Electrophysiological evidence that dentate hilar mossy cells are excitatory and innervate both granule cells and interneurons

1995 ◽  
Vol 74 (1) ◽  
pp. 179-194 ◽  
Author(s):  
H. E. Scharfman

1. The hypothesis that dentate hilar "mossy" cells are excitatory was tested by simultaneous intracellular recording in rat hippocampal slices. Mossy cells were recorded simultaneously with their potential targets, granule cells and interneurons. The gamma-amino-butyric acid-A (GABAA) receptor antagonist bicuculline was used in most experiments to block the normally strong inhibitory inputs to granule cells that could mask excitatory effects of mossy cells. Some cells were recorded with electrodes containing the marker Neurobiotin so that their identity could be confirmed morphologically. 2. A mossy cell action potential was immediately followed by a brief depolarization in a granule cell in 20 of 1,316 pairs (1.5%) that were recorded in the presence of bicuculline. The mean amplitude of depolarizations was 1.99 +/- 0.24 (SE) mV when the postsynaptic membrane potential was -55 to -65 mV. Depolarizations could trigger an action potential if the granule cell was depolarized from its resting potential so that its membrane potential was -50 to -60 mV. These data suggest that mossy cells excite granule cells monosynaptically. 3. Monosynaptic excitation of an interneuron by a mossy cell was recorded in 4 of 47 (8.5%) simultaneously recorded mossy cells and interneurons, also in the presence of bicuculline. The mean interneuron depolarization was 1.64 +/- 0.29 mV when the interneuron membrane potential was approximately -60 mV. When an interneuron was at its resting potential (-52 to -63 mV), action potentials were often triggered by the depolarizations. 4. Without bicuculline present, mossy cells had no apparent monosynaptic effects on granule cells, as has been previously reported. However, effects that appeared to be polysynaptic were observed in 5 of 92 pairs (5.4%). Specifically, a small, brief hyperpolarization occurred in granule cells 2.5-7.3 ms after the peak of a mossy cell action potential. Given the results indicating that mossy cells excite interneurons, and the long latency to onset of the hyperpolarization, one possible explanation for the hyperpolarization is that mossy cells excited interneurons that inhibited granule cells. 5. The results suggest that mossy cells are excitatory neurons. In addition, mossy cells appear to innervate both granule cells and interneurons that are located within several hundred micrometers of the mossy cell soma. The only detectable effect on granule cells in this area under normal conditions appears to be disynaptic and inhibitory. However, when GABAA-receptor-mediated inhibition is blocked, monosynaptic excitation of granule cells by mossy cells can be detected.

1995 ◽  
Vol 74 (2) ◽  
pp. 673-683 ◽  
Author(s):  
A. A. Oyelese ◽  
D. L. Eng ◽  
G. B. Richerson ◽  
J. D. Kocsis

1. The effects of axotomy on the electrophysiologic properties of adult rat dorsal root ganglion (DRG) neurons were studied to understand the changes in excitability induced by traumatic nerve injury. Nerve injury was induced in vivo by sciatic nerve ligation with distal nerve transection. Two to four weeks after nerve ligation, a time when a neuroma forms, lumbar (L4 and L5) DRG neurons were removed and placed in short-term tissue culture. Whole cell patch-clamp recordings were made 5–24 h after plating. 2. DRG neurons were grouped into large (43–65 microns)-, medium (34–42 microns)-, and small (20–32 microns)- sized classes. Large neurons had short duration action potentials with approximately 60% having inflections on the falling phase of their action potentials. In contrast, action potentials of medium and small neurons were longer in duration and approximately 68% had inflections. 3. Pressure microejection of gamma-aminobutyric acid (GABA, 100 microM) or muscimol (100 microM) onto voltage-clamped DRG neurons elicited a rapidly desensitizing inward current that was blocked by 200 microM bicuculline. To measure the peak conductance induced by GABA or muscimol, neurons were voltage-clamped at a holding potential of -60 mV, and pulses to -80 mV and -100 mV were applied at a rate of 2.5 or 5 Hz during drug application. Slope conductances were calculated from plots of whole cell current measured at each of these potentials. 4. GABA-induced currents and conductances of control DRG neurons increased progressively with cell diameter. The mean GABA conductance was 36 +/- 10 nS (mean +/- SE) in small neurons, 124 +/- 21 nS in medium neurons, and 527 +/- 65 nS in large neurons. 5. After axotomy, medium neurons had significantly larger GABA-induced conductances compared with medium control neurons (390 +/- 50 vs. 124 +/- 21; P < 0.001). The increase in GABA conductance of medium neurons was associated with a decrease in duration of action potentials. In contrast, small neurons had no change in GABA conductance or action potential duration after ligation. The GABA conductance of large control neurons was highly variable, and ligation resulted in an increase that was significant only for neurons > 50 microns. The mean action potential duration in large neurons was not significantly changed, but neurons with inflections on the falling phase of the action potential were less common after ligation. There was no difference in resting potential or input resistance between control and ligated groups, except that the resting potential was less negative in small cells after axotomy.(ABSTRACT TRUNCATED AT 400 WORDS)


1994 ◽  
Vol 72 (5) ◽  
pp. 2167-2180 ◽  
Author(s):  
H. E. Scharfman

1. Simultaneous intracellular recordings of area CA3 pyramidal cells and dentate hilar “mossy” cells were made in rat hippocampal slices to test the hypothesis that area CA3 pyramidal cells excite mossy cells monosynaptically. Mossy cells and pyramidal cells were differentiated by location and electrophysiological characteristics. When cells were impaled near the border of area CA3 and the hilus, their identity was confirmed morphologically after injection of the marker Neurobiotin. 2. Evidence for monosynaptic excitation of a mossy cell by a pyramidal cell was obtained in 7 of 481 (1.4%) paired recordings. In these cases, a pyramidal cell action potential was followed immediately by a 0.40 to 6.75 (mean, 2.26) mV depolarization in the simultaneously recorded mossy cell (mossy cell membrane potentials, -60 to -70 mV). Given that pyramidal cells used an excitatory amino acid as a neurotransmitter (Cotman and Nadler 1987; Ottersen and Storm-Mathisen 1987) and recordings were made in the presence of the GABAA receptor antagonist bicuculline (25 microM), it is likely that the depolarizations were unitary excitatory postsynaptic potentials (EPSPs). 3. Unitary EPSPs of mossy cells were prone to apparent “failure.” The probability of failure was extremely high (up to 0.72; mean = 0.48) if the effects of all presynaptic action potentials were examined, including action potentials triggered inadvertently during other spontaneous EPSPs of the mossy cell. Probability of failure was relatively low (as low as 0; mean = 0.24) if action potentials that occurred during spontaneous activity of the mossy cell were excluded. These data suggest that unitary EPSPs produced by pyramidal cells are strongly affected by concurrent synaptic inputs to the mossy cell. 4. Unitary EPSPs were not clearly affected by manipulation of the mossy cell's membrane potential. This is consistent with the recent report that area CA3 pyramidal cells innervate distal dendrites of mossy cells (Kunkel et al. 1993). Such a distal location also may contribute to the high incidence of apparent failures. 5. Characteristics of unitary EPSPs generated by pyramidal cells were compared with the properties of the unitary EPSPs produced by granule cells. In two slices, pyramidal cell and granule cell inputs to the same mossy cell were compared. In other slices, inputs to different mossy cells were compared. In all experiments, unitary EPSPs produced by granule cells were larger in amplitude but similar in time course to unitary EPSPs produced by pyramidal cells. Probability of failure was lower and paired-pulse facilitation more common among EPSPs triggered by granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)


1962 ◽  
Vol 45 (6) ◽  
pp. 1195-1216 ◽  
Author(s):  
Fred J. Julian ◽  
John W. Moore ◽  
David E. Goldman

A method similar to the sucrose-gap technique introduced be Stäpfli is described for measuring membrane potential and current in singly lobster giant axons (diameter about 100 micra). The isotonic sucrose solution used to perfuse the gaps raises the external leakage resistance so that the recorded potential is only about 5 per cent less than the actual membrane potential. However, the resting potential of an axon in the sucrose-gap arrangement is increased 20 to 60 mv over that recorded by a conventional micropipette electrode when the entire axon is bathed in sea water. A complete explanation for this effect has not been discovered. The relation between resting potential and external potassium and sodium ion concentrations shows that potassium carries most of the current in a depolarized axon in the sucrose-gap arrangement, but that near the resting potential other ions make significant contributions. Lowering the external chloride concentration decreases the resting potential. Varying the concentration of the sucrose solution has little effect. A study of the impedance changes associated with the action potential shows that the membrane resistance decreases to a minimum at the peak of the spike and returns to near its initial value before repolarization is complete (a normal lobster giant axon action potential does not have an undershoot). Action potentials recorded simultaneously by the sucrose-gap technique and by micropipette electrodes are practically superposable.


1986 ◽  
Vol 250 (1) ◽  
pp. C47-C54 ◽  
Author(s):  
P. Mollard ◽  
J. Mironneau ◽  
T. Amedee ◽  
C. Mironneau

Smooth muscle cells were isolated from the longitudinal layer of pregnant rat myometrium (18-19 days) and studied either freshly dissociated or during short-term primary culture (until 30 h) using intracellular microelectrode techniques and direct microscopic observation. The isolated myometrial cells excluded trypan blue vital stain and could repetitively contract in response to various stimuli. Electrophysiological studies at 37 degrees C showed normal resting potential (-54.5 +/- 7.5 mV, n = 71). Action potentials with overshoot (+7.8 +/- 4.6 mV, n = 71) could be elicited by intracellular stimulation. Moreover, the membrane potential was largely dependent on the external K+ concentration. The action potential was suppressed in a Ca2+-free solution [with 0.1 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid], and the overshoot amplitude was clearly Ca2+ dependent. The action potential was inhibited by Mn2+ ions (1 mM), Co2+ ions (1 mM), and D 600 (1 microM) but was unaffected by tetrodotoxin (2 microM) and external Na+ removal. Tetraethylammonium chloride (TEA, 10 mM) and 4-aminopyridine (4-AP, 10 mM) increased both overshoot amplitude and duration of the electrical responses. When the cell surface area was measured with light microscopy, the mean specific membrane resistance was 14.8 +/- 4.6 k omega . cm2 (n = 14), and the mean specific membrane capacitance was 2.3 +/- 0.7 microF/cm2 (n = 14). Outward-going rectification was consistently observed in all cells examined. This was either inhibited by TEA and 4-AP (10 mM each) or reduced in the presence of 1 mM Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)


1992 ◽  
Vol 67 (5) ◽  
pp. 1346-1358 ◽  
Author(s):  
K. J. Staley ◽  
T. S. Otis ◽  
I. Mody

1. Whole-cell and sharp electrode recordings from adult rat dentate gyrus GCs were performed in the 400-microns-thick hippocampal slice preparation maintained at 34 +/- 1 degrees C. Intrinsic membrane properties of granule cells (GCs) were evaluated with the use of a switching current-clamp amplifier. 2. With the whole-cell technique, the average resting membrane potential (RMP) of GCs was -85 mV when a potassium gluconate electrode solution was used versus -74 mV measured with potassium acetate-filled sharp microelectrodes. The membrane voltage response to injected current was linear over two membrane potential ranges, greater than 10 mV hyperpolarized from RMP and between 10 mV more negative than RMP and -62 mV. The average input resistances (RN) calculated over these ranges were 107 and 228 M omega in the whole-cell recordings versus 37 and 54 M omega in the sharp electrode recordings. There was no correlation between RMP and RN with either recording technique. The membrane time constant (tau m) determined at the RMP was 26.9 ms for whole-cell recordings and 13.9 ms for sharp electrode recordings. 3. There was no evidence of time-dependent changes in RMP, RN, and tau m in whole-cell recordings, although the slow inward rectification seen at hyperpolarized potentials decreased over 30-60 min. Addition of calcium buffers to the whole-cell recording solution did not result in a significant change in the average RMP, the average RN, or the average tau m. 4. Action potential threshold was comparable in whole-cell (-49 mV) and sharp electrode (-52 mV) recordings, but action potential amplitude was larger in whole-cell (126 mV) than in sharp electrode (106 mV) recordings. Spike frequency adaptation was present in the whole-cell recordings and could be abolished by addition of calcium buffers to the electrode solution. 5. We estimated rho, the ratio of dendritic to somatic conductance, to be 5.1 for the whole-cell records and 2.1 for sharp electrode recordings. The electrotonic length of the equivalent cylinder representing the cell processes was estimated to be 0.49 from the whole-cell data and 0.79 from the sharp electrode recordings. This implies that at rest there is only a 10% decrement in steady-state membrane voltage along the length of the dendrite due to shunting across the membrane resistance; small synaptic events occurring in the distal dendritic tree will therefore have a more substantial influence on the soma than previous analyses suggested.(ABSTRACT TRUNCATED AT 400 WORDS)


1957 ◽  
Vol 40 (6) ◽  
pp. 859-885 ◽  
Author(s):  
Ichiji Tasaki ◽  
Susumu Hagiwara

1. Intracellular injection of tetraethylammonium chloride (TEA) into a giant axon of the squid prolongs the duration of the action potential without changing the resting potential (Fig. 3). The prolongation is sometimes 100-fold or more. 2. The action potential of a giant axon treated with TEA has an initial peak followed by a plateau (Fig. 3). The membrane resistance during the plateau is practically normal (Fig. 4). Near the end of the action potential, there is an apparent increase in the membrane resistance (Fig. 5D and Fig. 6, right). 3. The phenomenon of abolition of action potentials was demonstrated in the squid giant axon treated with TEA (Fig. 7). Following an action potential abolished in its early phase, there is no refractoriness (Fig. 8). 4. By the method of voltage clamp, the voltage-current relation was investigated on normal squid axons as well as on axons treated with TEA (Figs. 9 and 10). 5. The presence of stable states of the membrane was demonstrated by clamping the membrane potential with two voltage steps (Fig. 11). Experimental evidence was presented showing that, in an "unstable" state, the membrane conductance is not uniquely determined by the membrane potential. 6. The effect of low sodium water was investigated in the axon treated with TEA (Fig. 12). 7. The similarity between the action potential of a squid axon under TEA and that of the vertebrate cardiac muscle was stressed. The experimental results were interpreted as supporting the view that there are two stable states in the membrane. Initiation and abolition of an action potential were explained as transitions between the two states.


1986 ◽  
Vol 56 (1) ◽  
pp. 32-49 ◽  
Author(s):  
R. Shingai ◽  
B. N. Christensen

External horizontal cells were enzymatically dissociated from intact catfish (Ictalurus punctatus) retina and pipetted onto a small chamber attached to the stage of an inverted phase-contrast microscope. Individual horizontal cells were recognized by their large size and restricted dendritic arborization. Low-resistance (3-12 M omega) patch-type electrodes were used to record intracellular potentials and to pass current across the cell membrane under either current or voltage-clamp conditions. The average resting potential of isolated horizontal cells was -67 V + 6.9 mV (mean +/- SD, n = 40). At the resting potential, the cell membrane appears to be mainly permeable to K. A depolarizing current step evoked an action potential in the cell. The maximum rate of rise of the action potential (dV/dt) in normal physiological solution was 6.5 +/- 1.8 V/s (means +/- SD, n = 24) and was reduced to 1.2 +/- 0.39 V/s (means +/- SD, n = 9) in 1-10 micron tetrodotoxin (TTX) and 3.2 +/- 1.4 V/s (means +/- SD, n = 6) in Ca-free solution. The maximum dV/dt was reduced in 10 mM extracellular K concentration [K]o to about half of that seen in standard saline, and values in 30 or 80 mM [K]o were similar to that measured in TTX. Following an action potential, the membrane potential reached a plateau potential of + 17.4 +/- 8.1 mV (means +/- SD, n = 17) and remained depolarized for variable periods of time lasting from less than a second to a few minutes. When the plateau potential was long lasting, the cell repolarized slowly and upon reaching zero rapidly repolarized to the original resting potential. The duration of the plateau potential decreased or was absent in saline containing one of the following calcium channel antagonists: La, Cd, Co, or Ni. The voltage-clamp technique was used to identify the membrane currents responsible for the membrane potential changes seen under current clamp. Experiments were carried out using either a single or two individual electrodes. Fast and steady-state inward currents were recorded from isolated horizontal cells in the voltage range between -20 and +20 mV. These currents were a result of increased membrane conductance to both Na and Ca ions. The Na channels are inactivated at depolarized potentials and are TTX sensitive. Ca channels are partially inactivated at depolarized potentials. The Ca conductance is decreased by Cd, Co, Ni, and La. Ba can substitute for Ca in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)


1967 ◽  
Vol 47 (2) ◽  
pp. 357-373
Author(s):  
Y. PICHON ◽  
J. BOISTEL

1. The use of very fine-tipped and mechanically strong microelectrodes has allowed reliable recordings of resting and action potentials to be made in cockroach giant axons in sheathed and desheathed nerve cords. 2. When the microelectrode was withdrawn from a giant axon in an intact connective the first positive change in the potential from the resting level, was in most cases followed by a negative deflexion to the original zero level, the ‘sheath potential’. The values of this ‘sheath potential’ together with the resting potential, the action potential, the maximum rate of rise and maximum rate of fall of the action potential have been measured in three different salines. 3. In normal saline, resting potentials were lower in sheathed preparations (58·1 ± 55·4 mV.) than in desheathed ones (67·4 ± 6·2 mV.), whereas action potentials were higher in the former (103±5·9 mV.) than in the latter (85·9±4·6 mV.). 4. Elevation of K+ and Ca2+ concentrations in the saline to the haemolymph level resulted in a decrease of resting and action potentials in desheathed cords, to 57·3±5·3 mV. and 36·5±7·6 mV. respectively. No alterations in the membrane potentials were recorded in intact connectives bathed in this saline, the mean resting potential being 55·6±4·2 mV. and the mean action potential 107·9±6·0 mV. Local desheathing of the nerve cord led only to local disturbance of the resting and action potentials, thus indicating that diffusion processes along the extracellular spaces were very slow. 5. The use of a saline in which cation concentrations have been elevated to the extracellular level resulted in normal resting potentials (64·6±3·3 mV.) and action potentials (90·9±7·2 mV.) in desheathed cords, despite the relatively high potassium concentration (17·1 mM./l.). 6. Recordings of the maximum rates of rise and rates of fall showed that there was no significant modification in the shape of the action potential in these different experimental conditions. 7. The values of the ‘sheath potential’ were very variable from one impalement to another and it is suggested that this potential might be related to variations of the microelectrode tip potential bathed in different ionic solutions. 8. The low resting potentials and high action potentials of giant axons in intact nerve cords may result from an excess of inorganic cations in the extracellular fluid.


1992 ◽  
Vol 68 (1) ◽  
pp. 197-212 ◽  
Author(s):  
K. J. Staley ◽  
I. Mody

1. Stimulation of the perforant path in the outer molecular layer of the adult rat dentate gyrus produced a depolarizing post-synaptic potential (DPSP) in granule cells when recorded using whole-cell techniques in the standard hippocampal slice preparation at 34 degrees C. The postsynaptic currents (PSCs) contributing to the DPSP were analyzed using specific receptor antagonists in current- and voltage-clamp recordings. 2. The DPSP reversal potential was dependent on the intracellular chloride concentration, and the amplitude of the DPSP was increased 55% after perfusion of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI). The GABAA receptor-mediated PSC reversed at -66 mV, which was 19 mV positive to the resting membrane potential (-85 mV) but hyperpolarized relative to action potential threshold. At -35 mV, the GABAA PSC had a latency to peak of 12.9 ms after the stimulus and decayed monoexponentially with an average time constant of 23.4 ms. 3. The component of the PSC blocked by the Quis/AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) had a latency to peak of 7.1 ms and decayed monoexponentially with a time constant of 9.9 ms at -35 mV. The N-methyl-D-aspartate (NMDA) receptor-mediated PSC, which was blocked by D-amino-5-phosphonovaleric acid (D-AP5), had a waveform that was similar to the GABAA PSC: the latency to peak was 16 ms and the decay was monoexponential with a time constant of 24.5 ms at -35 mV. 4. The ratio of the peak PSCs mediated by GABAA, Quis/AMPA, and NMDA receptors measured at -35 mV with cesium gluconate electrode solutions was 1:0.2:0.1. This ratio was essentially constant over the range of stimulus intensities that produced compound PSC amplitudes of 80-400 pA. 5. Measured at its reversal potential, the GABAA receptor-mediated postsynaptic conductance (GGABA-A) decreased the peak DPSP amplitude by 35%, shunted 50% of the charge transferred to the soma by the excitatory PSC, and completely inhibited the NMDA receptor-mediated component of the DPSP. 6. Simultaneous stimulation of presynaptic fibers from both the perforant path and interneurons results in a large depolarizing GGABA-A that inhibits the granule cell by shunting the excitatory PSCs. As predicted by models of shunting, the similar kinetics of the GABAA and NMDA PSCs leads to particularly effective inhibition of the NMDA PSC. The more rapid Quis/AMPA PSC is less affected by the GGABA-A, so that granule cell excitation under these conditions is primarily due to Quis/AMPA receptor activation.


1992 ◽  
Vol 70 (9) ◽  
pp. 1236-1246 ◽  
Author(s):  
Jean Marc Renaud ◽  
Peter Light

The effects of increasing the extracellular K+ concentration on the capacity to generate action potentials and to contract were tested on unfatigued muscle fibers isolated from frog sartorius muscle. The goal of this study was to investigate further the role of K+ in muscle fatigue by testing whether an increased extracellular K+ concentration in unfatigued muscle fibers causes a decrease in force similar to the decrease observed during fatigue. Resting and action potentials were measured with conventional microelectrodes. Twitch and tetanic force was elicited by field stimulation. At pHo (extracellular pH) 7.8 and 3 mmol K+∙L−1 (control), the mean resting potential was −86.6 ± 1.7 mV (mean ± SEM) and the mean overshoot of the action potential was 5.6 ± 2.5 mV. An increased K+ concentration from 3 to 8.0 mmol∙L−1 depolarized the sarcolemma to −72.2 ± 1.4 mV, abolished the overshoot as the peak potential during an action potential was −12.0 ± 3.9 mV, potentiated the twitch force by 48.0 ± 5.7%, but did not affect the tetanic force (maximum force) and the ability to maintain a constant force during the plateau phase of a tetanus. An increase to 10 mmol K+∙L−1 depolarized the sarcolemma to −70.1 ± 1.7 mV and caused large decreases in twitch (31.6 ± 26.1%) and tetanic (74.6 ± 12.1%) force. Between 3 and 9 mmol K+∙L−1, the effects of K+ at pHo 7.2 (a pHo mimicking the change in interstitial pH during fatigue) and 6.4 (a pHo known to inhibit force recovery following fatigue) on resting and action potentials as well as on the twitch and tetanic force were similar to those at pHo 7.8. Above 9 mmol K+∙L−1 significant differences were found in the effect of K+ between pHo 7.8 and 7.2 or 6.4. In general, the decrease in peak action potential and twitch and tetanic force occurred at higher K+ concentrations as the pHo was more acidic. The results obtained in this study do not support the hypothesis that an accumulation of K+ at the surface of the sarcolemma is sufficiently large to suppress force development during fatigue. The possibility that the K+ concentration in the T tubules reaches the critical K+ concentration necessary to cause a failure of the excitation–contraction coupling mechanism is discussed.Key words: excitation–contraction coupling, fatigue, potassium, tetanus, twitch.


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