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Physiological properties of supragranular cortical inhibitory interneurons expressing retrograde persistent firing.

Imbrosci B, Neitz A, Mittmann T - Neural Plast. (2015)

Bottom Line: Despite this well accepted notion, recent research has shown that, under certain circumstances, the axon can also generate APs independent of synaptic inputs at axonal sites distal from the soma.This unusual form of neuronal communication seems to preferentially occur in cortical inhibitory interneurons following a period of intense neuronal activity and might have profound implications for neuronal information processing.Here we show that trains of ectopically generated APs can be induced in a large portion of neocortical layer 2/3 GABAergic interneurons following a somatic depolarization inducing hundreds of APs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology, University Medical Center of the Johannes-Gutenberg University Mainz, 55128 Mainz, Germany ; Neurowissenschaftliches Forschungszentrum, Charité-Universitätsmedizin Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germany.

ABSTRACT
Neurons are polarized functional units. The somatodendritic compartment receives and integrates synaptic inputs while the axon relays relevant synaptic information in form of action potentials (APs) across long distance. Despite this well accepted notion, recent research has shown that, under certain circumstances, the axon can also generate APs independent of synaptic inputs at axonal sites distal from the soma. These ectopic APs travel both toward synaptic terminals and antidromically toward the soma. This unusual form of neuronal communication seems to preferentially occur in cortical inhibitory interneurons following a period of intense neuronal activity and might have profound implications for neuronal information processing. Here we show that trains of ectopically generated APs can be induced in a large portion of neocortical layer 2/3 GABAergic interneurons following a somatic depolarization inducing hundreds of APs. Sparsely occurring ectopic spikes were also observed in a large portion of layer 1 interneurons even in absence of prior somatic depolarization. Remarkably, we found that interneurons which produce ectopic APs display specific membrane and morphological properties significantly different from the remaining GABAergic cells and may therefore represent a functionally unique interneuronal subpopulation.

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Persistent firing layer 2/3 interneurons showed small soma size and heterologous gap junction coupling. (a) One representative biocytin-filled FS-nPF and nFS-nPF interneuron (top) and two representative biocytin-filled nFS-PF interneurons (bottom). (b) Size of soma in the three electrophysiologically characterized interneuronal classes (FS nonpersistent firing, FS-nPF; nFS nonpersistent firing, nFS-nPF; nFS persistent firing, nFS-PF). (c) Triple immunofluorescence staining for biocytin (red), GFP (green), and parvalbumin (blue). The white arrow points at one representative biocytin-filled nFS-PF neuron. Note the biocytin diffusion from the patched-PF interneuron (white arrow) into two neighboring GFP and parvalbumin-positive interneurons (presumably FS-nPF) (grey arrow heads), suggesting gap junction coupling among these cells.
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fig4: Persistent firing layer 2/3 interneurons showed small soma size and heterologous gap junction coupling. (a) One representative biocytin-filled FS-nPF and nFS-nPF interneuron (top) and two representative biocytin-filled nFS-PF interneurons (bottom). (b) Size of soma in the three electrophysiologically characterized interneuronal classes (FS nonpersistent firing, FS-nPF; nFS nonpersistent firing, nFS-nPF; nFS persistent firing, nFS-PF). (c) Triple immunofluorescence staining for biocytin (red), GFP (green), and parvalbumin (blue). The white arrow points at one representative biocytin-filled nFS-PF neuron. Note the biocytin diffusion from the patched-PF interneuron (white arrow) into two neighboring GFP and parvalbumin-positive interneurons (presumably FS-nPF) (grey arrow heads), suggesting gap junction coupling among these cells.

Mentions: In our patch-clamp recordings from GAD67-GFP mice we could distinguish between fast-spiking (FS) (20.7%, 24 of 116 neurons) and non-fast-spiking (nFS) interneurons (79.3%, 92 of 116 neurons) (Figure 3(a)) based on the strictly different firing behavior of these cell subtypes upon somatic current injection. Interneurons were considered as FS if upon saturating somatic current injection they could achieve a firing rate of at least 200 Hz (mean maximal firing rate for FS: 301.33 ± 14.73 Hz). The remaining interneurons were considered nFS and their mean maximal firing rate was well below 200 Hz (104.03 ± 2.91 Hz). Interestingly we were able to induce persistent firing in a relatively large portion of nFS interneurons (32.6%, 30 of 92 neurons) but only in one out of 24 FS interneurons (4.2%) (Figure 3(a)). This finding suggests that persistent firing is very unlikely to occur in FS cells or alternatively it may indicate the existence of a very rare but still functionally distinct neuronal type. We decided to not include this single FS, persistent firing cell in further analyses. To examine whether the phenomenon of persistent firing was expressed by a specific functional class of layer 2/3 cortical interneurons we further characterized different physiological and morphological properties in three different populations of recorded interneurons: FS, nonpersistent firing (FS-nPF) (20%, 23 of 116 cells), nFS, nonpersistent firing (nFS-nPF) (53%, 62 of 116 cells), and nFS, persistent firing (nFS-PF) (26%, 30 of 116 cells) (Figure 3(a)). Interestingly, nFS-PF neurons presented a resting Vm significantly more hyperpolarized (P < 0.05) and a spike threshold significantly more depolarized (P < 0.05) in comparison with nFS-nPF (Table 1). As a consequence the Δvoltage between resting Vm and spike threshold was significantly larger in the nFS-PF than in the nFS-nPF group (P < 0.01). This suggests that nFS-PF interneurons require larger Vm depolarization to transit from a resting into an active state. FS interneurons showed intermediate Δvoltage values which did not differ from either of nFS neuronal groups (P > 0.05 for both FS versus nFS-nPF and Fs versus nFS-PF) (Figure 3(b), Table 1). nFS-PF interneurons displayed particularly wide somatic APs. The spike half-width in nFS-PF interneurons was not only significantly larger than FS interneurons (P < 0.001), which are well described to have very narrow APs [15], but also highly significantly larger than nFS-nPF (P < 0.001) (Table 1). It remains to be disclosed if the broad spike width observed in PF interneurons can be attributed to the expression of a specific set of voltage-dependent K+ channels with lower kinetics [16] and whether it may have a causal role in the induction of PF. The input resistance did also strongly differ between interneuronal groups. nFS-nPF neurons showed a relatively high input resistance; meanwhile nFS-PF cells displayed significantly lower values (P < 0.001), similarly to Fs interneurons (Figure 3(c), Table 1). The reduced input resistance in PF interneurons was not a result of leak currents due to bad recording conditions, since the resting Vm of this neuronal class was not depolarized but even more hyperpolarized than the other neuronal groups. All together, these data suggest that much stronger excitatory inputs are needed to drive nFS-PF interneurons above the spike threshold. To better analyse the relation between neuronal input and output we measured the frequency of action potential firing upon a depolarizing somatic current injection of gradually increasing amplitude. As expected FS interneurons achieved the highest firing rate (in some cells up to 400 Hz) which was highly significantly different from both nFS-nPF and nFS-PF (from 200 to 850 pA P < 0.001, Figure 3(d)). Furthermore, nFS-PF interneurons showed a significantly reduced firing compared to nFS-nPF cells at relatively low current injection amplitude (between 150 and 450 pA) (Figure 3(e)). This resulted in a rightward shift of the firing rate versus current injection curve in persistent firing interneurons compared to nFS-nPF (Figure 3(e)). Taken together these findings indicate that interneurons displaying persistent firing possess peculiar membrane properties which make them particularly reluctant to synaptic recruitment. The activation of this neuronal class may require very strong excitatory synaptic inputs. However, neither the frequency nor the amplitude of sEPSCs in nFS-PF were different from nFS-nPF interneurons (Table 1) suggesting a similar functional excitatory connectivity in these two neuronal populations. All together the differences in membrane and firing properties observed between nFS-nPF and nFS-PF cells suggest that the here identified PF interneurons constitute a functional unique neuronal population. Future studies should further investigate the cellular mechanisms responsible for these peculiar intrinsic features. Biocytin-filling further revealed that nFS-PF displayed specific morphological features. nFS-PF neurons always displayed multiple neuronal processes extending from the soma (Figure 4(a), bottom). Interestingly, we also observed differences in the size of soma of the three populations of interneurons. FS interneurons had the largest cell bodies (versus nFS-nPF: P < 0.01; versus nFS-PF: P < 0.001). In contrast, the soma size of persistent firing interneurons was the smallest (versus nFS-nPF: P < 0.05) (Figures 4(a)-4(b)). One additional feature found exclusively in one out of six persistent firing interneurons, but never in nFS-nPF cells (0/21), was the diffusion of biocytin from one recorded PF cell into nearby located interneurons (Figure 4(c), red channel). In this example, the cell pointed by the white arrow was proven to be a PF interneuron. This neuron was the only one from which patch-clamp recordings were performed and therefore the only neuron directly filled with biocytin. It is conceivable that the two additional biocytin-positive neurons (grey arrow heads) were stained indirectly by the diffusion of biocytin from the recorded neuron via gap-junctions [17, 18]. In the second channel in green, the GFP staining confirmed that all three neurons were GAD67-positive inhibitory neurons [10]. The two cells indirectly filled with biocytin (grey arrow heads) but not the recorded one (white arrow) were also found to be immunopositive for parvalbumin (Figure 4(c), blue channel). Since parvalbumin was found to be expressed by FS but never by nFS interneurons (data not shown) it is very likely that the PF, parvalbumin-negative cell, and the two parvalbumin-positive neurons belong to two different neuronal subtypes (nFS-PF and FS-nPF, resp.). This result suggests heterologous gap-junctions coupling between nFS-PF and FS-nPF interneurons.


Physiological properties of supragranular cortical inhibitory interneurons expressing retrograde persistent firing.

Imbrosci B, Neitz A, Mittmann T - Neural Plast. (2015)

Persistent firing layer 2/3 interneurons showed small soma size and heterologous gap junction coupling. (a) One representative biocytin-filled FS-nPF and nFS-nPF interneuron (top) and two representative biocytin-filled nFS-PF interneurons (bottom). (b) Size of soma in the three electrophysiologically characterized interneuronal classes (FS nonpersistent firing, FS-nPF; nFS nonpersistent firing, nFS-nPF; nFS persistent firing, nFS-PF). (c) Triple immunofluorescence staining for biocytin (red), GFP (green), and parvalbumin (blue). The white arrow points at one representative biocytin-filled nFS-PF neuron. Note the biocytin diffusion from the patched-PF interneuron (white arrow) into two neighboring GFP and parvalbumin-positive interneurons (presumably FS-nPF) (grey arrow heads), suggesting gap junction coupling among these cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Persistent firing layer 2/3 interneurons showed small soma size and heterologous gap junction coupling. (a) One representative biocytin-filled FS-nPF and nFS-nPF interneuron (top) and two representative biocytin-filled nFS-PF interneurons (bottom). (b) Size of soma in the three electrophysiologically characterized interneuronal classes (FS nonpersistent firing, FS-nPF; nFS nonpersistent firing, nFS-nPF; nFS persistent firing, nFS-PF). (c) Triple immunofluorescence staining for biocytin (red), GFP (green), and parvalbumin (blue). The white arrow points at one representative biocytin-filled nFS-PF neuron. Note the biocytin diffusion from the patched-PF interneuron (white arrow) into two neighboring GFP and parvalbumin-positive interneurons (presumably FS-nPF) (grey arrow heads), suggesting gap junction coupling among these cells.
Mentions: In our patch-clamp recordings from GAD67-GFP mice we could distinguish between fast-spiking (FS) (20.7%, 24 of 116 neurons) and non-fast-spiking (nFS) interneurons (79.3%, 92 of 116 neurons) (Figure 3(a)) based on the strictly different firing behavior of these cell subtypes upon somatic current injection. Interneurons were considered as FS if upon saturating somatic current injection they could achieve a firing rate of at least 200 Hz (mean maximal firing rate for FS: 301.33 ± 14.73 Hz). The remaining interneurons were considered nFS and their mean maximal firing rate was well below 200 Hz (104.03 ± 2.91 Hz). Interestingly we were able to induce persistent firing in a relatively large portion of nFS interneurons (32.6%, 30 of 92 neurons) but only in one out of 24 FS interneurons (4.2%) (Figure 3(a)). This finding suggests that persistent firing is very unlikely to occur in FS cells or alternatively it may indicate the existence of a very rare but still functionally distinct neuronal type. We decided to not include this single FS, persistent firing cell in further analyses. To examine whether the phenomenon of persistent firing was expressed by a specific functional class of layer 2/3 cortical interneurons we further characterized different physiological and morphological properties in three different populations of recorded interneurons: FS, nonpersistent firing (FS-nPF) (20%, 23 of 116 cells), nFS, nonpersistent firing (nFS-nPF) (53%, 62 of 116 cells), and nFS, persistent firing (nFS-PF) (26%, 30 of 116 cells) (Figure 3(a)). Interestingly, nFS-PF neurons presented a resting Vm significantly more hyperpolarized (P < 0.05) and a spike threshold significantly more depolarized (P < 0.05) in comparison with nFS-nPF (Table 1). As a consequence the Δvoltage between resting Vm and spike threshold was significantly larger in the nFS-PF than in the nFS-nPF group (P < 0.01). This suggests that nFS-PF interneurons require larger Vm depolarization to transit from a resting into an active state. FS interneurons showed intermediate Δvoltage values which did not differ from either of nFS neuronal groups (P > 0.05 for both FS versus nFS-nPF and Fs versus nFS-PF) (Figure 3(b), Table 1). nFS-PF interneurons displayed particularly wide somatic APs. The spike half-width in nFS-PF interneurons was not only significantly larger than FS interneurons (P < 0.001), which are well described to have very narrow APs [15], but also highly significantly larger than nFS-nPF (P < 0.001) (Table 1). It remains to be disclosed if the broad spike width observed in PF interneurons can be attributed to the expression of a specific set of voltage-dependent K+ channels with lower kinetics [16] and whether it may have a causal role in the induction of PF. The input resistance did also strongly differ between interneuronal groups. nFS-nPF neurons showed a relatively high input resistance; meanwhile nFS-PF cells displayed significantly lower values (P < 0.001), similarly to Fs interneurons (Figure 3(c), Table 1). The reduced input resistance in PF interneurons was not a result of leak currents due to bad recording conditions, since the resting Vm of this neuronal class was not depolarized but even more hyperpolarized than the other neuronal groups. All together, these data suggest that much stronger excitatory inputs are needed to drive nFS-PF interneurons above the spike threshold. To better analyse the relation between neuronal input and output we measured the frequency of action potential firing upon a depolarizing somatic current injection of gradually increasing amplitude. As expected FS interneurons achieved the highest firing rate (in some cells up to 400 Hz) which was highly significantly different from both nFS-nPF and nFS-PF (from 200 to 850 pA P < 0.001, Figure 3(d)). Furthermore, nFS-PF interneurons showed a significantly reduced firing compared to nFS-nPF cells at relatively low current injection amplitude (between 150 and 450 pA) (Figure 3(e)). This resulted in a rightward shift of the firing rate versus current injection curve in persistent firing interneurons compared to nFS-nPF (Figure 3(e)). Taken together these findings indicate that interneurons displaying persistent firing possess peculiar membrane properties which make them particularly reluctant to synaptic recruitment. The activation of this neuronal class may require very strong excitatory synaptic inputs. However, neither the frequency nor the amplitude of sEPSCs in nFS-PF were different from nFS-nPF interneurons (Table 1) suggesting a similar functional excitatory connectivity in these two neuronal populations. All together the differences in membrane and firing properties observed between nFS-nPF and nFS-PF cells suggest that the here identified PF interneurons constitute a functional unique neuronal population. Future studies should further investigate the cellular mechanisms responsible for these peculiar intrinsic features. Biocytin-filling further revealed that nFS-PF displayed specific morphological features. nFS-PF neurons always displayed multiple neuronal processes extending from the soma (Figure 4(a), bottom). Interestingly, we also observed differences in the size of soma of the three populations of interneurons. FS interneurons had the largest cell bodies (versus nFS-nPF: P < 0.01; versus nFS-PF: P < 0.001). In contrast, the soma size of persistent firing interneurons was the smallest (versus nFS-nPF: P < 0.05) (Figures 4(a)-4(b)). One additional feature found exclusively in one out of six persistent firing interneurons, but never in nFS-nPF cells (0/21), was the diffusion of biocytin from one recorded PF cell into nearby located interneurons (Figure 4(c), red channel). In this example, the cell pointed by the white arrow was proven to be a PF interneuron. This neuron was the only one from which patch-clamp recordings were performed and therefore the only neuron directly filled with biocytin. It is conceivable that the two additional biocytin-positive neurons (grey arrow heads) were stained indirectly by the diffusion of biocytin from the recorded neuron via gap-junctions [17, 18]. In the second channel in green, the GFP staining confirmed that all three neurons were GAD67-positive inhibitory neurons [10]. The two cells indirectly filled with biocytin (grey arrow heads) but not the recorded one (white arrow) were also found to be immunopositive for parvalbumin (Figure 4(c), blue channel). Since parvalbumin was found to be expressed by FS but never by nFS interneurons (data not shown) it is very likely that the PF, parvalbumin-negative cell, and the two parvalbumin-positive neurons belong to two different neuronal subtypes (nFS-PF and FS-nPF, resp.). This result suggests heterologous gap-junctions coupling between nFS-PF and FS-nPF interneurons.

Bottom Line: Despite this well accepted notion, recent research has shown that, under certain circumstances, the axon can also generate APs independent of synaptic inputs at axonal sites distal from the soma.This unusual form of neuronal communication seems to preferentially occur in cortical inhibitory interneurons following a period of intense neuronal activity and might have profound implications for neuronal information processing.Here we show that trains of ectopically generated APs can be induced in a large portion of neocortical layer 2/3 GABAergic interneurons following a somatic depolarization inducing hundreds of APs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology, University Medical Center of the Johannes-Gutenberg University Mainz, 55128 Mainz, Germany ; Neurowissenschaftliches Forschungszentrum, Charité-Universitätsmedizin Berlin, Campus Charité Mitte, Charitéplatz 1, 10117 Berlin, Germany.

ABSTRACT
Neurons are polarized functional units. The somatodendritic compartment receives and integrates synaptic inputs while the axon relays relevant synaptic information in form of action potentials (APs) across long distance. Despite this well accepted notion, recent research has shown that, under certain circumstances, the axon can also generate APs independent of synaptic inputs at axonal sites distal from the soma. These ectopic APs travel both toward synaptic terminals and antidromically toward the soma. This unusual form of neuronal communication seems to preferentially occur in cortical inhibitory interneurons following a period of intense neuronal activity and might have profound implications for neuronal information processing. Here we show that trains of ectopically generated APs can be induced in a large portion of neocortical layer 2/3 GABAergic interneurons following a somatic depolarization inducing hundreds of APs. Sparsely occurring ectopic spikes were also observed in a large portion of layer 1 interneurons even in absence of prior somatic depolarization. Remarkably, we found that interneurons which produce ectopic APs display specific membrane and morphological properties significantly different from the remaining GABAergic cells and may therefore represent a functionally unique interneuronal subpopulation.

Show MeSH
Related in: MedlinePlus