Ventilatory behavior after hypoxia in C57BL/6J and A/J mice.

Given the environmental forcing by extremes in hypoxia-reoxygenation, there might be no genetic effect on posthypoxic short-term potentiation of ventilation. Minute ventilation (VE), respiratory frequency (f), tidal volume (VT), and the airway resistance during chemical loading were assessed in unanesthetized unrestrained C57BL/6J (B6) and A/J mice using whole body plethysmography. Static pressure-volume curves were also performed. In 12 males for each strain, after 5 min of 8% O2 exposure, B6 mice had a prominent decrease in VE on reoxygenation with either air (-11%) or 100% O2 (-20%), due to the decline of f. In contrast, A/J animals had no ventilatory undershoot or f decline. After 5 min of 3% CO2-10% O2 exposure, B6 exhibited significant decrease in VE (-28.4 vs. -38.7%, air vs. 100% O2) and f (-13.8 vs. -22.3%, air vs. 100% O2) during reoxygenation with both air and 100% O2; however, A/J mice showed significant increase in VE (+116%) and f (+62.2%) during air reoxygenation and significant increase in VE (+68.2%) during 100% O2 reoxygenation. There were no strain differences in dynamic airway resistance during gas challenges or in steady-state total respiratory compliance measured postmortem. Strain differences in ventilatory responses to reoxygenation indicate that genetic mechanisms strongly influence posthypoxic ventilatory behavior.     

7-nitroindazole and posthypoxic ventilatory behavior in the A/J and C57BL/6J mouse strains.

Periodic breathing (PB) is a fundamental breathing pattern in many common cardiopulmonary illnesses. The finding of PB in C57BL/6J (B6) mice was previously ascribed to strain differences in posthypoxic ventilatory and frequency decline in the B6 mice (Han F, Subramanian S, Price ER, Nadeau J, and Strohl KP. J Appl Physiol 92: 1133-1140, 2002). We tested whether the induction of posthypoxic frequency decline in A/J mice, through administration of a neuronal nitric oxide synthase blocker [7-nitroindazole (7-NI); 60 mg/kg], would cause A/J mice to exhibit PB and/or alter PB expression in the B6 strain. Recordings of ventilatory behavior by the plethysmography method were made when unanesthetized B6 (n = 10) or A/J (n = 6) animals were reoxygenated with 100% O2 or room air after exposure to 8% O2. Before undergoing gas challenges, mice were given an intraperitoneal injection of either peanut oil alone (vehicle) or 7-NI suspended in peanut oil. Compared with vehicle, both strains of mice exhibited posthypoxic frequency decline and the absence of short-term potentiation with 7-NI administration. B6 mice continued to exhibit posthypoxic PB; however, the PB was characterized by longer cycle and apnea length. In contrast, A/J mice did not show increased tendency toward posthypoxic PB with 7-NI. We conclude that 7-NI further differentiates the A/J and B6 strains in terms of PB and that strain-related differences in posthypoxic frequency decline are not primary determinants of this strain difference in the occurrence of PB. Metabolism was not associated with either the expression of posthypoxic ventilatory decline or PB. Furthermore, neuronal nitric oxide may be an organizing feature in the presence, length, and/or cycle length of apnea in the susceptible strain.     

Acetazolamide protects against posthypoxic unstable breathing in the C57BL/6J mouse.

Acetazolamide (Acz), a carbonic anhydrase inhibitor, is used to manage periodic breathing associated with altitude and with heart failure. We examined whether Acz would alter posthypoxic ventilatory behavior in the C57BL/6J (B6) mouse model of recurrent central apnea. Experiments were performed with unanesthetized, awake adult male B6 mice (n = 9), ventilatory behavior was measured using flow-through whole body plethysmography. Mice were given an intraperitoneal injection of either vehicle or Acz (40 mg/kg), and 1 h later they were exposed to 1 min of 8% O(2)-balance N(2) (poikilocapnic hypoxia) or 12% O(2)-3% CO(2)-balance N(2) (isocapnic hypoxia) followed by rapid reoxygenation (100% O(2)). Hypercapnic response (8% CO(2)-balance O(2)) was examined in six mice. With Acz, ventilation, including respiratory frequency, tidal volume, and minute ventilation, in room air was significantly higher and hyperoxic hypercapnic ventilatory responsiveness was generally lower compared with vehicle. Poikilocapnic and isocapnic hypoxic ventilatory responsiveness were similar among treatments. One minute after reoxygenation, animals given Acz exhibited posthypoxic frequency decline, a lower coefficient of variability for frequency, and no tendency toward periodic breathing, compared with vehicle treatment. We conclude that Acz improves unstable breathing in the B6 model, without altering hypoxic response or producing short-term potentiation, but with some blunting of hypercapnic responsiveness.     

Strain differences in murine ventilatory behavior persist after urethane anesthesia.

Differences in breathing pattern between awake C57BL/6J (B6) and A/J mice are such that A/J mice breathe slower, deeper, and with greater variability than B6. We theorized that urethane anesthesia, by affecting cortical and subcortical function, would test the hypothesis that strain differences require a fully functional neuroaxis. We anesthetized B6 and A/J mice with urethane, placed them in a whole-body plethysmograph, and measured the durations of inspiration and expiration, respiratory frequency (Fr), and peak amplitude during exposure to room air (21% O2), hyperoxia (5 min, 100% O2), hypoxia (5 min, 8% O2), and posthypoxic reoxygenation (5 min, 100% O2). Breathing variability was assessed by calculating the coefficient of variation (CV) and by applying spatial statistics to Poincaré plots constructed from the timing and amplitude data. Even though Fr in anesthetized B6 and A/J mice was greater than that for unanesthetized animals, anesthetized A/J mice still breathed slower, deeper, and with greater variability than B6 mice at rest and during hyperoxia. During the fourth minute of hypoxia, Fr and its CV were not significantly different between strains. Even though Fr was similar between strains immediately after hypoxia, its CV was significantly greater for B6 than A/J mice. Posthypoxic Fr was significantly less than baseline Fr in B6 but not A/J mice, and the CV for posthypoxic Fr was greater for B6 but less for AJ mice compared with baseline CV. This difference in patterning was confirmed by spatial statistical analysis. We conclude that strain-specific differences in respiratory pattern and its variability are robust genetic traits. The neural substrate for these differences, at least partially, exists within subcortical structures generating the breathing pattern.     

Periodic breathing in the mouse.

The hypothesis was that unstable breathing might be triggered by a brief hypoxia challenge in C57BL/6J (B6) mice, which in contrast to A/J mice are known not to exhibit short-term potentiation; as a consequence, instability of ventilatory behavior could be inherited through genetic mechanisms. Recordings of ventilatory behavior by the plethsmography method were made when unanesthetized B6 or A/J animals were reoxygenated with 100% O(2) or air after exposure to 8% O(2) or 3% CO(2)-10% O(2) gas mixtures. Second, we examined the ventilatory behavior after termination of poikilocapnic hypoxia stimuli in recombinant inbred strains derived from B6 and A/J animals. Periodic breathing (PB) was defined as clustered breathing with either waxing and waning of ventilation or recurrent end-expiratory pauses (apnea) of > or = 2 average breath durations, each pattern being repeated with a cycle number > or = 3. With the abrupt return to room air from 8% O(2), 100% of the 10 B6 mice exhibited PB. Among them, five showed breathing oscillations with apnea, but none of the 10 A/J mice exhibited cyclic oscillations of breathing. When the animals were reoxygenated after 3% CO(2)-10% O(2) challenge, no PB was observed in A/J mice, whereas conditions still induced PB in B6 mice. (During 100% O(2) reoxygenation, all 10 B6 mice had PB with apnea.) Expression of PB occurred in some but not all recombinant mice and was not associated with the pattern of breathing at rest. We conclude that differences in expression of PB between these strains indicate that genetic influences strongly affect the stability of ventilation in the mouse.     

Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior

BTBR mice are potentially useful tools for autism research because their behavior parallels core social interaction impairments and restricted-repetitive behaviors. Altered regulation of central serotonin (5-HT) neurotransmission may underlie such behavioral deficits. To test this, we compared 5-HT transporter (SERT), 5-HT(1A) and 5-HT(2A) receptor densities among BTBR and C57 strains. Autoradiographic [(3) H] cyanoimipramine (1 nM) binding to SERT was 20-30% lower throughout the adult BTBR brain as compared to C57BL/10J mice. In hippocampal membrane homogenates, [(3) H] citalopram maximal binding (B(max) ) to SERT was 95 ± 13 fmol/mg protein in BTBR and 171 ± 20 fmol/mg protein in C57BL/6J mice, and the BTBR dissociation constant (K(D) ) was 2.0 ± 0.3 nM versus 1.1 ± 0.2 in C57BL/6J mice. Hippocampal 5-HT(1A) and 5-HT(2A) receptor binding was similar among strains. However, 8-OH-DPAT-stimulated [(35) S] GTPγS binding in the BTBR hippocampal CA(1) region was 28% higher, indicating elevated 5-HT(1A) capacity to activate G-proteins. In BTBR mice, the SERT blocker, fluoxetine (10 mg/kg) and the 5-HT(1A) receptor partial-agonist, buspirone (2 mg/kg) enhanced social interactions. The D(2) /5-HT(2) receptor antagonist, risperidone (0.1 mg/kg) reduced marble burying, but failed to improve sociability. Overall, altered SERT and/or 5-HT(1A) functionality in hippocampus could contribute to the relatively low sociability of BTBR mice.     

Curcumin prevents corticosterone-induced neurotoxicity and abnormalities of neuroplasticity via 5-HT receptor pathway

Curcumin, a major active component of Curcuma longa, possesses antioxidant and neuroprotective activities. The present study explores the mechanisms underlying the neuroprotective effect of curcumin against corticosterone and its relation to 5-hydroxy tryptamine (5-HT) receptors. Exposure of cortical neurons to corticosterone results in decreased mRNA levels for three 5-HT receptor subtypes, 5-HT(1A), 5-HT(2A) and 5-HT(4), but 5-HT(1B,) 5-HT(2B), 5-HT(2C), 5-HT(6) and 5-HT(7) receptors remain unchanged. Pre-treatment with curcumin reversed this effect on mRNA for the 5-HT(1A) and 5-HT(4) receptors, but not for the 5-HT(2A) receptor. Moreover, curcumin exerted a neuroprotective effect against corticosterone-induced neuronal death. This observed effect of curcumin was partially blocked by either 5-HT(1A) receptor antagonist p-MPPI or 5-HT(4) receptor antagonist RS 39604 alone; whereas, the simultaneous application of both antagonists completely reversed the effect. Curcumin was also found to regulate corticosterone-induced morphological changes such as increases in soma size, dendritic branching and dendritic spine density, as well as elevate synaptophysin expression in cortical neurons. p-MPPI and RS 39604 reversed the effect of curcumin-induced change in neuronal morphology and synaptophysin expression of corticosterone-treated neurons. In addition, an increase in cyclic adenosine monophosphate (cAMP) level was observed after curcumin treatment, which was further prevented by RS 39604, but not by p-MPPI. However, curcumin-induced elevation in protein kinase A activity and phosphorylation of cAMP response element-binding protein levels were inhibited by both p-MPPI and RS 39604. These findings suggest that the neuroprotection and modulation of neuroplasticity exhibited by curcumin might be mediated, at least in part, via the 5-HT receptor-cAMP-PKA-CREB signal pathway.     

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.     

Endogenous 5-HT2B receptor activation regulates neonatal respiratory activity in vitro

An implication of 5-HT(2B) receptors in central nervous system has not yet been clearly elucidated. We studied the role of different 5-HT(2) receptor subtypes in the medullary breathing center, the pre-B?tzinger complex, and on hypoglossal motoneurons in rhythmically active transversal slice preparations of neonatal rats and mice. Local microinjection of 5-HT(2) receptor agonists revealed tonic excitation of hypoglossal motoneurons. Excitatory effects of the 5-HT(2B) receptor agonist BW723C86 could be blocked by bath application of LY272015, a highly selective 5-HT(2B) receptor antagonist. Excitatory effects of the 5-HT(2A/B/C) receptor agonist alpha-methyl 5-HT could be blocked by the preferential 5-HT(2A) receptor antagonist ketanserin. Therefore, 5-HT-induced excitation of hypoglossal motoneurons is mediated by convergent activation of 5-HT(2A) and 5-HT(2B) receptors. Local microinjection of BW723C86 in the pre-B?tzinger complex increased respiratory frequency. Bath application of LY272015 blocked respiratory activity, whereas ketanserin had no effect. Therefore, endogenous 5-HT appears to support tonic action on respiratory rhythm generation via 5-HT(2B) receptors. In preparations of 5-HT(2B) receptor-deficient mice, respiratory activity appeared unaltered. Whereas BW723C86 and LY272015 had no effects, bath application of ketanserin disturbed and blocked rhythmic activity. This demonstrates a stimulatory role of endogenous 5-HT(2B) receptor activation at the pre-B?tzinger complex and hypoglossal motoneurons that can be taken up by 5-HT(2A) receptors in the absence of 5-HT(2B) receptors. The presence of functional 5-HT(2B) receptors in the neonatal medullary breathing center indicates a potential convergent regulatory role of 5-HT(2B) and -(2A) receptors on the central respiratory network.     

Neonatal buspirone modulates the intermale aggression in adult mice

Mice of two strains with different levels of male aggression (RSB and RLB) were subjected to daily injections of 5-HT1A receptor agonist buspirone (25 microg) on the 2nd - 6th postnatal days. This neonatal treatment augmented the aggressive behavior (tested in the dyadic contests with non-aggressive A/Sn males) in aggressive RSB mice and reduced aggression in less aggressive RLB. Correlations with different signs were found between the 5-HT and 5-HIAA levels in the neocortex, hippocampus, and hypothalamus and behavioral indices of aggression in RSB and RLB males. The remote effects of neonatal buspirone in these two mice strains presumably depend on genotype-related features of ontogeny of the 5-HT system.     

Differential lung mechanics are genetically determined in inbred murine strains.

Genetic determinants of lung structure and function have been demonstrated by differential phenotypes among inbred mice strains. For example, previous studies have reported phenotypic variation in baseline ventilatory measurements of standard inbred murine strains as well as segregant and nonsegregant offspring of C3H/HeJ (C3) and C57BL/6J (B6) progenitors. One purpose of the present study is to test the hypothesis that a genetic basis for differential baseline breathing pattern is due to variation in lung mechanical properties. Quasi-static pressure-volume curves were performed on standard and recombinant inbred strains to explore the interactive role of lung mechanics in determination of functional baseline ventilatory outcomes. At airway pressures between 0 and 30 cmH2O, lung volumes are significantly (P < 0.01) greater in C3 mice relative to the B6 and A/J strains. In addition, the B6C3F1/J offspring demonstrate lung mechanical properties significantly (P < 0.01) different from the C3 progenitor but not distinguishable from the B6 progenitor. With the use of recombinant inbred strains derived from C3 and B6 progenitors, cosegregation analysis between inspiratory timing and measurements of lung volume and compliance indicate that strain differences in baseline breathing pattern and pressure-volume relationships are not genetically associated. Although strain differences in lung volume and compliance between C3 and B6 mice are inheritable, this study supports a dissociation between differential inspiratory time at baseline, a trait linked to a putative genomic region on mouse chromosome 3, and differential lung mechanics among C3 and B6 progenitors and their progeny.     

Cannabinoid-serotonin interactions in alcohol-preferring vs. alcohol-avoiding mice

Because cannabinoid and serotonin (5-HT) systems have been proposed to play an important role in drug craving, we investigated whether cannabinoid 1 (CB1) and 5-HT(1A) receptor ligands could affect voluntary alcohol intake in two mouse strains, C57BL/6 J and DBA/2 J, with marked differences in native alcohol preference. When offered progressively (3-10% ethanol) in drinking water, in a free-choice procedure, alcohol intake was markedly lower (approximately 70%) in DBA/2 J than in C57BL/6 J mice. In DBA/2 J mice, chronic treatment with the cannabinoid receptor agonist WIN 55,212-2 increased alcohol intake. WIN 55,212-2 effect was prevented by concomitant, chronic CB1 receptor blockade by rimonabant or chronic 5-HT(1A) receptor stimulation by 8-hydroxy-2-(di-n-propylamino)-tetralin, which, on their own, did not affect alcohol intake. In C57BL/6 J mice, chronic treatment with WIN 55,212-2 had no effect but chronic CB1 receptor blockade or chronic 5-HT(1A) receptor stimulation significantly decreased alcohol intake. Parallel autoradiographic investigations showed that chronic treatment with WIN 55,212-2 significantly decreased 5-HT(1A)-mediated [35S]guanosine triphosphate-gamma-S binding in the hippocampus of both mouse strains. Conversely, chronic rimonabant increased this binding in C57BL/6 J mice. These results show that cannabinoid neurotransmission can exert a permissive control on alcohol intake, possibly through CB1-5-HT(1A) interactions. However, the differences between C57BL/6 J and DBA/2 J mice indicate that such modulations of alcohol intake are under genetic control.     

Ventilatory behavior during sleep among A/J and C57BL/6J mouse strains.

The pattern of breathing during sleep could be a heritable trait. Our intent was to test this genetic hypothesis in inbred mouse strains known to vary in breathing patterns during wakefulness (Han F, Subramanian S, Dick TE, Dreshaj IA, and Strohl KP. J Appl Physiol 91: 1962-1970, 2001; Han F, Subramanian S, Price ER, Nadeau J, and Strohl KP, J Appl Physiol 92: 1133-1140, 2002) to determine whether such differences persisted into non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Measures assessed in C57BL/6J (B6; Jackson Laboratory) and two A/J strains (A/J Jackson and A/J Harlan) included ventilatory behavior [respiratory frequency, tidal volume, minute ventilation, mean inspiratory flow, and duty cycle (inspiratory time/total breath time)], and metabolism, as performed by the plethsmography method with animals instrumented to record EEG, electromyogram, and heart rate. In all strains, there were reductions in minute ventilation and CO2 production in NREM compared with wakefulness (P < 0.001) and a further reduction in REM compared with NREM (P < 0.001), but no state-by-stain interactions. Frequency showed strain (P < 0.0001) and state-by-strain interactions (P < 0.0001). The A/J Jackson did not change frequency in REM vs. NREM [141 +/- 15 (SD) vs. 139 +/- 14 breaths/min; P = 0.92], whereas, in the A/J Harlan, it was lower in REM vs. NREM (168 +/- 14 vs. 179 +/- 12 breaths/min; P = 0.0005), and, in the B6, it was higher in REM vs. NREM (209 +/- 12 vs. 188 +/- 13 breaths/min; P < 0.0001). Heart rate exhibited strain (P = 0.003), state (P < 0.0001), and state-by-strain interaction (P = 0.017) and was lower in NREM sleep in the A/J Harlan (P = 0.035) and B6 (P < 0.0001). We conclude that genetic background affects features of breathing during NREM and REM sleep, despite broad changes in state, metabolism, and heart rate.     

Interactions in hypoxic and hypercapnic breathing are genetically linked to mouse chromosomes 1 and 5.

The genetic basis for differences in the regulation of breathing is certainly multigenic. The present paper builds on a well-established genetic model of differences in breathing using inbred mouse strains. We tested the interactive effects of hypoxia and hypercapnia in two strains of mice known for variation in hypercapnic ventilatory sensitivity (HCVS); i.e., high gain in C57BL/6J (B6) and low gain in C3H/HeJ (C3) mice. Strain differences in the magnitude and pattern of breathing were measured during normoxia [inspired O(2) fraction (Fi(O(2))) = 0.21] and hypoxia (Fi(O(2)) = 0.10) with mild or severe hypercapnia (inspired CO(2) fraction = 0.03 or 0.08) using whole body plethysmography. At each level of Fi(O(2)), the change in minute ventilation (Ve) from 3 to 8% CO(2) was computed, and the strain differences between B6 and C3 mice in HCVS were maintained. Inheritance patterns showed potentiation effects of hypoxia on HCVS (i.e., CO(2) potentiation) unique to the B6C3F1/J offspring of B6 and C3 progenitors; i.e., the change in Ve from 3 to 8% CO(2) was significantly greater (P < 0.01) with hypoxia relative to normoxia in F1 mice. Linkage analysis using intercross progeny (F2; n = 52) of B6 and C3 progenitors revealed two significant quantitative trait loci associated with variable HCVS phenotypes. After normalization for body weight, variation in Ve responses during 8% CO(2) in hypoxia was linked to mouse chromosome 1 (logarithm of the odds ratio = 4.4) in an interval between 68 and 89 cM (i.e., between D1Mit14 and D1Mit291). The second quantitative trait loci linked differences in CO(2) potentiation to mouse chromosome 5 (logarithm of the odds ratio = 3.7) in a region between 7 and 29 cM (i.e., centered at D5Mit66). In conclusion, these results support the hypothesis that a minimum of two significant genes modulate the interactive effects of hypoxia and hypercapnia in this genetic model.     

Compensatory airway dilation and additive ventilatory augmentation mediated by dorsomedial medullary 5-hydroxytryptamine 2 receptor activity and hypercapnia

5-HT2 receptor activity in the hypoglossal nucleus and hypercapnia is associated with airway dilation. 5-HT neurons in the medullary raphe and hypercapnia are responsible for tidal volume change. In this study, the effects of 5-HT2 receptors in the dorsomedial medulla oblongata (DMM), which receives projections from the medullary raphe, and hypercapnia on airway resistance and respiratory variables were studied in mice while monitoring 5-HT release in the DMM. A microdialysis probe was inserted into the DMM of anesthetized adult mice. Each mouse was placed in a double-chamber plethysmograph. After recovery from anesthesia, the mice were exposed to stepwise increases in CO(2) inhalation (5%, 7%, and 9% CO(2) in O(2)) at 8-min intervals with a selective serotonin reuptake inhibitor, fluoxetine, or fluoxetine plus a 5-HT2 receptor antagonist, LY-53857 in the DMM. In response to fluoxetine plus LY-53857 coperfusion, specific airway resistance was increased, and tidal volume and minute ventilation were decreased. CO(2) inhalation with fluoxetine plus LY-53857 coperfusion in the DMM largely decreased airway resistance and additively increased minute ventilation. Thus, 5-HT2 receptor activity in the DMM increases basal levels of airway dilation and ventilatory volume, dependent on central inspiratory activity and the volume threshold of the inspiratory off-switch mechanism. Hypercapnia with low 5-HT2 receptor activity in the DMM largely recovers airway dilation and additively increases ventilatory volume. Interaction between 5-HT2 receptor activity in the DMM and CO(2) drive may elicit a cycle of hyperventilation with airway dilation and hypoventilation with airway narrowing.     

Molecular dynamics of buspirone analogues interacting with the 5-HT1A and 5-HT2A serotonin receptors

Three-dimensional (3-D) models of the human serotonin 5-HT1A and 5-HT2A receptors were constructed, energy refined, and used to study the interactions with a series of buspirone analogues. For both receptors, the calculations showed that the main interactions of the ligand imide moieties were with amino acids in transmembrane helix (TMH) 2 and 7, while the main interactions of the ligand aromatic moieties were with amino acids in TMH5, 6 and 7. Differences in binding site architecture in the region of highly conserved serine and tyrosine residues in TMH7 gave slightly different binding modes of the buspirone analogues at the 5-HT1A and 5-HT2A receptors. Molecular dynamics simulations of receptor-ligand interactions indicated that the buspirone analogues did not alter the interhelical hydrogen bonding patterns upon binding to the 5-HT2A receptor, while interhelical hydrogen bonds were broken and others were formed upon ligand binding to the 5-HT1A receptor. The ligand-induced changes in interhelical hydrogen bonding patterns of the 5-HT1A receptor were followed by rigid body movements of TMH2, 4 and 6 relative to each other and to the other TMHs, which may reflect the structural conversion into an active receptor structure.     

Structural and functional differences of the carotid body between DBA/2J and A/J strains of mice.

In a previous study, DBA/2J and A/J inbred mice showed extremely different hypoxic ventilatory responses, suggesting variations in their carotid bodies. We have assessed the morphological and functional differences of the carotid bodies in these mice. Histological examination revealed a clearly delineated carotid body only in the DBA/2J mice. Many typical glomus cells and glomeruli appeared in the DBA/2J but not in the A/J mice. The size of the carotid body in the DBA/2J and A/J mice was 6.3 +/- 0.5 x 10(6) and 1.5 +/- 0.3 x 10(6) micro m(3), respectively. The area immunostained for tyrosine hydroxylase, an estimation of the glomus cell quantity, was four times larger in the DBA/2J mice than in the A/J mice. The individual data points in the DBA/2J mice segregated from those in the A/J mice. ACh increased intracellular Ca(2+) in most clusters (81%) of cultured carotid body cells from the DBA/2J mice, but only in 18% of clusters in the A/J mice. These data suggest that genetic determinants account for the strain differences in the structure and function of the carotid body.     

Anxiety and increased 5-HT1A receptor response in NCAM null mutant mice.

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety-like behavior of homozygous (NCAM-/-) and heterozygous (NCAM/-) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety-like behavior was reduced in both NCAM+/+ and NCAM-/- mice by systemic administration of the benzodiazepine agonist diazepam and the 5-HT1A receptor agonists buspirone and 8-OH-DPAT. However, NCAM-/- mice showed anxiolytic-like effects at lower doses of buspirone and 8-OH-DPAT than NCAM+/+ mice. Such increased response to 5-HT1A receptor stimulation suggests a functional change in the serotonergic system of NCAM-/- mice, likely involved in the control of anxiety and aggression. However, 5-HT1A receptor binding and tissue content of serotonin and its metabolite 5-hydroxyindolacetic acid were found unaltered in every brain area of NCAM-/- mice investigated, indicating that expression of 5-HT1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM-/- mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5-HT1A receptors and inwardly rectifying K+ channels as the respective effector systems.