High threshold, proximal initiation, and slow conduction velocity of action potentials in dentate granule neuron mossy fibers

Abstract

Dentate granule neurons give rise to some of the smallest unmyelinated fibers in the mammalian CNS, the hippocampal mossy fibers. These neurons are also key regulators of physiological and pathophysiological information flow through the hippocampus. We took a comparative approach to studying mossy fiber action potential initiation and propagation in hippocampal slices from juvenile rats. Dentate granule neurons exhibited axonal action potential initiation significantly more proximal than CA3 pyramidal neurons. This conclusion was suggested by phase plot analysis of somatic action potentials and by local tetrodotoxin application to the axon and somatodendritic compartments. This conclusion was also verified by immunostaining for voltage-gated sodium channel alpha subunits and by direct dual soma/axonal recordings. Dentate neurons exhibited a significantly higher action potential threshold and slower axonal conduction velocity than CA3 neurons. We conclude that while the electrotonically proximal axon location of action potential initiation allows granule neurons to sensitively detect and integrate synaptic inputs, the neurons are sluggish to initiate and propagate an action potential.

FIG. 1.

Morphological comparison of dentate granule and CA3 pyramidal neurons, 2 cell types giving rise to unmyelinated intrahippocampal fibers. A: confocal 3-dimensional reconstruction of a mature dentate granule neuron filled with Alexa Fluor 488, scale bar: 50 μm. Gray arrowhead points to the small diameter dentate granule axon, the mossy fiber. B: confocal 3-dimensional reconstruction of a mature CA3 pyramidal neuron filled with Alexa Fluor 488, scale bar 50 μm. Gray arrowhead indicates the larger-diameter CA3 pyramidal axon. Fluorescent debris in the field labeled during the approach of the whole cell pipette has been digitally removed from the panel. C and D: immunohistochemistry for ankyrin G from a dentate granule and CA3 subfield, respectively, scale bar 25 μm.

FIG. 2.

Phase plot analysis suggests action potential initiation site to be more proximal to the soma in dentate granule neurons when compared with CA3 pyramidal neurons. A: dentate granule somatic membrane potential record of an action potential. The action potential was elicited by sustained current injection of 50 pA. B: CA3 somatic membrane potential record of an action potential in response to sustained current injection (325 pA). In both A and B, a bias current was used to maintain a similar baseline membrane potential before depolarizing current injection (near −84 mV). In both cases, an action potential developed within 150–200 ms from the start of the current injection. C and D: phase plots, the 1st derivative of the somatic membrane voltage (dV/dt) vs. membrane voltage (V_m), for a dentate granule (C) and CA3 pyramidal neuron (D). ↓, the threshold for the action potential. After the action potential threshold, 2 inflection points are indicated with (*, ▿). See also Fig. 5, A3 and B3, arrow and plus sign. E and F: threshold estimated from phase plots (C and D), and differences in initial phase plot slope from the 2 cell types. Examination of action potential threshold at 10 mV ms⁻¹ (···). , data points used to calculate the slope and average threshold from the phase plots. Baseline dV/dt due to the passive response to current injection has been subtracted from the plots. G: summary of action potentials thresholds at 10 mV ms⁻¹ in both dentate granule (DG) and CA3 neurons (n = 11 and 9, respectively). *, P < 0.0001. H: summary of phase plot slopes (inflection rate) at 10 mV ms⁻¹ in DG and CA3 neurons. *, P < 0.00001. Dentate granule neurons exhibit a shallower phase plot slope at threshold than CA3 pyramidal neurons.

FIG. 3.

Pharmacological evidence for robust action potential initiation in the proximal mossy fiber. A: effect of local TTX application (gray traces) on phase plots of action potentials elicited by just-suprathreshold current injection (minimal stimulation, 80 pA). Baseline traces (absence of TTX) are black. A shift in threshold is indicated by a shift of first inflection of the phase plot along the x-axis. Distal axon local application was 70–90 μm from the soma; proximal axon local application was 10–30 μm from the soma; proximal dendrite local application was at the primary dendrite adjacent to the soma. B: strong (1.0 nA) stimulation was used to elicit the action potential. Again, the proximal axon was the site at which local TTX application elicited a significant change in threshold. C and D: summary of the change in threshold taken from phase plots at a slope of 10 mV ms⁻¹ from 4 to 5 dentate granule neurons in each condition. *P < 0.05.

FIG. 4.

Voltage-gated sodium channel and initial segment visualization with combined cell fills and immunohistochemistry. A1: representative Alexa Fluor 488 filled dentate granule neuron. Scale bar is 30 μm. A2: corresponding voltage-gated sodium channel alpha subunit (PanNav) immunoreactivity. The arrows in A1 and A2 denote the soma/hillock junction from which measurements of staining were made. B1–B3: axonal stretched side views (see methods) of the 1st 100 μm from the representative mossy fiber from A. This rotated view highlights the axonal localization of PanNav staining within the filled axon. In these images the x axis represents combined x and y planes from the confocal stack. The y axis represents the z plane, the depth within the confocal stack. B1: side view of Alexa Fluor 488 filled mossy fiber (green) Scale bar: y = 5 μm. B2: corresponding side view of PanNav immunoreactivity (magenta) B3: overlay of B1 and B2 shows overlapping region (white) of Alexa Fluor 488 fill and PanNav staining. C: quantification of fluorescence pixel intensity for PanNav along the 1st 100 μm of the representative mossy fiber from B1, shown by circles over the binned area. D: summary of average voltage-gated sodium channel localization from 10 granule neurons, squares plus error bars represent SE. The red line is an exponential decay function, fit from the peak through the falling phase of the mean PanNav staining. Values above the dotted line at 0 pixel intensity denote voltage-gated sodium channel immunoreactivity above that found on the proximal dentate granule neuron dendrite. E1–E3: side views of ankyrin G immunoreactivity. E1: side view of another representative mossy fiber Alexa Fluor 488 fill (green). E2: corresponding side view of ankyrin G immunoreactivity (magenta). E3: overlay of side views E1 and E2 shows localization of ankryirin G immunoreactivity on the mossy fiber (white). F: quantification of fluorescence pixel intensity of the representative mossy fiber from E1, represented by diamonds over the binned regions. G: summary of average ankyrin G staining compiled from 7 mossy fibers. Red line illustrates an exponential decay fit from the peak through the falling phase of the mean ankyrin G staining along the initial 100 μm of mossy fiber.

FIG. 5.

Latency analysis from simultaneous axon and soma recordings. A1–A7: data from a representative dentate granule neuron. A1: the somatic membrane potential (V_m) during an action potential. Scale bar is 25 mV. A2: 1st derivative of the somatic membrane potential (dV/dt). Scale bar is 100 mV ms⁻¹. A3: 2nd derivative of the somatic membrane potential (d²V/dt²) exhibits 2 inflection points (indicated by the ↓ and +) occurring after the action potential threshold (- - -). Scale bar is 0.5 × 10⁶ mV ms⁻². A4: 3rd derivative of the membrane potential (d³V/dt³) is used as an aid to determine the first peak inflection on the d²V/dt² trace. Scale bar is 1 × 10¹⁰ mV ms⁻³. A5: axonally recorded extracellular signal at 280 μm from the soma. Scale bar is 10 pA. A6: 1st derivative of the axonal signal (dI/dt). Scale bar is 70 pA ms⁻¹. A7:. 2 latencies are indicated. Time 1 represents the time from the action potential threshold until the membrane potential reaches the 1st (nonstationary) inflection point of d²V/dt² (at the ↓). The nonstationary inflection in the 2nd derivative coincides with a 3rd derivative value that does not pass through 0 in A4 (↓). Time 2 represents the time from the action potential threshold until the peak in the 1st derivative in the axonal action potential signal (A6). B1–B4: data from a representative CA3 pyramidal neuron. B1: the CA3 somatic membrane potential (V_m) during an action potential. Scale bar is 25 mV. B2: 1st derivative of the somatic membrane potential (dV/dt). Scale bar is 100 mV ms⁻¹. B3: 2nd derivative of the somatic membrane potential (d²V/dt²) shows a waveform with 2 inflection points (indicated with ↓ and +) occurring after the action potential threshold (- - -). ··· through the 1st peak indicates a stationary inflection point in the 1st component. Scale bar is 0.5 × 10⁶ mV ms⁻². B4: 3rd derivative of the membrane potential (d³V/dt³) is used as an aid to determine the 2nd derivative stationary inflection point (3rd derivative = 0). Scale bar 1 × 10¹⁰ mV ms⁻³. C: a summary of time 1 from 31 paired recordings to show that the time between 1st threshold crossing and the development/invasion of the action potential into the axon hillock is not dependent on the axonal recording location. —, mean value for time 1 is 0.172 ± 0.004 ms; - - -, SE. D: a summary of time 2 from 31 paired recordings shows that the time between the 1st threshold crossing and the detection of the action potential in the axon ≤400 μm from the soma varied linearly with distance. Regression line (—) is plotted from 70 to 450 μm of axon recording distance (R² = 0.83). - - -, the 95% confidence bands of all possible straight lines through the data points. E: summary of the average conduction velocity over a distance of 400 μm from the soma on mossy fibers. Note the average conduction velocity is calculated from the velocity from the action potential initiation site to the axon recording site and the velocity from the action potential initiation site to the soma.