaine (37 mM) to ATR pretreated animals increased the incidence of cocaine induced respiratory arrest to more than twofold greater than when cocaine was administered in the absence of pretreatment. The ATR pretreated animals that did not experience inspiratory arrest after cocaine were shown to exhibit significant decrements in f and V(E) as a consequence of prolonged inspiratory pauses. The reduction in MABP after cocaine in ATR pretreated animals was also significant. These results suggest that ATR enhances the central respiratory toxicity of cocaine by acting synergistically at CVLM chemosensitive sites.

Decreased CSF pH at ventral brain stem induces widespread c-Fos immunoreactivity in rat brain neurons.

Physiological evidence has indicated that central respiratory chemosensitivity may be ascribed to neurons located at the ventral medullary surface (VMS); however, in recent years, multiple sites have been proposed. Because c-Fos immunoreactivity is presumed to identify primary cells as well as second- and third-order cells that are activated by a particular stimulus, we hypothesized that activation of VMS cells using a known adequate respiratory stimulus, H(+), would induce production of c-Fos in cells that participate in the central pH-sensitive respiratory chemoreflex loop. In this study, stimulation of rostral and caudal VMS respiratory chemosensitive sites in chloralose-urethane-anesthetized rats with acidic (pH 7.2) mock cerebrospinal fluid induced c-Fos protein immunoreactivity in widespread brain sites, such as VMS, ventral pontine surface, retrotrapezoid, medial and lateral parabrachial, lateral reticular nuclei, cranial nerves VII and X nuclei, A(1) and C(1) areas, area postrema, locus coeruleus, and paragigantocellular nuclei. At the hypothalamus, the c-Fos reaction product was seen in the dorsomedial, lateral hypothalamic, supraoptic, and periventricular nuclei. These results suggest that 1) multiple c-Fos-positive brain stem and hypothalamic structures may represent part of a neuronal network responsive to cerebrospinal fluid pH changes at the VMS, and 2) VMS pH-sensitive neurons project to widespread regions in the brain stem and hypothalamus that include respiratory and cardiovascular control sites.

Cardiorespiratory effects of cocaine and procaine at the ventral brainstem.

The caudal ventrolateral medulla (CVLM) is an area of the brainstem, in the vicinity of the hypoglossal nerve roots, where cholinergic and adrenergic neurons participate in respiratory and vasomotor control. Cardiorespiratory depression has been produced by topical application of cocaine to the CVLM. In the present studies, the effects of topical pretreatments of the CVLM with alpha-adrenergic blockers (prazosin 4.8 mM) and beta-adrenergic blockers (propranolol 11.3 mM) on the cardiorespiratory responses to topically administered cocaine (37 mM) were investigated in urethane anesthetized cats. Both prazosin and propranolol failed to produce ventilatory responses and to counteract cocaine-induced apneustic breathing. However, the cocaine-induced decrement in mean arterial blood pressure (MABP) following pretreatment of the CVLM with propranolol was found to be 11 +/- 5%, compared to the 18 +/- 5% decrement produced by cocaine alone. These differences were not statistically significant. Procaine (37 mM) in equimolar doses to cocaine, produced a small statistically significant decrement in MABP (P < 0.05) without ventilatory effects. Topical administration of procaine (73.3 mM), at approximately twice the equimolar dosage of cocaine, produced apneustic breathing that was indistinguishable from that produced by cocaine. The neurotoxic properties of cocaine that produce apneustic breathing appear to be similar to that produced by the anesthetic agent procaine, and the alpha- and beta-adrenoceptor blockers prazosin and propranolol do not appear to antagonize the vasomotor depression induced by cocaine at the CVLM.

Effects of cholinomimetics on cocaine-induced hypotension and apneusis at a ventral brainstem cardiorespiratory control site.

The current study was undertaken to evaluate the effects of cholinomimetic drugs on cocaine-induced central cardiorespiratory depression. Cats anesthetized by urethane (2.0 g/kg) were subjected to topical application at the caudal ventrolateral medullary surface (cVMS) of cocaine and two cholinomimetic pretreatment drugs. The following drug regimens were tested: 37 mM cocaine 1) given alone; 2) given 5 min after 2.7 mM carbachol pretreatment; and 3) given 5 min after 3.6 mM physostigmine pretreatment. In 7 of 11 cats, pretreatment with physostigmine decreased the incidence of cocaine-induced apneusis and hypoventilation significantly (p < 0.05); these animals showed no significant change in the mean arterial blood pressure during the 5-min pretreatment before administration of cocaine. In 4 of 11 cats, the physostigmine pretreatment produced a significant decrease in mean arterial blood pressure followed by lethal cardiorespiratory arrest when cocaine was administered. Pretreatment with carbachol resulted in cardiorespiratory responses which were not significantly different from those produced by cocaine alone. In anesthetized cats not exhibiting hypotensive responses to physostigmine, pretreatment may ameliorate cocaine-induced respiratory failure by ventral brainstem control mechanisms.

Central cocaine neurotoxicity at brainstem cardiorespiratory control sites.

Cocaine hydrochloride was applied topically to the ventrolateral medullary surface (VMS) where chemosensitive respiratory and vasomotor control sites are colocalized. Cats (n = 16) were anesthetized with urethane (2.0 g/kg, 80 percent of dose titrated over 60 min). The trachea of each animal was cannulated and the VMS was surgically exposed. Tidal volume (VT), frequency of breathing (f), systolic and diastolic blood pressure (SBP and DBP, respectively), and heart rate (HR) were measured. Cocaine (62.5 micrograms per site) administered at the VMS control sites decreased f, SBP, and DBP significantly (p < 0.05), without changing HR or VT values. This cocaine-induced hypoventilation was associated with brief intervals of inspiratory cramp (apneusis). Central cocaine neurotoxicity may result from interaction of cocaine with VMS sites, producing increased inspiratory drive and decreased vasomotor tone.

Cholinergic-opioid interactions at brainstem respiratory chemosensitive areas in cats.

Central respiratory chemosensitivity has been ascribed to CO2-sensitive neurons located on the ventral brainstem surface. The effects of cholinergic mechanisms on CO2-sensitive neuronal activity recorded extracellularly at the brainstem respiratory chemosensitive area at the caudal ventral medullary surface (cVMS) were investigated in cats (n = 14) anesthetized with chloralose-urethane. The neurons increased their firing rate from 10.4 +/- 1.6 Hz to 33.9 +/- 5.2 Hz when the mock cerebrospinal fluid (mCSF) superfusing buffer solution was changed from pH 7.4 (control) to pH 7.0 (acidic). Atropine (ATR) applied topically to the cVMS depressed the H(+)-ion-induced increase in neuronal frequency from 32.8 +/- 4.8 Hz to 13.4 +/- 2.2 Hz. ATR also depressed the inspired-CO2-induced increase in neuronal activity from 33.2 +/- 8.3 Hz to 18.9 +/- 4.9 Hz, suggesting the possibility of a muscarinic cholinergic involvement in cVMS neuronal responses to changes in PCO2 and mCSF-pH. Acetylcholine (ACh) increased the activity of cVMS CO2-sensitive neurons by 237.5% +/- 34.9%, and naloxone applied topically to the cVMS augmented the ACh responsiveness to 338.6% +/- 52.7%. Physostigmine (PHY) increased neuronal activity by 254.3% +/- 42.9%, and this increase was augmented to 435.4% +/- 61.2% by naloxone. Although responses of the CO2-sensitive neurons to PHY were biphasic, the depressant phase failed to appear whenever the cVMS was pretreated with naloxone. Naloxone also augmented the responsiveness of cVMS neurons to increased H+ ion superfusion. These findings suggest that the endogenous opiates may be involved in the central regulation of respiration by interaction with CO2-sensitive cholinergic structures at the cVMS.(ABSTRACT TRUNCATED AT 250 WORDS)

Naloxone application to the ventrolateral medulla enhances the respiratory response to inspired carbon dioxide.

Previous studies have shown that systemic administration of the opiate antagonist naloxone potentiates the ventilatory response to inspired carbon dioxide. The present study was designed to localize the site of action of naloxone for increasing the respiratory chemosensitivity to inhaled carbon dioxide (CO2) in cats. Naloxone applied topically to the caudal chemosensitive area on the ventral medullary surface (VMS) during hypercapnic breathing produced a 75% greater increase in minute ventilation than hypercapnic breathing alone. Furthermore, hypercapnic breathing produced a 200% increase in neuronal activity of VMS chemosensitive cells; this was further increased 120% by naloxone. It is concluded that naloxone increases the sensitivity of neurons in the caudal respiratory chemosensitive area of cats to hypercapnia, and that endogenous opiates may act as modulators at VMS chemosensitive sites during hypercapnic breathing.

Age differences in responsiveness of brainstem chemosensitive neurons to extracellular pH changes.

The sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata (VMS) to extracellular pH changes was examined in newborn and young developing kittens and compared to that of adult cats. The pH was varied by superfusion of the VMS with mock cerebrospinal fluid (CSF) of pH 7.4 (control), 7.0 (acid) and 7.8 (alkaline). A total of 97 neuronal units in the three age groups changed their firing rates inversely in response to extracellular fluid (ECF) pH changes. The greatest sensitivity was found in the adult group where acid superfusion caused an increase in neuronal activity. The least sensitivity was observed in the newborn group (1-6 days old), whereas the young kitten group (4-6 weeks old) exhibited an intermediate sensitivity. Neurons of kittens older than 7 weeks of age demonstrated a response pattern characteristic of the adult group. Neurons of neonates older than seven days, exhibited a response pattern characteristic of the young kitten group.

The role of hypoxia in organophosphorus nerve agent intoxication.

Effectiveness of the standard therapy for organophosphorus (OP) anticholinesterase poisoning has been questioned because of the relative resistance of the cholinesterase (ChE) enzymes to reactivation by oximes (Harris et al., 1968). Because tissue hypoxia may be a significant lethal component of OP nerve agent intoxication, oxygen therapy should be beneficial. Although there have been no systematic investigations of the value of oxygen therapy in OP poisoning, it may improve the efficiency of ChE reactivation by the standard therapies of cholinolytics and oximes. Because tissue hypoxia may be generated by arterial hypoxemia due to respiratory failure, oxygen therapy should limit concomitant metabolic disturbances associated with anaerobiosis. This review addresses the interspecies variations and experimental model developments that should challenge neuroscientists to adopt an integrated, physiological approach to understanding the precise role of hypoxia in neurotoxicity. The significance of tissue hypoxia to protection and recovery of humans from OP nerve agent intoxication is also discussed.

Vasopressor responses and hypoxemia with acutely increased intracranial pressure.

We studied anesthetized dogs subjected to graded increases in intracranial pressure (ICP) to assess the role of the systemic vasopressor (Cushing) response in the arterial hypoxemia associated with increased ICP. The arterial PO2 decrement was significantly greater with rapidly increased ICP compared to slowly increased ICP (P less than 0.01). Systemic vasopressor responses generated in cats by direct electrical stimulation of the vasomotor center resulted in arterial hypoxemia during controlled ventilation. Therefore, arterial hypoxemia was coincident with increased systemic blood pressure produced by either elevation of ICP or electrical stimulation of the vasomotor center.

Amelioration of hypoxemia by neuromuscular blockade following brain injury.

Brain injury has been commonly associated with respiratory failure and uncontrolled skeletal muscle activity. In the present study, neuromuscular (NM) blockade induced by injection of succinylcholine hydrochloride was used to block uncontrolled muscle contractions in dogs with brain injury caused by rapid elevation of intracranial pressure (ICP). Decerebrate posturing, a decrease in value (mean +/- SEM) of arterial oxygen tension (Pa02) of 26 +/- 1 torr, and an increase in arterial carbon dioxide tension (PaCO2) of 11 +/- 1 torr occurred in the dogs, which were supported by mechanical ventilation. The arterial hypoxemia developed independently of the decerebration; however, dogs that demonstrated decerebrate posturing exhibited significantly larger decreases in Pa02 than dogs that did not (P less than 0.01). NM blockade ameliorated the effects of elevated ICP on the arterial blood gases; i.e., the amount of hypoxemia in decerebrate dogs was significantly less in dogs subjected to NM blockade than in dogs not subjected to NM blockade. It is concluded that uncontrolled skeletal muscle activity that exacerbates arterial hypoxemia associated with brain injury is ameliorated by use of NM blockade as a therapeutic adjunct to mechanical ventilation.

Neurophysiological studies on superficial medullary chemosensitive area for respiration.

In cats anesthetized with chloralose-urethane (40 mg/kg chloralose; 200 mg/kg urethane) pH sensitivity of neurons in the caudal chemosensitive area on the ventrolateral surface of the medulla oblongata was examined while monitoring phrenic nerve activity simultaneously. pH was varied by superfusion of the ventral medullary surface with mock cerebrospinal fluid (CSF) of different pH (pH 7.4 control, 7.0 and 7.8). A total of 130 units from 21 cats changed their firing rate in response to CSF-pH changes. These were subdivided into 3 groups. In Group I, 31 respiratory pH sensitive units increased their firing rate in response to decreased mock CSF-pH, noxious pinch, joint movement in contralateral forelimb, and increased inspired CO2. These responses may have originated from respiratory center neurons. Group II consisted of 59 non-respiratory pH sensitive units whose firing rate changed in an inverse manner with CSF-pH changes. Of these, 30 responded to contralateral distal forelimb movement, 15 to hair manipulation, 9 to heavy pressure and 5 to noxious pinch. Increased inspired CO2 (rebreathing) did not modify activity. The response to pH is believed to be from non-specific neurons. Group III consisted of 40 non-respiratory pH sensitive units responding to CSF-pH changes and to increased inspired CO2. The firing rate was irregular, the interval distribution approaching an exponential function. It may be postulated that the impulse frequency of chemosensitive impulses may be irregular at the site of impulse generation, the irregularity decreasing by convergence during transmission to the respiratory centers. The time course of Group III chemosensitive units was similar to phrenic nerve responses.

Morphological observations on superficial medullary CO2--chemosensitive areas.

Physiological investigations have indicated that the ventrolateral surface of the medulla oblongata is involved in the chemical drive to respiration. In this investigation, light and electron microscopic investigations of the 3 chemosensitive regions reveal the following. (1) Evaginations of the ventral surface abut the overlying pia mater thereby delimiting discrete compartments; invaginations of the surface delimit wide cisternae lined with basement membrane. Neuronal elements with numerous synapses, were found scattered among astrocytic processes of the marginal glia in intermediate and caudal chemosensitive areas Microvasculature are conspicuously absent from the marginal glia. Intramedullary vessels are surrounded by perivascular spaces and the endothelium shows zonulae occludentes at cell junctions. (2) Horseradish peroxidase (HRP) applied to the ventral surface diffused throughout the interstitial and perivascular compartments, into synaptic clefts and neuronal soma. Diffusion of HRP into blood vessels was blocked at zonulae occludentes. Following intravenous injection of HRP, no reaction product was found outside cerebral vasculature in chemosensitive areas. (3) In spontaneously breathing cats, 2% procaine applied to the caudal chemosensitive area resulted in respiratory depression which began with the second breath. It is proposed, that substances which stimulate or depress respiration, when applied to the ventral medullary surface, produce their effects on superficial neurons located in the intermediate and caudal chemosensitive areas after diffusion through interstitial spaces.

Analysis of the sexual dimorphism in the basioccipital portion of the dog's skull.

Sexual differences in the basioccipital portion of the skull of dogs have been described and an index is presented which reliably predicts the sex of the skull. 92 dolichocephalic skull (44 male, 48 female) from mongrel dogs were used. In the basioccipital region of the male skulls, a triangular area, which extends from the basion to a line joining the medialmost points of the two jugular foramina, appears narrow and elevated. The pharyngeal tubercle is also prominent. In female skulls the rostral angle of this triangle is wider and the triangular area seems smoother and flat. The anterior half of the female basioccipital portion is wider and smoother than in the male skull. In order to quantify the observed differences, four indices were calculated. All of the indices were statistically significant. However, only index IV could be reliably used in predicting the sex of an unknown skull: (formula: see text) was derived from breadth IV (distance between the two temporo-occipital fissures at their lateralmost points) and length II (distance between the basion and a line drawn between the two medialmost points of the jugular foramina in the midline). Values of less than 123 for male and greater than 136 for female placed the skulls in their proper sex group. Skulls that had intermediate values could be placed in correct sex grouping only in conjunction with strong observational criteria. It is suggested that in absence of such strong observational criteria these skulls may belong to immature or castrate animals.