Postsynaptic Mechanisms Are Essential for Forskolin-Induced Potentiation of Synaptic Transmission

2006 ◽  
Vol 95 (4) ◽  
pp. 2570-2579 ◽  
Author(s):  
Irina V. Sokolova ◽  
Henry A. Lester ◽  
Norman Davidson
Keyword(s):  
Synaptic Transmission ◽  
Ampa Receptors ◽  
Internal Solution ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Nmda Receptor Blockade ◽  
Mepsc Frequency ◽  
Pka Pathway ◽  
Activity Dependent

It has been demonstrated that stimulation of protein kinase A (PKA) results in enhanced synaptic transmission in the hippocampus and other brain areas. To investigate mechanisms of the PKA-mediated potentiation of synaptic transmission, we used rat hippocampal embryonic cultures. In low-density cultures, paired recordings under the perforated patch demonstrated that 15-min forskolin treatment produced long-lasting potentiation of evoked excitatory postsynaptic currents (eEPSCs) mediated by the cAMP/PKA pathway. eEPSC amplitudes increased to 240 ± 10% of baseline after 15 min of forskolin treatment (early). After forskolin washout, eEPSCs declined to a potentiated level. Potentiation was sustained for ≥85 min after forskolin washout and, 60 min after forskolin washout, constituted 152 ± 7% of baseline (late potentiation). Disruption of presynaptic processes with the whole cell configuration and internal solution containing PKA inhibitor peptide did not affect forskolin-induced potentiation. Disruption of postsynaptic processes, in contrast, impaired early potentiation and abolished late potentiation. Study of mEPSCs confirmed the contribution of postsynaptic mechanisms. Forskolin-induced enhancement of mEPSC frequency observed under the perforated patch was attenuated by the whole cell configuration. Forskolin also induced an increase of mEPSC amplitudes in the perforated patch, but not in the whole cell, experiments. Potentiation of eEPSCs was not activity dependent, persisting in the absence of stimulation. NMDA receptor blockade did not abolish forskolin-induced potentiation. In summary, we demonstrate that forskolin-induced potentiation of eEPSCs was mediated by postsynaptic mechanisms, presumably by upregulation of AMPA receptors by phosphorylation.

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations

1994 ◽  
Vol 267 (4) ◽  
pp. C1152-C1159 ◽  
Author(s):  
S. Grinstein ◽  
R. Romanek ◽  
O. D. Rotstein
Keyword(s):  
Single Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Pipette Solution ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Patch Configuration ◽  
In Cells

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

scholarly journals Multiple Forms of Endocytosis In Bovine Adrenal Chromaffin Cells

1997 ◽  
Vol 139 (4) ◽  
pp. 885-894 ◽  
Author(s):  
Corey Smith ◽  
Erwin Neher
Keyword(s):  
Chromaffin Cells ◽  
Initial Rate ◽  
Secretory Activity ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Adrenal Chromaffin ◽  
Cell Capacitance

We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent “compensatory” retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of ∼6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed “excess” retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of ∼248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.

Rapidly Deactivating AMPA Receptors Determine Excitatory Synaptic Transmission to Interneurons in the Nucleus Tractus Solitarius From Rat

1997 ◽  
Vol 78 (1) ◽  
pp. 82-91 ◽  
Author(s):  
Stefan Titz ◽  
Bernhard U. Keller
Keyword(s):  
Synaptic Transmission ◽  
Ampa Receptor ◽  
Decay Time ◽  
Ampa Receptors ◽  
Time Course ◽  
Nucleus Tractus Solitarius ◽  
Synaptic Cleft ◽  
Excitatory Synaptic Transmission ◽  
Membrane Properties ◽  
Time Constants

Titz, Stefan and Bernhard U. Keller. Rapidly deactivating AMPA receptors determine excitatory synaptic transmission to interneurons in the nucleus tractus solitarius from rat. J. Neurophysiol. 78: 82–91, 1997. Excitatory synaptic transmission was investigated in interneurons of the parvocellular nucleus tractus solitarius (pNTS) by performing patch-clamp experiments in thin slice preparations from rat brain stem. Stimulation of single afferent fibers evoked excitatory postsynaptic currents (EPSCs) mediated by glutamate receptors of the dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) and N-methyl-d-aspartate types. AMPA-receptor-mediated EPSCs displayed decay time constants of 3.5 ± 1.2 (SD) ms (13 cells), which were slow compared with EPSC decay time constants in neurons of the cerebellum or hippocampus. Slow EPSC decay was not explained by dendritic filtering, because the passive membrane properties of pNTS interneurons provided favorable voltage-clamp conditions. Also, the slowness of EPSC decay did not result from slow deactivation of AMPA receptors (0.7 ± 0.2 ms, 5 cells), which was investigated during rapid application of agonist to outside-out patches. Comparison of AMPA receptor kinetics with EPSC decay time constants suggested that the slow time course of EPSCs resulted from the prolonged presence of glutamate in the synaptic cleft.

Desensitization and resensitization rates of glutamate-activated channels may regulate motoneuron excitability

1991 ◽  
Vol 66 (4) ◽  
pp. 1166-1175 ◽  
Author(s):  
D. O. Smith ◽  
C. Franke ◽  
J. L. Rosenheimer ◽  
F. Zufall ◽  
H. Hatt
Keyword(s):  
Ampa Receptors ◽  
Single Channel ◽  
Kainate Receptors ◽  
Whole Cell ◽  
Solution Exchange ◽  
Channel Opening ◽  
Fast Solution ◽  
Agonist Concentration ◽  
Kinetics Of ◽  
Channel Properties

1. Single-channel properties of desensitizing glutamate-activated channels were analyzed in outside-out patch-clamp recordings from a motoneuron-enriched cell fraction from embryonic chick. A piezo-driven device was used to achieve fast solution exchange at the electrode tip, resulting in maximum activation within 2 ms. 2. Quisqualate/AMPA receptors, with a 13-pS conductance, desensitized rapidly; the desensitization rate depended on agonist concentration but not on membrane potential. When quisqualate was applied slowly, the quisqualate-activated channels desensitized without prior channel opening, indicating desensitization from the closed state. After a 10-ms refractory period, resensitization of all channels required up to 300 ms; resensitization rate did not depend on the duration of the preceding quisqualate application. 3. At agonist concentrations less than or equal to 1 mM, kainate receptors, with a 20-pS conductance, did not desensitize. At kainate concentrations greater than or equal to 1 mM, though, kainate receptors desensitized to a low steady-state conductance within approximately 200 ms. Resensitization of all channels required as long as 3 s, which could render kainate receptors inexcitable during high-frequency activation. 4. Desensitization rates of whole-cell currents were similar to those observed in outside-out mode. Glutamate- and quisqualate-activated responses were similar, suggesting that the rapidly desensitizing quisqualate-sensitive receptor type may dominate the kinetics of whole-cell excitatory postsynaptic currents (EPSCs) in this preparation. 5. It may be concluded that the efficacy of glutamate-mediated synaptic transmission is modulated by differences in the rates of desensitization and resensitization.

scholarly journals Membrane responses to norepinephrine in cultured brown fat cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 523-544 ◽  
Author(s):  
M T Lucero ◽  
P A Pappone
Keyword(s):  
Brown Fat ◽  
Fat Cells ◽  
Adrenergic Agonists ◽  
Whole Cell ◽  
K Channels ◽  
Inward Current ◽  
General Physiology ◽  
Perforated Patch ◽  
Beta Adrenergic Agonists ◽  
Outward Currents

We used the "perforated-patch" technique (Horn, R., and A. Marty, 1988. Journal of General Physiology. 92:145-159) to examine the effects of adrenergic agonists on the membrane potentials and membrane currents in isolated cultured brown fat cells from neonatal rats. In contrast to our previous results using traditional whole-cell patch clamp, 1-23-d cultured brown fat cells clamped with the perforated patch consistently showed vigorous membrane responses to both alpha- and beta-adrenergic agonists, suggesting that cytoplasmic components essential for the thermogenic response are lost in whole-cell experiments. The membrane responses to adrenergic stimulation varied from cell to cell but were consistent for a given cell. Responses to bath-applied norepinephrine in voltage-clamped cells had three possible components: (a) a fast transient inward current, (b) a slower outward current carried by K+ that often oscillated in amplitude, and (c) a sustained inward current largely by Na+. The fast inward and outward currents were activated by alpha-adrenergic agonists while the slow inward current was mediated by beta-adrenergic agonists. Oscillating outward currents were the most frequently seen response to norepinephrine stimulation. Activation of this current, termed IK,NE, was independent of voltage and seemed to be carried by Ca2(+)-activated K channels since the current oscillated in amplitude at constant membrane potential and gradually decreased when the cells were bathed with calcium-free external solution. IK,NE had a novel pharmacology in that it could be blocked by 4-aminopyridine, tetraethylammonium, apamin, and charybdotoxin. Both IK,NE and the voltage-gated K channels also present in brown fat (Lucero, M. T., and P. A. Pappone, 1989a. Journal of General Physiology. 93:451-472) may play a role in maintaining cellular homeostasis in the face of the high metabolic activity involved in thermogenesis.

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