Biomedical subjects

J S Erlichman

Publications and source records attributed to J S Erlichman.

14 recordsLinked to original sources

PubMed2001

Developmental changes in intracellular pH regulation in medullary neurons of the rat.

We examined intracellular pH (pH(i)) regulation in the retrotrapezoid nucleus (RTN), a CO(2)-sensitive site, and the hypoglossal nucleus, a nonchemosensitive site, during development (postnatal days 2-18) in rats. Respiratory acidosis [10% CO(2), extracellular pH (pH(o)) 7.18] caused acidification without pH(i) recovery in the RTN at all ages. In the hypoglossal nucleus, pH(i) recovered in young animals, but as animal age increased, the slope of pH(i) recovery diminished. In animals older than postnatal day 11, the pH(i) responses to hypercapnia were identical in the hypoglossal nucleus and the RTN, but hypoglossal nucleus and RTN neurons could regulate pH(i) during intracellular acidification at constant pH(o) at all ages. Recovery of pH(i) from acidification in the RTN depended on extracellular Na+ and was inhibited by amiloride but was unaffected by DIDS, suggesting a role for Na+/H+ exchange. Hence, pH(i) regulation during acidosis is more effective in the hypoglossal nucleus in younger animals, possibly as a requirement of development, but in older juvenile animals (older than postnatal day 11), pH(i) regulation is relatively poor in chemosensitive (RTN) and nonchemosensitive nuclei (hypoglossal nucleus).

Acidosis↗

PubMed2001

Ventilatory effects of impaired glial function in a brain stem chemoreceptor region in the conscious rat.

Glia are thought to regulate ion homeostasis, including extracellular pH; however, their role in modulating central CO2 chemosensitivity is unclear. Using a push-pull cannula in chronically instrumented and conscious rats, we administered a glial toxin, fluorocitrate (FC; 1 mM) into the retrotrapezoid nucleus (RTN), a putative chemosensitive site, during normocapnia and hypercapnia. FC exposure significantly increased expired minute ventilation (VE) to a value 38% above the control level during normocapnia. During hypercapnia, FC also significantly increased both breathing frequency and expired VE. During FC administration, maximal ventilation was achieved at approximately 4% CO2, compared with 8-10% CO2 during control hypercapnic trials. RTN perfusion of control solutions had little effect on any ventilatory measures (VE, tidal volume, or breathing frequency) during normocapnic or hypercapnic conditions. We conclude that unilateral impairment of glial function in the RTN of the conscious rat results in stimulation of respiratory output.

Animals↗

PubMed1998

Diethyl pyrocarbonate (DEPC) inhibits CO2 chemosensitivity in Helix aspersa.

Central CO2 chemoreceptors in poikilothermic vertebrates may not regulate ventilation at a particular pH setpoint; central chemoreceptor responses may more accurately reflect the relative charge state (alpha) of the imidazole of histidine. We have tested the alphastat hypothesis in the terrestrial, air breathing, pulmonate snail, Helix aspersa, by chemically modifying histidine residues in the central CO2 chemoreceptor area of this animal using diethyl pyrocarbonate (DEPC). After focal application of 20 mM DEPC to the central CO2 chemoreceptor region, the pneumostome, a respiratory, CO2 responsive organ in the snail, no longer responded to hypercapnic, acidotic stimulation of the central chemoreceptor area. However, pneumostomal responses to hypoxic stimulation of the pneumostome and to focal stimulation of the central chemoreceptor area with sodium nitroprusside, a respiratory stimulant in H. aspersa, remained intact after DEPC treatment. Furthermore, DEPC treatment of the central chemoreceptor area blocked pneumostomal responses to ammonia pre-pulse treatment, which changes intracellular pH, while extracellular pH is held constant. These results resemble mammalian responses to DEPC treatment and indicate that central chemoreceptor responses in H. aspersa may originate from changes in the alpha of intracellular histidine residues.

Ammonium Chloride↗

PubMed1998

Ventilatory effects of glial dysfunction in a rat brain stem chemoreceptor region.

Glia are thought to be important in brain extracellular fluid ion and pH regulation, but their role in brain stem sites that sense pH and stimulate breathing is unknown. Using a diffusion pipette, we administered the glial toxin, fluorocitrate (FC; 1 mM) into one such brain stem region, the retrotrapezoid nucleus (RTN) for 45-60 min. This dose and time period were chosen so that the effects of FC would be largely reversible. Within minutes, tissue pH decreased, and respiratory output increased. Both recovered almost completely after cessation of FC administration. The response to systemic CO2 stimulation was unaffected by FC treatment compared with that following control diffusion. Anatomic analysis showed, at the center of FC administration, some small (mean diameter = 5.1 micrometer) cells that stained for DEAD Red, a marker for altered cell membrane permeability, and some fragmented glia (glial fibrillary acidic protein immunohistochemistry). The average RTN tissue volume that contained such DEAD Red-positive cells was 271 nl, approximately 23% of the volume of one RTN region. Reversible disruption of glia in the RTN, a region known to contain central chemoreception, results in an acidic local pH and in stimulation of respiratory output.

Animals↗

PubMed1998

Brain stem lesion size determined by DEAD red or conjugation of neurotoxin to fluorescent beads.

Neurotoxin microinjected into the retrotrapezoid nucleus of anesthetized rats decreases phrenic activity and eliminates the response to CO2. In unanesthetized rats, such treatment has no effect on awake, resting breathing and decreases CO2 sensitivity by 40% (M. Akilesh, M. Kamper, A. Li, and E. E. Nattie. J. Appl. Physiol. 82: 469-479, 1997). One important factor in explaining these disparate results is the actual size of the anatomic lesion. In the present study, we injected ibotenic acid into the retrotrapezoid nucleus of anesthetized rats and evaluated lesion size by using two new approaches: 1) DEAD red, a fluorescent probe that enters impaired cells through leaky membranes and binds to nucleic acids, and 2) conjugation of toxin to fluorescent beads. With the use of DEAD red, the region containing labeled dying cells was 313 +/- 104 nl (n = 4), six times larger than the initial injected volume, and the physiological effects on phrenic amplitude, the CO2 response, and blood pressure began within minutes and were substantial. With conjugated toxin, in theory, neuronal damage would be limited to the region of detectable fluorescence (49 +/- 10 nl; n = 4). Effects on phrenic amplitude, CO2 sensitivity, and blood pressure were absent until approximately 2 h postinjection. Control experiments, with 2 h of in vitro incubation of the neurotoxin-microbead conjugate and injection of the supernatant after centrifugation, showed similar results that suggest release of conjugated neurotoxin. We conclude that DEAD red provides a useful means to monitor neuronal impairment in acute studies in vivo. Conjugation of neurotoxin to microbeads may be less reliable in this regard.

Animals↗

PubMed1997

Comparative aspects of central CO2 chemoreception.

We compare and contrast the putative mechanisms underlying CO2 chemoreceptor function in air breathing vertebrates and terrestrial pulmonate snails. We discuss the role of intracellular pH (pHi) in central respiratory responses to CO2 and describe a variety of patterns of pHi regulation in chemosensory areas. One pattern, in which pHi retains a fixed relationship to the CO2 stimulus over time, seems well suited to chemoreceptor cells. Alphastat regulation of ventilation is apparent in both air breathing vertebrates and terrestrial pulmonate snails. Diethyl pyrocarbonate inhibits respiratory responses to hypercapnia in both groups of animals. The neuronal basis of chemosensitivity is similar, in that putative chemoreceptor cells depolarize during hypercapnic stimulation, but the ionic basis of excitability appears to be a potassium conductance in the vertebrates studied to date and a calcium conductance in the snails. Despite divergent evolutionary histories, chemosensory responses and mechanisms are remarkably similar in air breathing vertebrates and terrestrial pulmonate snails.

Animals↗

PubMed1997

Cell-cell coupling occurs in dorsal medullary neurons after minimizing anatomical-coupling artifacts.

Dye (Lucifer Yellow) and tracer (Biocytin) coupling, referred to collectively as anatomical coupling, were identified in 20% of the solitary complex neurons tested in medullary tissue slices (120-350 microm) prepared from rat, postnatal day 1-18, using a modified amphotericin B-perforated patch recording technique. Ten per cent of the neurons sampled in nuclei outside the solitary complex were anatomically coupled. Fifty-eight per cent of anatomically coupled neurons exhibited electrotonic postsynaptic potential-like activity, which had peak-to-peak amplitudes of < or = 7 mV, with the same polarity as action potentials; increased and decreased in frequency during depolarizing and hyperpolarizing current injection; was maintained during high Mg2+-low Ca2+ chemical synaptic blockade; and was measured only in anatomically coupled neurons. The high correlation between anatomical coupling and electrotonic postsynaptic potential-like activity suggests that Lucifer Yellow, Biocytin and ionic current used the same pathways of intercellular communication, which were presumed to be gap junctions. Anatomical coupling was attributed solely to the junctional transfer of Lucifer Yellow and Biocytin since potential sources of non-junctional staining were minimized. Specifically, combining 0.26 mM amphotericin B and 0.15-0.5% Lucifer Yellow produced a hydrophobic, viscous solution that did not leak from the pressurized pipette tip < or = 3 microm outer diameter) submerged in artificial cerebral spinal fluid. Moreover, unintentional contact of the pipette tip with adjacent neurons that resulted in accidental staining, another source of non-junctional staining, wits averted by continuously visualizing the tip prior to tight seal formation with infrared video microscopy, used here for the first time with Hoffman modulation contrast optics. During perforated patch recording which typically lasted for 1-3 h. Lucifer Yellow was confined to the pipette, indicating that the amphotericin B patch was intact. However, once the patch was intentionally ruptured at the end of recording, the viscous, lipophilic solution entered the neuron resulting in double labeling. Placing a mixture of amphotericin B, Biocytin and Lucifer Yellow directly into the pipette tip did not compromise tight seal formation with an exposed, cleaned soma, and resulted in immediate (<1 min) steady-state perforation at 22-25 degrees C. This adaptation of conventional perforated patch recording was termed "rapid perforated patch recording". The possible functional implication of cell-cell coupling in the dorsal medulla oblongata in central CO2/H+ chemoreception for the cardiorespiratory control systems is discussed in the second paper of this set [Huang et al. (1997) Neuroscience 80, 41-57].

Action Potentials↗

PubMed1997

Cell-cell coupling between CO2-excited neurons in the dorsal medulla oblongata.

Anatomically coupled neurons (17 of 137) and non-coupled neurons (120 of 137), in and near the nucleus tractus solitarius and dorsal motor nucleus (i.e. solitary complex), were studied by rapid perforated patch recording in slices (rat, 150-350 microm thick, postnatal day 0-21) before, during and after exposure to hypercapnic acidosis. Anatomical coupling refers to the intercellular transfer of Lucifer Yellow and Biocytin into adjoining neurons, presumably via gap junctions [see Dean et al. (1997) Neuroscience 80, 21-40]. Eighty-six per cent of the anatomically coupled neurons (12 of 14) were depolarized by hypercapnic acidosis, a response referred to as CO2 excitation or CO2 chemosensitivity. In all, 28% (12 of 43) of the CO2-excited neurons were anatomically coupled to at least one other neuron. None (0 of 39) of the CO2-inhibited neurons were anatomically coupled, and only 4% (two of 46) of the CO2-insensitive neurons were anatomically coupled. Increasing the fractional concentration of CO2 from five to 10 and 15% in constant bicarbonate (26 mM) decreased intracellular pH (control 7.3 7.4, 22-25 degrees C) by approximately 1.0 and 1.5 pH units, respectively, as measured using the pH-sensitive fluorescent dye, 2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein. Nine of the anatomically coupled neurons (six CO2-excited, one CO2-insensitive and two unidentified) exhibited spontaneous electrotonic postsynaptic potential-like activity, suggesting that they were also electrotonically coupled. During hypercapnic acidosis, the amplitudes of electrotonic postsynaptic potentials were unchanged, concomitant with small changes in input resistance. The frequency of electrotonic postsynaptic potentials increased during hypercapnic acidosis in many anatomically coupled neurons (eight of nine), indicating that both neurons of the coupled pair were stimulated. Cell-cell coupling occurred preferentially in and between CO2-excited neurons of the solitary complex. Further, CO2-excited neurons were not electrotonically uncoupled during intracellular acidosis, in contrast to the effect that decreased intracellular pH has on many other types of coupled cells. It was not determined whether anatomical coupling was affected by hypercapnic acidosis since dye mixture was always administered under normocapnic conditions. The high correlation between anatomical coupling, electrotonic coupling activity and CO2-induced depolarization suggests that cell-cell coupling is an important electroanatomical feature in CO2-excited neurons of the solitary complex. CO2-excited neurons have been hypothesized to function in central chemoreception for the cardiorespiratory control systems, suggesting that cell cell coupling may contribute in part to central chemoreception of CO2 and H+.

Action Potentials↗

PubMed1996

A fluorescence technique to measure intracellular pH of single neurons in brainstem slices.

We have developed a technique to measure the pH, of single neurons in brainstem slices using a fluorescence imaging system. Slices were loaded with the pH-sensitive fluorescent dye BCECF and fluorescence was visualized by exciting the slices alternately at 500 and 440 nm. The emitted fluorescence at 530 nm was directed through an MTI GenIISys image intensifier and MT1 CCD72 camera. The images were processed by image-1/FL software. The ratio of emitted fluorescence at excitation wavelengths of 500 and 440 nm was measured and converted to pH by constructing a calibration curve using high K+/nigericin solutions at pH values ranging from 5.8 to 8.6. BCECF-loaded slices showed distinct spheres of intense fluorescence and diffuse background fluorescence. Slices labeled with a neuron-specific antibody, neuron-specific enolase, showed staining that correlated with the spheres of intense fluorescence of BCECF-loaded cells. Slices labeled with a glial-specific antibody, glial fibrillary acidic protein, showed a diffuse, background staining. Neurons that were retrograde-labeled with rhodamine beads fluoresced as large spheres that exactly correlated with the fluorescence from BCECF-loaded cells. Further, large fluorescent spheres had membrane potentials of about -60 mV and generated action potentials. These findings indicate that the large fluorescent spheres are neurons. pHi was measured in these large spheres (neurons) in the dorsal and ventral medullary chemosensitive regions, and was 7.32 +/- 0.02 (n = 110) and 7.38 +/- 0.02 (n = 85), respectively.

Animals↗

PubMed1994

Carbonic anhydrase and CO2 chemoreception in the pulmonate snail Helix aspersa.

We have studied the effects of carbonic anhydrase inhibition on the hypercapnic ventilatory response of the pulmonate snail, Helix aspersa, in an isolated brain-pneumostome preparation. We found that the cell permeant carbonic anhydrase inhibitor, acetazolamide (ACTZ), increased pneumostomal opening and ventilation during normocapnia (2-3% CO2) and decreased the rate of pneumostomal response to step changes in CO2 (4.5%), but did not change the steady-state ventilatory response to elevated CO2 (4.5%) compared to the inactive ACTZ analogue, N2-substituted 2-acetylamino-1,3,4-thiadiazole (Cl 13850). In contrast, the cell impermeant carbonic anhydrase inhibitor, quartenary ammonium sulfonilamide (QAS), had no effect on the pneumostomal response to CO2 compared to Cl 13850. Using Hansson's histochemical technique to stain for carbonic anhydrase activity, we identified a small number of neurons in the subesophageal ganglia that exhibited carbonic anhydrase activity. Some of these cells were in the region of CO2-sensitivity. In conclusion, carbonic anhydrase inhibition slows the ventilatory response to rapid changes in CO2, but does not affect the intrinsic ability of H. aspersa to respond to CO2. The ventilatory effects of carbonic anhydrase inhibition may be attributed to the intracellular actions of the carbonic anhydrase enzyme.

Animals↗

PubMed1994

Central chemoreceptor stimulus in the terrestrial, pulmonate snail, Helix aspersa.

We studied the effect of hypercapnic and fixed acid central chemoreceptor stimulation on the pneumostome in the pulmonate snail, Helix aspersa. We found that focal stimulation of the central chemoreceptor area of the pulmonate snail brain with hypercapnic solutions more effectively increased the pneumostomal area than did fixed acid stimulation at the same extracellular pH. Disrupting intracellular pH regulation by inhibiting Cl- transport, either pharmacologically (DIDS) or by ion substitution (Cl(-)-free perfusate), enhanced pneumostomal responses to CO2. While maintaining a constant perfusate pH, addition of NH4Cl to the perfusate resulted in pneumostomal closure; whereas removal of NH4Cl from the bath resulted in pneumostomal opening. In conclusion, the ventilatory response to CO2 in H. aspersa does not require Cl- transport or conductance. Furthermore, changing pHi alone is an adequate stimulus for the central chemoreceptors in the snail.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

CO2 chemoreception in the pulmonate snail, Helix aspersa.

We have studied the response of the pneumostome to CO2, O2 and combined CO2 and O2 in intact snails. We found that pneumostomal opening increases in response to both hypercapnia and mild hypoxia. We determined which neural structures were essential for the pneumostomal response to CO2 by eliminating parts of the nervous system: the subesophageal ganglia and an intact anal nerve were necessary and sufficient elements for the CO2 response. Within the subesophageal ganglia, we identified a discrete region on the medial margin of the visceral ganglion that was capable of increasing pneumostomal area when focally stimulated with 6% CO2. Ion substitution experiments indicated that pneumostomal responses to hypercapnia were not mediated by the pneumostomal motor neurons themselves, but rather by interneurons connected polysynaptically to the motor neurons controlling pneumostomal function. In conclusion, intact H. aspersa have a ventilatory response to CO2, and this response is mediated by CO2 sensitive cells located in a small area of the central nervous system.

Animals↗

PubMed1993

Control of segmental upper airway resistance in patients with obstructive sleep apnea.

We sought to determine if the upper airway response to an added inspiratory resistive load (IRL) during wakefulness could be used to predict the site of upper airway collapse in patients with obstructive sleep apnea (OSA). In 10 awake patients with OSA, we investigated the relationship between resistance in three segments of the upper airway (nasal, nasopharyngeal, and oropharyngeal) and three muscles known to influence these segments (alae nasi, tensor veli palatini, and genioglossus) while the patient breathed with or without a small IRL (2 cmH2O.l-1.s). During IRL, patients with OSA exhibited increased nasopharyngeal resistance and no significant increase in either the genioglossus or tensor veli palatini activities. Neither nasal resistance nor alae nasi EMG activity was affected by IRL. We contrasted this to the response of five normal subjects, in whom we found no change in the resistance of either segment of the airway and no change in the genioglossus EMG but a significant activation of the tensor palatini. In six patients with OSA, we used the waking data to predict the site of upper airway collapse during sleep and we had limited success. The most successful index (correct in 4 of 6 patients) incorporated the greatest relative change in segmental resistance during IRL at the lowest electromyographic activity. We conclude, in patients with OSA, IRL narrows the more collapsible segment of the upper airway, in part due to inadequate activation of upper airway muscles. However, it is difficult to predict the site of upper airway collapse based on the waking measurements where upper airway muscle activity masks the passive airway characteristics.

Adult↗

PubMed1991

Nasal and pharyngeal resistance after topical mucosal vasoconstriction in normal humans.

Phenylephrine, an alpha-adrenergic agonist, increases pharyngeal cross-sectional area when applied topically to the nasal and pharyngeal mucosa, as determined by magnetic resonance imaging. In this study, we examined the possibility that the increase in area results from either a decrease in transmural collapsing pressure, as a result of a decrease in upstream (nasal) resistance, or an increase in upper airway muscle activity. In eight normal, awake men we measured inspiratory pharyngeal and nasal resistance and the electrical activity of the genioglossus (EMGGG) and alae nasi (EMG(AN) before and after pharyngeal and nasal + pharyngeal instillation of 1 ml of either 0.25% phenylephrine or normal saline; phenylephrine and saline were tested on separate days. Under control eucapnic conditions, pharyngeal resistance was 0.43 +/- 0.03 cm H2O/L/s, and nasal resistance was 2.43 +/- 0.14 cm H2O/L/s. Pharyngeal resistance was 0.29 +/- 0.03 cm H2O/L/s after nasal + pharyngeal instillation of phenylephrine and 0.98 +/- 0.13 cm H2O/L/s after saline; nasal resistance was 2.18 +/- 0.13 cm H2O/L/s after nasal + pharyngeal instillation of phenylephrine and 3.15 +/- 0.21 cm H2O/L/s after saline. Thus, phenylephrine decreased both nasal and pharyngeal inspiratory resistance. The change in pharyngeal resistance was not dependent on the change in nasal resistance. Eucapnic EMGGG and EMGAN activities did not change after phenylephrine or saline. We conclude that phenylephrine decreased pharyngeal resistance independent of a change in nasal resistance of upper airway muscle activity, and we believe that the changes in resistance we observed reflect a direct effect of phenylephrine on the pharyngeal mucosa and a consequent enlargement of pharyngeal size.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Topical↗