SUMMARY1. O2-,K+-and pH-sensitive microelectrodes were used to measure extracellular oxygen pressure (Po2), K' activity (aK.) and pH (pH.) in ventral regions of the medulla oblongata containing respiratory neurons in the in vitro brainstem-spinal cord preparation from 0 to 4-day-old rats.2. The location of respiratory neurons was mapped by extracellular recordings with conventional microelectrodes, or with the reference barrel of ion-sensitive microelectrodes. The major populations of respiratory neurons were distributed in the ventrolateral reticular formation near the nucleus ambiguus at depths of 300-600 ,um. In this area, aK. baseline increased from 3-2 to 3-8 mm whereas steadystate values of Po2 and pHo fell from 120 to 7 mmHg and from 6-9 to 6-7, respectively.3. During rhythmic inspiratory discharges recorded with suction electrodes from ventral roots of spinal (C3-C5) and cranial (IX, X, XII) nerves, aKo transiently increased by up to 100 /M, and Po2 fell maximally by 0 4 mmHg. During episodes of non-rhythmic neuronal discharge, aKo increased by as much as 0-4 mm and Po2 decreased by about 10 mmHg. In contrast, no variations in pHo could be detected during such activities.4. Activation of medullary neurons by tetanic electrical stimulation of axonal tracts in the ventrolateral column of the spinal cord at the level of the phrenic motoneuron pool produced aKo elevations of up to 5 mm, decreases of Po2 by up to 50 mmHg, and pHo increases by a maximum of 0 07 pH units. These aKo and Po2 transients were reduced by more than 80 % during blockade of synaptic transmission with 5 mm manganese (Mn2+) and completely blocked by 1 ,tm tetrodotoxin (TTX).5. The tissue Po2 gradient as well as activity-related decreases of P2 were completely abolished after block of oxidative cellular metabolism by addition of 2-10 mm cyanide (CN-) to the bathing solution.6. Inhibition of the Na+-K+ pump by addition of 3-50 /M ouabain (3-10 min)caused a reversible increase of aKo by 0-8-3 mm, a delayed recovery of stimulusinduced aKo elevations, and produced a disturbance of the respiratory rhythm.7. The sensitivity of the respiratory network to oxygen depletion was tested by superfusing the neuraxis with hypoxic solutions gassed with N2 instead of 02(5-20 min). The response of the respiratory network to such hypoxic exposure MS 9830 J. BROCKHA US AND OTHERS consisted of an initial increase in frequency of respiratory motor output, followed by a depression of respiratory activity that terminated in a reversible loss of the respiratory rhythm.8 10. It is concluded that the oxygen supply of the ventral respiratory network is sufficient to maintain aerobic neuronal metabolism and metabolically driven Na+-K+ pump activity which allows respiratory rhythmogenesis and network function in the in vitro neonatal rat brainstem-spinal cord preparation.

1. The role of excitatory amino acids (EAAs) in the bulbospinal transmission of inspiratory drive was studied by intracellular and single-electrode voltage-clamp recordings from phrenic motoneurons in the in vitro neonatal rat brain stem spinal cord. 2. In all brain stem-spinal cord preparations there were spontaneously generated rhythmic membrane depolarizations and associated spiking of phrenic motoneurons during the inspiratory phase of the respiratory cycle. The envelope of the motoneuron drive potential had a rapid onset to peak (50 ms) followed by a plateau/declining phase that lasted 400-700 ms. The peak potential was approximately 10-20 mV above base-line potential. The drive current under voltage clamp had a similar shape and duration to the drive potential with a peak current greater than 1.5 nA. 3. The involvement of EAAs in the bulbospinal transmission of inspiratory drive was demonstrated by checking the effects of various EAA receptor antagonists on the phrenic motoneuron inspiratory drive. When kynurenic acid (KYN), an antagonist acting on all three subtypes of EAA receptors, was applied to the solution bathing the spinal cord, the motoneuron action potentials were abolished, and the amplitude of inspiratory drive potential was significantly reduced. To further classify the role of the different EAA receptor subtypes in the synaptic transmission of inspiratory drive, the effects on the drive potential of either 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a specific non-N-methyl-D-aspartic acid (non-NMDA) receptor antagonist, or DL-2-amino-5-phosphonovaleric acid (AP5), DL-2-amino-7-phosphonoheptanoic acid (AP7), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin emaleate (MK-801), NMDA receptor antagonists, were investigated. Bath or local application of CNQX induced a dose-dependent decrease of the inspiratory drive potential without changing intrinsic motoneuron membrane properties. On the other hand, application of AP7 or MK 801 had a small effect on the inspiratory drive potential or the inspiratory drive current when the motoneuron membrane potential was clamped near end-expiratory potentials (-60 to -75 mV). 4. To establish the presence of EAA receptors on the phrenic motoneuronal membrane and to provide information on the available receptor subtypes for action of the endogenously released transmitter, we tested the effects of agonists for the major EAA receptor subtypes after blocking synaptic transmission (produced by axonal action potentials) by bath application of tetrodotoxin (TTX).(ABSTRACT TRUNCATED AT 400 WORDS)

Summary Eating is a learned process. Our desires for specific foods arise through experience. Both electrical stimulation and optogenetic studies have shown that increased activity in the lateral hypothalamus (LH) promotes feeding. Current dogma is that these effects reflect a role for LH neurons in the control of the core motivation to feed, and their activity comes under control of forebrain regions to elicit learned food-motivated behaviors. However, these effects could also reflect the storage of associative information about the cues leading to food in LH itself. Here we present data from several studies that are consistent with a role for LH in learning. In the first experiment, we used a novel GAD-Cre rat to show that optogenetic inhibition of LH GABA neurons restricted to cue presentation disrupts the rats’ ability to learn that a cue predicts food without affecting subsequent food consumption. In the second experiment, we show that this manipulation also disrupts the ability of a cue to promote food seeking after learning. Finally, we show that inhibition of the terminals of the LH GABA neurons in VTA facilitates learning about reward-paired cues. These results suggest that the LH GABA neurons are critical for storing and later disseminating information about reward-predictive cues.

Salt cluster ions of alkali metal chlorides ACl (A = Li(+), Na(+), K(+), Rb(+) and Cs(+)) and sodium salts NaB (B = I(-), HCOO(-), CH(3)COO(-), NO(2)(-), and NO(3)(-)), formed by electrospray ionization, were studied systematically by mass spectrometry. The influences on the total positive ion and negative ion currents of variation of solvent, solution concentration, desolvation temperature, solution flow-rate, capillary voltage and cone voltage were investigated. Only cone voltage was found to influence dramatically the distribution of salt cluster ions in the mass spectra observed. Under conditions of normal cone voltage of approximately 70 V, cluster ions having magic numbers of molecules are detected with high relative signal intensity. Under conditions of low cone voltage of approximately 10 V, the distribution of cluster ions detected is characterized by a relatively low average mass/charge ratio due to the presence of multiply charged cluster ions; in addition, there is a marked reduction in cluster ions having a magic number of molecules. Product ion mass spectra obtained by tandem mass spectrometry of cluster ions are characterized by a base peak having a magic number of molecules that is less than and closest to the number of molecules in the precursor ion. Structures have been proposed for some dications and some quadruply charged ions. At pH 3 and 11, the mass spectra of NaCl clusters show the presence of mixed clusters of NaCl with HCl and NaOH, respectively. The effects of ionic radius on 20 distributions of cluster ions for 10 salts were investigated; however, the fine structure of these effects is not readily discerned.

A novel mass spectrometry (MS)-based lipidomics strategy that exposes glycerophospholipids to an ethereal solution of diazomethane and acid, derivatizing them to contain a net fixed, permanent positive charge, is described. The sensitivity of modified lipids to MS detection is enhanced via improved ionization characteristics as well as consolidation of ion dissociation to form one or two strong, characteristic polar headgroup fragments. Our strategy has been optimized to enable a priori prediction of ion fragmentation patterns for four subclasses of modified glycerophospholipid species. Our method enables analyte ionization regardless of proton affinity, thereby decreasing ion suppression and permitting predictable precursor ion-based quantitation with improved sensitivity in comparison to MS-based methods that are currently used on unmodified lipid precursors.