The main olfactory bulb (MOB) receives a dense projection from the pontine nucleus locus coeruleus (LC), the largest collection of norepinephrine (NE)-containing cells in the brain. LC is the sole source of NE innervation of MOB. Previous studies of the actions of exogenously applied NE on mitral cells, the principal output neurons of MOB, are contradictory. The effect of synaptically released NE on mitral cell activity is not known, nor is the influence of NE on responses of mitral cells to olfactory nerve inputs. The goal of the present study was to assess the influence of LC activation on spontaneous and olfactory nerve-evoked activity of mitral cells. In methoxyflurane-anesthetized rats, intracoerulear microinfusions of acetyicholine (ACh) (200 mM; 90-120 nl) evoked a four- to fivefold increase in LC neuronal discharge, and a transient EEG desynchronization and decrease in mitral cell discharge. LC activation increased excitatory responses of mitral cells evoked by weak (i.e., perithreshold) nasal epithelium shocks (1.0 Hz) in 17/18 cells (mean Increase = 67%). The discharge rate of mitral cells at the time that epithelium-evoked responses were increased did not differ significantly from pre-LC activation baseline values. Thus, changes in mitral baseline activity do not account for the increased response to epithelium stimulation. These findings suggest that increased activity in LC-NE projections to MOB may enhance detection of relatively weak odors.

Injection of GABA into the midbrain periaqueductal gray (PAG) activates medullary neurons that are involved in pain inhibition and potentiates morphine-induced analgesia. These observations suggest that GABAergic mechanisms in the PAG may modulate the descending pain inhibitory system that arises from this structure. In the present study, the effects of GABA and GABA antagonists on membrane properties and baseline activity of PAG neurons were examined using both in vitro and in vivo preparations. Application of bicuculline methiodide (BICM), at a dose that blocked the response to GABA, potently increased the baseline firing rate in 53% of cells recorded in vitro and 74% of cells recorded in the intact preparation. Application of BICM often yielded multiple or burst spiking episodes in both preparations. In 69% of cells the effect of BICM was diminished or totally abolished when the slice was perfused with high-magnesium, calcium-free, physiological saline solution. Intracellular recordings revealed that bicuculline caused depolarization of the membrane (70% of cells), increased the firing frequency (94% of cells) and increased the frequency of excitatory postsynaptic potentials (18% of cells). The effect of bicuculline on membrane resistance was not pronounced and in 64% of neurons it did not cause any measurable change in the resting membrane resistance. PAG neurons responsive to GABA and its antagonists were observed in all regions of the PAG. However, the highest number of neurons that responded to GABA and its antgonists was found in the medial and medioventral parts of the PAG. These results indicate that PAG may contain a tonically active GABAergic network that operates, at least in part, through GABAA receptors. This GABAergic system may modulate activity in descending pain inhibitory pathways emanating from PAG.

The medial preoptic area (MPO), a sexually dimorphic region, plays a pivotal role in neuroendocrine function and reproductive behavior. We recently reported that MPO projects heavily to the midbrain periaqueductal gray (PAG). We also noted that MPO projects to the dorsolateral pontine tegmentum. Here we identified the cells of origin of the MPO-->tegmental projection and delineated the terminal organization of MPO projections to Barrington's nucleus, locus coeruleus (LC), and the rostromedial pericoerulear region (pLCrm). Correlative cyto- and chemoarchitectonic studies were done to define better the nuclear groups of the dorsolateral pontine tegmentum. Retrograde tracing revealed that MPO neurons projecting to the dorsolateral pontine tegmentum are preferentially distributed in distinct subregions of MPO, including the sexually dimorphic medial preoptic nucleus (MPN). Anterograde tracing with wheat germ agglutinin-horseradish peroxidase or Phaseolus vulgaris leucoagglutinin demonstrated considerable target specificity in projections from MPO to the dorsolateral pontine tegmentum. Barrington's nucleus receives a dense focal input along its entire rostrocaudal axis. In addition, pLCrm is heavily targeted by MPO inputs; pLCrm contains a concentrated plexus of extranuclear dendrites of LC neurons. The lateral dorsal tegmental (LDT) nucleus and LC proper receive only sparse input from MPO. MPO projections to Barrington's nucleus could regulate micturition reflexes during reproductive behavior. The MPO-->pLCrm projection could influence noradrenergic LC neurons in relation to reproductive and/or gonadal steroid function. Given the strong established connections from olfactory structures to MPO, it is possible that the MPO-->LC pathway provides an anatomical substrate for olfactory modulation of arousal.

IntroductionThe KCl cotransporter (KCC) is a member of the cation-chloride cotransporter family of proteins that mediate electroneutral net transport of salt to effect cell volume regulation, epithelial solute secretion and absorption, and control of intracellular ion concentrations. 1 In red blood cells (RBCs), KCC functions during reticulocyte maturation to establish the steady-state volume and mean corpuscular hemoglobin concentration (MCHC) of the mature RBC. 2,3 RBC KCC activity is stimulated in vitro by cell swelling (low MCHC), 4,5 acid pH, 6,7 and urea, 8 but the physiologic stimuli that effect reticulocyte volume reduction in vivo are not clear.KCC activation is controlled by phosphatase/kinase equilibria. Activation involves dephosphorylation of a serine (threonine) residue, probably by protein phosphatase 1 (PP1) 9,10 or PP2A. 11 There is also evidence for control by tyrosine phosphorylation; in fact, certain tyrosine kinase inhibitors activate KCC, 12,13 whereas others inhibit, 14,15 suggesting multiple control points, including PP1 itself. 16,17 Activation by cell swelling appears to involve inhibition of a putative volume-sensitive serine/threonine kinase, which maintains the system in a phosphorylated, quiescent state. 18 It is unknown whether control is mediated through phosphorylation of the transporter itself or other regulatory protein(s).KCC is also activated by sulfhydryl alkylation with Nethylmaleimide (NEM) 5,[19][20][21] and oxidation by diamide 22 or hydrogen peroxide. 23 CDNB (chloro-dinitrobenzine) stimulates KCC without a direct oxidant effect by enzymatic coupling to reduced glutathione (GSH). [24][25][26][27] Although the target of these agents is not known, it is clear that KCC activity and regulation are modulated by the oxidation state of RBC sulfhydryls.KCC activity in sickle (SS) RBCs is greatly elevated compared with normal (AA) RBCs, regardless of the activating stimulus. Some of this elevated activity is a consequence of the high percentage of reticulocytes in SS blood. 2,28 Nevertheless, there is evidence that the presence of sickle hemoglobin (Hb S, ␤ 6glu3val ) or hemoglobin C (Hb C, ␤ 6glu3lys ) directly affects the activity and properties of KCC in RBCs and RBC ghosts. [29][30][31]7 demonstrated acid stimulation of KCC in SS RBCs and showed that acidification of SS RBCs produced an increase in cell density. 6,7 Bookchin et al 32 showed that SS reticulocytes were susceptible to acid-induced dehydration and suggested that most of the dense cells in sickle blood arose from a subpopulation of SS reticulocytes with high sensitivity to KCC-mediated dehydration. Franco et al 33 found that SS reticulocytes that were dense in vivo were more susceptible to acid-induced dehydration via KCC than reticulocytes that were normally hydrated in vivo. These studies demonstrated the capacity of KCC to mediate SS RBC dehydration in vitro, but since direct comparisons with AA reticulocytes have not been made, the abnormal volume regulatory response of KCC in SS reticulocytes has not been f...

KCl cotransport activated by swelling of sickle red blood cells (SS RBC) is inhibited by deoxygenation. Yet recent studies found a Cl−-dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl−-dependent component of the deoxygenation-induced net K+efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl−-dependent K+ efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+ at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+ or Rb+ minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+exchange activity, known to be modulated by cytosolic Ca2+ elevation and cell shrinkage, remained silent under deoxygenation conditions.