pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats

M. O. Bevensee, Theodore Cummins, G. G. Haddad, W. F. Boron, G. Boyarsky

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Abstract

1. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to study the regulation of intracellular pH (pH(i)) in single pyramidal neurons freshly isolated from the hippocampal CA1 region of immature (2- to 10-day-old) and more mature (21- to 30-day-old) rats. 2. Whether isolated from immature or mature rats, neurons had a broad range of initial pH(i) values (6.3-7.7) when the cells were examined in solutions buffered with Hepes and no CO2/HCO3-. The initial pH(i) distribution for neurons isolated from immature rats was best fitted with a Gaussian distribution with a mean of 6.95. In contrast, the initial pH(i) distribution for neurons isolated from mature rats was best fitted with the sum of two Gaussian distributions with means of 6.68 and 7.32. 3. When neurons with a relatively low initial pH(i) in Hepes-buffered solutions were acid loaded, pH(i) recovered very slowly. Neurons with a relatively high initial pH(i) recovered rapidly. The rate constant for the exponential pH(i) recovery increased with initial pH(i). All pH(i) recoveries required Na+. 4. Both for neurons with a relatively high (≤ 7.05) and a relatively low (<7.05 initial pH(i), net acid extrusion rates (J(total) = dpH(i)/dt x buffering power) decreased linearly with increasing pH(i). Compared with the line for neurons with a relatively low initial pH(i), that for neurons with a relatively high pH(i) had a significantly greater slope and was alkaline shifted by 0.6-0.7 pH units. 5. Removing external Na+ in the absence of CO2/HCO3- caused pH(i) to decrease by ~ 0.3 in neurons with a relatively low initial pH(i), and by ~ 0.5 in neurons with a relatively high initial pH(i). This initial acidification was followed by a slower, partial pH(i) recovery in ~ 32% of neurons with a relatively low initial pH(i), but only ~ 14% of neurons with a relatively high pH(i). 6. When exposed to CO2/HCO3- all neurons initially acidified. Neurons with a relatively low initial pH(i) recovered to a pH(i) ~ 0.2 pH units greater than the initial value. Among neurons with higher initial pH(i) values, some did not recover at all, whereas others recovered to a value similar to or above the initial pH(i). On average, the final CO2/HCO3- pH(i) for neurons with a relatively high initial pH(i), was similar to the pH(i) in Hepes buffer. Neurons with a relatively high pH(i) in Hepes buffer continued to be more alkaline (by ~ 0.2 pH units) in CO2/HCO3-. 7. When neurons with a relatively high initial pH(i) in Hepes (≤ 7.25) were exposed to CO2/HCO3- and then acid loaded, J(total) values were more than twice the highest values observed in neurons with lower initial pH(i) values. Neurons with a moderate initial pH(i) in Hepes (7.05-7.24) had J(total) values, at comparable pH(i) values, that were ~ 2-fold greater than for neurons with a relatively low initial pH(i) (<7 0.5). 8. Thus, freshly isolated CA1 neurons of both mature and immature rats have a wide range of acid-base properties. Those with higher initial pH(i) values in a Hepes buffer tend to have greater J(total) values in both Hepes and CO2/HCO3- and tend to have higher steady-state pH(i) values in CO2/HCO3-.

Original languageEnglish (US)
Pages (from-to)315-328
Number of pages14
JournalJournal of Physiology
Volume494
Issue number2
StatePublished - Jul 15 1996
Externally publishedYes

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Hippocampus
Neurons
Buffers
Acids
Normal Distribution

ASJC Scopus subject areas

  • Physiology

Cite this

Bevensee, M. O., Cummins, T., Haddad, G. G., Boron, W. F., & Boyarsky, G. (1996). pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats. Journal of Physiology, 494(2), 315-328.

pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats. / Bevensee, M. O.; Cummins, Theodore; Haddad, G. G.; Boron, W. F.; Boyarsky, G.

In: Journal of Physiology, Vol. 494, No. 2, 15.07.1996, p. 315-328.

Research output: Contribution to journalArticle

Bevensee, MO, Cummins, T, Haddad, GG, Boron, WF & Boyarsky, G 1996, 'pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats', Journal of Physiology, vol. 494, no. 2, pp. 315-328.
Bevensee, M. O. ; Cummins, Theodore ; Haddad, G. G. ; Boron, W. F. ; Boyarsky, G. / pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats. In: Journal of Physiology. 1996 ; Vol. 494, No. 2. pp. 315-328.
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abstract = "1. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to study the regulation of intracellular pH (pH(i)) in single pyramidal neurons freshly isolated from the hippocampal CA1 region of immature (2- to 10-day-old) and more mature (21- to 30-day-old) rats. 2. Whether isolated from immature or mature rats, neurons had a broad range of initial pH(i) values (6.3-7.7) when the cells were examined in solutions buffered with Hepes and no CO2/HCO3-. The initial pH(i) distribution for neurons isolated from immature rats was best fitted with a Gaussian distribution with a mean of 6.95. In contrast, the initial pH(i) distribution for neurons isolated from mature rats was best fitted with the sum of two Gaussian distributions with means of 6.68 and 7.32. 3. When neurons with a relatively low initial pH(i) in Hepes-buffered solutions were acid loaded, pH(i) recovered very slowly. Neurons with a relatively high initial pH(i) recovered rapidly. The rate constant for the exponential pH(i) recovery increased with initial pH(i). All pH(i) recoveries required Na+. 4. Both for neurons with a relatively high (≤ 7.05) and a relatively low (<7.05 initial pH(i), net acid extrusion rates (J(total) = dpH(i)/dt x buffering power) decreased linearly with increasing pH(i). Compared with the line for neurons with a relatively low initial pH(i), that for neurons with a relatively high pH(i) had a significantly greater slope and was alkaline shifted by 0.6-0.7 pH units. 5. Removing external Na+ in the absence of CO2/HCO3- caused pH(i) to decrease by ~ 0.3 in neurons with a relatively low initial pH(i), and by ~ 0.5 in neurons with a relatively high initial pH(i). This initial acidification was followed by a slower, partial pH(i) recovery in ~ 32{\%} of neurons with a relatively low initial pH(i), but only ~ 14{\%} of neurons with a relatively high pH(i). 6. When exposed to CO2/HCO3- all neurons initially acidified. Neurons with a relatively low initial pH(i) recovered to a pH(i) ~ 0.2 pH units greater than the initial value. Among neurons with higher initial pH(i) values, some did not recover at all, whereas others recovered to a value similar to or above the initial pH(i). On average, the final CO2/HCO3- pH(i) for neurons with a relatively high initial pH(i), was similar to the pH(i) in Hepes buffer. Neurons with a relatively high pH(i) in Hepes buffer continued to be more alkaline (by ~ 0.2 pH units) in CO2/HCO3-. 7. When neurons with a relatively high initial pH(i) in Hepes (≤ 7.25) were exposed to CO2/HCO3- and then acid loaded, J(total) values were more than twice the highest values observed in neurons with lower initial pH(i) values. Neurons with a moderate initial pH(i) in Hepes (7.05-7.24) had J(total) values, at comparable pH(i) values, that were ~ 2-fold greater than for neurons with a relatively low initial pH(i) (<7 0.5). 8. Thus, freshly isolated CA1 neurons of both mature and immature rats have a wide range of acid-base properties. Those with higher initial pH(i) values in a Hepes buffer tend to have greater J(total) values in both Hepes and CO2/HCO3- and tend to have higher steady-state pH(i) values in CO2/HCO3-.",
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T1 - pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats

AU - Bevensee, M. O.

AU - Cummins, Theodore

AU - Haddad, G. G.

AU - Boron, W. F.

AU - Boyarsky, G.

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N2 - 1. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to study the regulation of intracellular pH (pH(i)) in single pyramidal neurons freshly isolated from the hippocampal CA1 region of immature (2- to 10-day-old) and more mature (21- to 30-day-old) rats. 2. Whether isolated from immature or mature rats, neurons had a broad range of initial pH(i) values (6.3-7.7) when the cells were examined in solutions buffered with Hepes and no CO2/HCO3-. The initial pH(i) distribution for neurons isolated from immature rats was best fitted with a Gaussian distribution with a mean of 6.95. In contrast, the initial pH(i) distribution for neurons isolated from mature rats was best fitted with the sum of two Gaussian distributions with means of 6.68 and 7.32. 3. When neurons with a relatively low initial pH(i) in Hepes-buffered solutions were acid loaded, pH(i) recovered very slowly. Neurons with a relatively high initial pH(i) recovered rapidly. The rate constant for the exponential pH(i) recovery increased with initial pH(i). All pH(i) recoveries required Na+. 4. Both for neurons with a relatively high (≤ 7.05) and a relatively low (<7.05 initial pH(i), net acid extrusion rates (J(total) = dpH(i)/dt x buffering power) decreased linearly with increasing pH(i). Compared with the line for neurons with a relatively low initial pH(i), that for neurons with a relatively high pH(i) had a significantly greater slope and was alkaline shifted by 0.6-0.7 pH units. 5. Removing external Na+ in the absence of CO2/HCO3- caused pH(i) to decrease by ~ 0.3 in neurons with a relatively low initial pH(i), and by ~ 0.5 in neurons with a relatively high initial pH(i). This initial acidification was followed by a slower, partial pH(i) recovery in ~ 32% of neurons with a relatively low initial pH(i), but only ~ 14% of neurons with a relatively high pH(i). 6. When exposed to CO2/HCO3- all neurons initially acidified. Neurons with a relatively low initial pH(i) recovered to a pH(i) ~ 0.2 pH units greater than the initial value. Among neurons with higher initial pH(i) values, some did not recover at all, whereas others recovered to a value similar to or above the initial pH(i). On average, the final CO2/HCO3- pH(i) for neurons with a relatively high initial pH(i), was similar to the pH(i) in Hepes buffer. Neurons with a relatively high pH(i) in Hepes buffer continued to be more alkaline (by ~ 0.2 pH units) in CO2/HCO3-. 7. When neurons with a relatively high initial pH(i) in Hepes (≤ 7.25) were exposed to CO2/HCO3- and then acid loaded, J(total) values were more than twice the highest values observed in neurons with lower initial pH(i) values. Neurons with a moderate initial pH(i) in Hepes (7.05-7.24) had J(total) values, at comparable pH(i) values, that were ~ 2-fold greater than for neurons with a relatively low initial pH(i) (<7 0.5). 8. Thus, freshly isolated CA1 neurons of both mature and immature rats have a wide range of acid-base properties. Those with higher initial pH(i) values in a Hepes buffer tend to have greater J(total) values in both Hepes and CO2/HCO3- and tend to have higher steady-state pH(i) values in CO2/HCO3-.

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