Focal acidosis in the pre-Botzinger complex area of awake goats induces a mild tachypnea.

There are widespread chemosensitive areas in the brain with varying effects on breathing. In the awake goat, microdialyzing (MD) 50% CO(2) at multiple sites within the medullary raphe increases pulmonary ventilation (Vi), blood pressure, heart rate, and metabolic rate (Vo(2)) (11), while MD in the rostral and caudal cerebellar fastigial nucleus has a stimulating and depressant effect, respectively, on these variables (17). In the anesthetized cat, the pre-Bötzinger complex (preBötzC), a hypothesized respiratory rhythm generator, increases phrenic nerve activity after an acetazolamide-induced acidosis (31, 32). To gain insight into the effects of focal acidosis (FA) within the preBötzC during physiological conditions, we tested the hypothesis that FA in the preBötzC during wakefulness would stimulate breathing, by increasing respiratory frequency (f). Microtubules were bilaterally implanted into the preBötzC of 10 goats. Unilateral MD of mock cerebral spinal fluid equilibrated with 6.4% CO(2) did not affect Vi, tidal volume (Vt), or f. Unilateral MD of 25 and 50% CO(2) significantly increased Vi and f by 10% (P < 0.05, n = 10, 17 trials), but Vt was unaffected. Bilateral MD of 6.4, 25, or 50% CO(2) did not significantly affect Vi, Vt, or f (P > 0.05, n = 6, 6 trials). MD of 80% CO(2) caused a 180% increase in f and severe disruptions in airflow (n = 2). MD of any level of CO(2) did not result in any significant changes in mean arterial blood pressure, heart rate, or Vo(2). Thus the data suggest that the preBötzC area is chemosensitive, but the responses to FA at this site are unique compared with other chemosensitive sites.

Normal breathing pattern and arterial blood gases in awake and sleeping goats after near total destruction of the presumed pre-Botzinger complex and the surrounding region.

Abrupt neurotoxic destruction of >70% of the pre-Bötzinger complex (preBötzC) in awake goats results in respiratory and cardiac failure (Wenninger JM, Pan LG, Klum L, Leekley T, Bastastic J, Hodges MR, Feroah TR, Davis S, Forster HV. J Appl Physiol 97: 1629-1636, 2004). However, in reduced preparations, rhythmic respiratory activity has been found in other areas of the brain stem (Huang Q, St. John WM. J Appl Physiol 64: 1405-1411, 1988; Janczewski WA, Feldman JL. J Physiol 570: 407-420, 2006; Lieske SP, Thoby-Brisson M, Telgkamo P, Ramierz JM. Nature Neurosci 3: 600-607, 2000; St. John WM, Bledsoe TA. J Appl Physiol 59: 684-690, 1985); thus we hypothesized that, when the preBötzC is destroyed incrementally over weeks, time-dependent plasticity within the respiratory network will result in a respiratory rhythm capable of maintaining normal blood gases. Microtubules were bilaterally implanted into the presumed preBötzC of seven goats. After recovery from surgery, studies were completed to establish baseline values for respiratory parameters. At weekly intervals, increasing volumes (in order 0.5, 1, 5, and 10 microl) of ibotenic acid (IA; 50 mM) were then injected into the preBötzC. All IA injections resulted in an acute tachypnea and dysrhythmia featuring augmented breaths, apneas, and increased breath-to-breath variation in breathing. In studies at night, apneas were nearly all central and occurred in the awake state. Breath-to-breath variation in breathing was greater (P < 0.05) during wakefulness than during non-rapid eye movement sleep. However, one week after the final IA injection, the breathing pattern, breath-to-breath variation, and arterial blood gases and pH were unchanged from baseline, but there was a 20% decrease in respiratory frequency (f) and CO(2) sensitivity (P < 0.05), as well as a 40% decrease in the ventilatory response to hypoxia (P < 0.001). In subsequent histological analysis of the presumed preBötzC region of lesioned goats, it was determined that there was a 90 and 92% reduction from control goats in total and neurokinin-1 receptor neurons, respectively. Therefore, it was concluded that 1) the dysrhythmic effects on breathing are state dependent; and 2) after incremental, near total destruction of the presumed preBötzC region, time-dependent plasticity within the respiratory network provides a rhythm capable of sustaining normal arterial blood gases.

Defining the Normal Dorsal Contour of the Corpus Callosum with Time.

BACKGROUND AND PURPOSE: Morphological changes of the corpus callosum have been associated with a large number of congenital neurocognitive and psychiatric disorders. Focal defects or notches of the dorsal surface of the corpus callosum have not been well characterized. Our purpose was the following; 1) to characterize the dorsal contour of the corpus callosum during the life span, 2) to characterize the relationship of contour deviations to neighboring vessels, and 3) to determine whether contour deviations are congenital or acquired. MATERIALS AND METHODS: We retrospectively reviewed normal sagittal T1-weighted brain MR images. A "notch" was defined as a concavity in the dorsal surface at least 1 mm in depth. The corpus callosum was considered to be "undulating" if there were >2 notches, including an anterior and posterior notch. The presence of a pericallosal artery and its relationship to a notch were assessed. RESULTS: We reviewed 1639 MR imaging studies, spanning 0-89 years of age. A total of 1102 notches were identified in 823 studies; 344 (31%) were anterior, 660 (60%) were posterior, and 98 (9%), undulating. There was a positive correlation between the prevalence (P < .001) and depth (P = .028) of an anterior notch and age and a negative correlation between the prevalence of a posterior notch and age (P < .001). There was no difference between patient sex and corpus callosum notching (P = .884). Of the 823 studies with notches, 490 (60%) were associated with a pericallosal artery (P < .001). CONCLUSIONS: The prevalence and depth of notches in the anterior corpus callosum increase significantly with age; this finding suggests that most notches are acquired. There is a significant positive association between the presence of a corpus callosum notch and adjacent pericallosal arteries, suggesting that this may play a role in notch formation.

The active site of Serratia endonuclease contains a conserved magnesium-water cluster.

Serratia endonuclease is an important member of a class of magnesium dependent nucleases that are widely distributed in nature. Here, we describe the location and geometry of a magnesium-water cluster within the active site of this enzyme. The sole protein ligand of the magnesium atom is Asn119; this metal ion is also associated with five water molecules to complete an octahedral coordination complex. These water molecules are very well ordered and there is no evidence of rotational disorder or motion. Glu127 and His89 are located nearby and each is hydrogen bonded to water molecules in the coordination sphere. Asp86 is not chelated to the magnesium or its surrounding water molecules. Results of kinetics and site-specific mutagenesis experiments suggest that this metal-water cluster contains the catalytic metal ion of this enzyme. All residues which hydrogen bond to the water molecules that coordinate the magnesium atom are conserved in nucleases homologous to Serratia endonuclease, suggesting that the water cluster is a conserved feature of this family of enzymes. We offer a detailed structural comparison to one other nuclease, the homing endonuclease I-PpoI, that has recently been shown, in spite of a lack of sequence homology, to share a similar active site geometry to Serratia endonuclease. Evidence from both of these structures suggests that the magnesium of Serratia nuclease participates in catalysis via an inner sphere mechanism.

2.5 A structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate.

In an X-ray diffraction study using the method of multiple isomorphous replacement, the structure of aspartate carbamoyltransferase (EC 2.1.3.2) complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) has been solved to 2.5 A. Ten rounds of model building and 123 cycles of restrained reciprocal space refinement have resulted in a model containing 94.4% of the theoretical atoms of the protein-inhibitor complex with an R-factor of 0.231. The fit of the model to the density is excellent, except for occasional side-chains and two sections of the regulatory chains that may be disordered. The electron density for the PALA molecule is readily identifiable for both catalytic (c) chains of the asymmetric unit and bonding interactions with several important residues including Ser52, Arg54, Thr55, Ser80, Lys84, Arg105, His134, Arg165, Arg229 and Gln231 are apparent. The carboxylate groups of the PALA molecule are in a nearly cis conformation. Gross quaternary changes between the T and R forms are noted and in agreement with earlier work from this laboratory. Namely, in the new structure the catalytic trimers move apart by 12 A along the 3-fold axis of the enzyme and relocate by 10 degrees relative to each other, adopting a more eclipsed position. The regulatory (r) chains in the new structure reorient about their 2-fold axis by 15 degrees. Large tertiary changes that include domain migration and rearrangement are also present between these two forms. In the R form both domains of the catalytic chain relocate closer to each other in order to bind to the inhibitor. The polar domain seems to bind primarily to the carbamoyl phosphate moiety of PALA, and the equatorial domain binds primarily to the L-aspartate moiety. Other changes in tertiary structure bring the 80s loop (from an adjacent catalytic chain) and the 240s loop into a position to interact with the PALA molecule. Changes have been searched for in all interface regions of the enzyme. While the C1-C4 and C1-R4 regions have been completely altered, most of the other interchain interfaces are similar in the T and R forms. The intrachain interfaces, between domains of the same catalytic chains, have undergone some reorganization as these domains move closer to each other when the inhibitor is bound. This new structure allows a reinterpretation of genetic and chemical modification studies done to date.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens.

Crystals have been obtained of the extracellular endonuclease from the bacterial pathogen Serratia marcescens. This magnesium-dependent enzyme is equally active against single and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia nuclease is not homologous with staphylococcal nuclease, the only other broad specificity endonuclease for which a structure exists, nor is it homologous with other nucleases that have been solved by X-ray diffraction. The structure of this enzyme should, therefore, provide new information about this class of enzyme. At present we have succeeded in obtaining large, high quality crystals using ammonium sulfate. They crystallize in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 106.7 A, b = 74.5 A, c = 68.9 A, and diffract to beyond 2 A. Low-resolution native data sets have been recorded and a search is under way for heavy-atom derivatives.

Crystallization and preliminary crystallographic analysis of NADPH:FMN oxidoreductase from Vibrio harveyi.

Crystals of NADPH:FMN oxidoreductase from Vibrio harveyi have been obtained and characterized by X-ray diffraction. This enzyme plays a role in the generation of light in luminescent bacteria by providing reduced FMN to luciferase. Large, high quality crystals were grown using polyethylene glycol 6000 at pH 7.0. They crystallize in the monoclinic space group P2(1) with cell dimensions a = 51.2 A, b = 85.9 A, c = 58.1 A, beta = 109.3 degrees, and diffract to 1.8 A. We expect two molecules per asymmetric unit. High resolution data sets have been recorded and a search is under way for heavy-atom derivatives.

Identification of the Serratia endonuclease dimer: structural basis and implications for catalysis.

The Serratia endonuclease is an extracellularly secreted enzyme capable of cleaving both single- and double-stranded forms of DNA and RNA. It is the first member of a large class of related and usually dimeric endonucleases for which a structure is known. Using X-ray crystallography, the structure of monomer of this enzyme was reported by us previously (Miller MD et al., 1994, Nature Struct Biol 1:461-468). We now confirm the dimeric nature of this enzyme through light-scattering experiments and identify the physiologic dimer interface through crystal packing analysis. This dimerization occurs through an isologous twofold interaction localized to the carboxy-terminal subdomain of the enzyme. The dimer is a prolate ellipsoid with dimensions 30 A x 35 A x 90 A. The dimer interface is flat and contains four salt links, several hydrogen bonds, and nonpolar interactions. Buried water is prominent in this interface and it includes an unusual "cubic" water cluster. The position of the two active sites in the dimer suggests that they can act independently in their cleavage of DNA, but have a geometrical advantage in attacking substrate relative to the monomer.

Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: evidence for an uncoupled R state.

In the R form of ATCase complexed with the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, large temperature factors are reported for the allosteric domains of the regulatory chains. We studied the conformational flexibility of the holoenzyme with molecular dynamics simulations and rigid body (TLS) analysis. The results of the molecular dynamics simulations suggest that, although local atomic fluctuations account for the temperature factors of the catalytic and zinc domains, they do not account for the large temperature factors of the allosteric regions. However, the temperature factors of the allosteric domains can be satisfactorily analyzed using a rigid body model. The simulations and rigid body analysis support the idea that the allosteric regions are mechanically uncoupled from the rest of the enzyme in the PALA structure. Implications of this uncoupling for allosteric regulation are discussed.

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).

Cleavage experiments with deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate) suggest that the homing endonuclease I-PpoI follows the same mechanism of phosphodiester bond hydrolysis as the non-specific Serratia nuclease.

We show here that two nucleases, Serratia nuclease and I-PpoI, with contrasting specificities, i.e. non-specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I-PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg2+ bound to an asparagine residue as a Lewis acid in phosphodiester bond cleavage.

Prevalence of penicillin-resistant pneumococci in Houston, Texas.

Pneumococci resistant to penicillin have been reported in increasing numbers world-wide. A survey for resistance to penicillin G in 222 clinical isolates taken from patients in Houston is reported here. Thirteen (5.9%) of the isolates screened exhibited moderate levels of resistance with MIC's ranging from 0.125-0.5 microgram/ml. The authors recommend routine screening for pneumococcal resistance in isolates from blood, cerebrospinal fluid and tissue exudates.

Characterization of the alanine racemases from two mycobacteria.

D-Alanine is a necessary precursor in the biosynthesis of the bacterial peptidoglycan. The naturally occurring L-alanine isomer is racemized to its D-form through the action of a class of enzymes called alanine racemases. These enzymes are ubiquitous among prokaryotes, and with very few exceptions are absent in eukaryotes, making them a logical target for the development of novel antibiotics. The alanine racemase gene from both Mycobacterium tuberculosis and M. avium was amplified by PCR and cloned in Escherichia coli. Overexpression of the proteins in the E. coli BL21 system, both as native and as His-tagged recombinant products, has been achieved. The proteins have been purified to electrophoretic homogeneity and analyzed biochemically. A D-alanine requiring double knock-out mutant of E. coli (alr, dadX) was constructed and the cloned genes were able to complement its deficiencies.

Contributions of the Kölliker-Fuse nucleus to coordination of breathing and swallowing.

Herein we compare the effects of perturbations in the Kölliker-Fuse nucleus (KFN) and the lateral (LPBN) and medial (MPBN) parabrachial nuclei on the coordination of breathing and swallowing. Cannula was chronically implanted in goats through which ibotenic acid (IA) was injected while awake. Swallows in late expiration (E) always reset while swallows in early inspiration (I) never reset the respiratory rhythm. Before cannula implantation, all other E and I swallows did not reset the respiratory rhythm, and had small effects on E and I duration and tidal volume (VT). However, after cannula implantation in the MPBN and KFN, E and I swallows reset the respiratory rhythm and increased the effects on I and E duration and VT. Subsequent injection of IA into the KFN eliminated the respiratory phase resetting of swallows but exacerbated the effects on I and E duration and VT. We conclude that the KFN and to a lesser extent the MPBN contribute to coordination of breathing and swallowing.

The effects of lesions in the dorsolateral pons on the coordination of swallowing and breathing in awake goats.

The purpose of this retrospective study was to gain insight into the contribution of the dorsolateral pons to the coordination of swallowing and breathing in awake goats. In 4 goats, cannulas were chronically implanted bilaterally through the lateral (LPBN) and medial (MPBN) parabrachial nuclei just dorsal to the Kölliker-Fuse nucleus (KFN). After >2weeks recovery from this surgery, the goats were studied for 5½h on a control day, and on separate days after receiving 1 and 10µl injections of ibotenic acid (IA) separated by 1week. The frequency of swallows did not change during the control and 1µl IA studies, but after injection of 10µl IA, there was a transient 65% increase in frequency of swallows (P<0.05). Under control conditions swallows occurred throughout the respiratory cycle, where late-E swallows accounted for 67.6% of swallows. The distribution of swallow occurrence throughout the respiratory cycle was unaffected by IA injections. Consistent with the concept that swallowing is dominant over breathing, we found that swallows increased inspiratory (T(I)) and expiratory (T(E)) time and decreased tidal volume (V(T)) of the breath of the swallow (n) and/or the subsequent (n+1) breath. Injections of 10µl IA attenuated the normal increases in T(I) and T(E) and further attenuated V(T) of the n breath. Additionally, E and I swallows reset respiratory rhythm, but injection of 1 or 10µl IA progressively attenuated this resetting, suggesting a decreased dominance over respiratory motor output with increasing IA injections. Post mortem histological analysis revealed about 50% fewer (P<0.05) neurons remained in the KFN, LPBN, and MPBN in lesioned compared to control goats. We conclude that dorsolateral pontine nuclei have a modulatory role in a hypothesized holarchical neural network regulating swallowing and breathing particularly contributing to the normal dominance of swallowing over breathing in both rhythm and motor pattern generation.

Site-specific effects on respiratory rhythm and pattern of ibotenic acid injections in the pontine respiratory group of goats.

To probe further the contributions of the rostral pons to eupneic respiratory rhythm and pattern, we tested the hypothesis that ibotenic acid (IA) injections in the pontine respiratory group (PRG) would disrupt eupneic respiratory rhythm and pattern in a site- and state-specific manner. In 15 goats, cannulas were bilaterally implanted into the rostral pontine tegmental nuclei (RPTN; n = 3), the lateral (LPBN; n = 4) or medial parabrachial nuclei (MPBN; n = 4), or the Kölliker-Fuse nucleus (KFN; n = 4). After recovery from surgery, 1- and 10-microl injections (1 wk apart) of IA were made bilaterally through the implanted cannulas during the day. Over the first 5 h after the injections, there were site-specific ventilatory effects, with increased (P < 0.05) breathing frequency in RPTN-injected goats, increased (P < 0.05) pulmonary ventilation (Vi) in LPBN-injected goats, no effect (P < 0.05) in MPBN-injected goats, and a biphasic Vi response (P < 0.05) in KFN-injected goats. This biphasic response consisted of a hyperpnea for 30 min, followed by a prolonged hypopnea and hypoventilation with marked apneas, apneusis-like breathing patterns, and/or shifts in the temporal relationships between inspiratory flow and diaphragm activity. In the awake state, 10-15 h after the 1-microl injections, the number of apneas was greater (P < 0.05) than during other studies at night. However, there were no incidences of terminal apneas. Breathing rhythm and pattern were normal 22 h after the injections. Subsequent histological analysis revealed that for goats with cannulas implanted into the KFN, there were nearly 50% fewer neurons (P < 0.05) in all three PRG subnuclei than in control goats. We conclude that in awake goats, 1) IA injections into the PRG have site-specific effects on breathing, and 2) the KFN contributes to eupneic respiratory pattern generation.

A role for the Kolliker-Fuse nucleus in cholinergic modulation of breathing at night during wakefulness and NREM sleep.

For many years, acetylcholine has been known to contribute to the control of breathing and sleep. To probe further the contributions of cholinergic rostral pontine systems in control of breathing, we designed this study to test the hypothesis that microdialysis (MD) of the muscarinic receptor antagonist atropine into the pontine respiratory group (PRG) would decrease breathing more in animals while awake than while in NREM sleep. In 16 goats, cannulas were bilaterally implanted into rostral pontine tegmental nuclei (n = 3), the lateral (n = 3) or medial (n = 4) parabrachial nuclei, or the Kölliker-Fuse nucleus (KFN; n = 6). After >2 wk of recovery from surgery, the goats were studied during a 45-min period of MD with mock cerebrospinal fluid (mCSF), followed by at least 30 min of recovery and a second 45-min period of MD with atropine. Unilateral and bilateral MD studies were completed during the day and at night. MD of atropine into the KFN at night decreased pulmonary ventilation and breathing frequency and increased inspiratory and expiratory time by 12-14% during both wakefulness and NREM sleep. However, during daytime studies, MD of atropine into the KFN had no effect on these variables. Unilateral and bilateral nighttime MD of atropine into the KFN increased levels of NREM sleep by 63 and 365%, respectively. MD during the day or at night into the other three pontine sites had minimal effects on any variable studied. Finally, compared with MD of mCSF, bilateral MD of atropine decreased levels of acetylcholine and choline in the effluent dialysis fluid. Our data support the concept that the KFN is a significant contributor to cholinergically modulated control of breathing and sleep.

The pontine respiratory group, particularly the Kölliker-Fuse nucleus, mediates phases of the hypoxic ventilatory response in unanesthetized goats.

The objective of the present study was to test the hypothesis that, in the in vivo awake goat model, perturbation/lesion in the pontine respiratory group (PRG) would decrease the sensitivity to hypercapnia and hypoxia. The study reported herein was part of two larger studies in which cholinergic modulation in the PRG was attenuated by microdialysis of atropine and subsequently ibotenic acid injections neurotoxically lesioned the PRG. In 14 goats, cannula were bilaterally implanted into either the lateral (n=4) or medial (n=4) parabrachial nuclei or the Kölliker-Fuse nucleus (KFN, n=6). Before and after cannula implantation, microdialysis of atropine, and injection of ibotenic acid, hypercapnic and hypoxic ventilatory sensitivities were assessed. Hypercapnic sensitivity was assessed by three 5-min periods at 3, 5, and 7% inspired CO2. In all groups of goats, CO2 sensitivity was unaffected (P>0.05) by any PRG perturbations/lesions. Hypoxic sensitivity was assessed with a 30-min period at 10.8% inspired O2. The response to hypoxia was typically triphasic, with a phase 1 increase in pulmonary ventilation, a phase 2 roll-off, and a phase 3 prolonged increase associated with shivering and increased metabolic rate and body temperature. In all groups of goats, the phase 1 of the hypoxic ventilatory responses was unaffected by any PRG perturbations/lesions, and there were no consistent effects on the phase 2 responses. However, in the KFN group of goats, the phase 3 ventilatory, shivering, metabolic rate, and temperature responses were markedly attenuated after the atropine dialysis studies, and the attenuation persisted after the ibotenic acid studies. These findings support an integrative or modulatory role for the KFN in the phase 3 responses to hypoxia.

Differences between three inbred rat strains in number of K+ channel-immunoreactive neurons in the medullary raphé nucleus.

Ventilatory sensitivity to hypercapnia is greater in Dahl salt-sensitive (SS) rats than in Fawn Hooded hypertensive (FHH) and Brown Norway (BN) inbred rats. Since pH-sensitive potassium ion (K(+)) channels are postulated to contribute to the sensing and signaling of changes in CO(2)-H(+) in chemosensitive neurons, we tested the hypothesis that there are more pH-sensitive K(+) channel-immunoreactive (ir) neurons within the medullary raphé nuclei of the highly chemosensitive SS rats than in the other two strains. Medullary tissues from male and female BN, FHH, and SS rats were stained with cresyl violet or with antibodies targeting TASK-1, K(v)1.4, and Kir2.3 channels. K(+) channel-ir neurons were quantified and compared with the total neurons in the region. The total number of neurons in the medullary raphé 1) was greater in male FHH than the other male rats, 2) did not differ among the female rats, and 3) did not differ between sexes. The average number of K(+) channel-ir neurons per section was 30-60 neurons higher in the male SS than in the other rat strains. In contrast, for the females, the number of K(+) channel-ir neurons was greatest in the BN. We also found significant differences in the number of K(+) channel-ir neurons between sexes in SS (males > females) and BN (females > males) rats, but not the FHH strain. Our findings support the hypothesis for males but not for females, suggesting that both genetic background and sex are determinants of K(+) channel immunoreactivity of medullary raphé neurons, and that the expression of pH-sensitive K(+) channels in the medullary raphé does not correlate with the ventilatory sensitivity to hypercapnia.