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Presynaptic calcium signalling in cerebellar mossy fibres.

Thomsen LB, Jörntell H, Midtgaard J - Front Neural Circuits (2010)

Bottom Line: A paired-pulse depression of the calcium signal lasting more than 1 s affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists.While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells.This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Pharmacology, University of Copenhagen Copenhagen, Denmark.

ABSTRACT
Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX)-sensitive fast Na(+) spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers. Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon appeared to be isolated from one another in terms of calcium signalling. CGP55845 application showed that GABA B receptors mediated presynaptic inhibition of the calcium signal over the entire firing frequency range of mossy fibres. A paired-pulse depression of the calcium signal lasting more than 1 s affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists. While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells. This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette.

No MeSH data available.


Related in: MedlinePlus

Anatomical and electrophysiological identification of mossy fibres.  (A) Montage of a mossy fibre trajectory in the whole-cerebellum preparation. The axon was injected with biocytin and reacted with DAB. The cerebellum is viewed from the ventral (ependymal) side. The cerebellar outline is indicated, including the cerebellar peduncles (right). The caudal tip (to the left) has been cut in two with a small sagittal incision to facilitate flattening of the tissue during fixation. The axon was traced using Neurolucida and the position of each identifiable rosette is indicated by red oval (complex rosette) or green circle (simple rosette) superimposed on the cerebellar outline. Below the cerebellar profile is the vertical projection of the Neurolucida tracing to show the dorsal–ventral variations in the mossy fibre trajectory. Colour pictures show representative rosettes. Some of the complex rosettes cannot be resolved in detail due to closely packed branching. All dimensions are measured in the mounted, dehydrated tissue. The 5-μm scale applies to the simple rosettes above and below the scale bar; the 10-μm scale applies to the remaining rosettes. (B) 3-D diagram of Neurolucida mossy fibre tracing from (A). Elevated (ventral) view approximately from the position indicated by “*” in (A). Complex rosettes indicated by red symbols, simple rosettes indicated by green symbols. (C) Montage of bright field image and fluorescence picture of mossy fibre rosette patch recording in a cerebellar slice. Axon injected with Lucifer Yellow. (See also Video in Supplementary Material). (D) I–V curves for axon in (B). Inset below shows rebound depolarization following the largest hyperpolarizing pulse in (D). (E) Local, subthreshold responses (arrowheads) and spike at the beginning of depolarizing pulse in (D). Spike amplitude in (D) and (E) low-pass filtered by recording electrode. (F) Summary graph of I–V responses in (D) at steady state (circles) and at 100 ms after pulse-onset for depolarizing pulses (squares). In the hyperpolarizing direction the peak values are indicated by squares. Data points from additional sweeps are included in (F), omitted for clarity from (D). Resting membrane potential −46 mV (horizontal, dashed line); spike current threshold +100 pA (vertical, dotted line).
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Figure 1: Anatomical and electrophysiological identification of mossy fibres. (A) Montage of a mossy fibre trajectory in the whole-cerebellum preparation. The axon was injected with biocytin and reacted with DAB. The cerebellum is viewed from the ventral (ependymal) side. The cerebellar outline is indicated, including the cerebellar peduncles (right). The caudal tip (to the left) has been cut in two with a small sagittal incision to facilitate flattening of the tissue during fixation. The axon was traced using Neurolucida and the position of each identifiable rosette is indicated by red oval (complex rosette) or green circle (simple rosette) superimposed on the cerebellar outline. Below the cerebellar profile is the vertical projection of the Neurolucida tracing to show the dorsal–ventral variations in the mossy fibre trajectory. Colour pictures show representative rosettes. Some of the complex rosettes cannot be resolved in detail due to closely packed branching. All dimensions are measured in the mounted, dehydrated tissue. The 5-μm scale applies to the simple rosettes above and below the scale bar; the 10-μm scale applies to the remaining rosettes. (B) 3-D diagram of Neurolucida mossy fibre tracing from (A). Elevated (ventral) view approximately from the position indicated by “*” in (A). Complex rosettes indicated by red symbols, simple rosettes indicated by green symbols. (C) Montage of bright field image and fluorescence picture of mossy fibre rosette patch recording in a cerebellar slice. Axon injected with Lucifer Yellow. (See also Video in Supplementary Material). (D) I–V curves for axon in (B). Inset below shows rebound depolarization following the largest hyperpolarizing pulse in (D). (E) Local, subthreshold responses (arrowheads) and spike at the beginning of depolarizing pulse in (D). Spike amplitude in (D) and (E) low-pass filtered by recording electrode. (F) Summary graph of I–V responses in (D) at steady state (circles) and at 100 ms after pulse-onset for depolarizing pulses (squares). In the hyperpolarizing direction the peak values are indicated by squares. Data points from additional sweeps are included in (F), omitted for clarity from (D). Resting membrane potential −46 mV (horizontal, dashed line); spike current threshold +100 pA (vertical, dotted line).

Mentions: For anterograde labelling of mossy fibres, the calcium indicator Calcium Green-1 dextran (molecular weight 5 kD; Molecular Probes, OR, USA) was dissolved in physiological saline at a concentration of 40 mM. Two microlitres of this solution was pressure-injected into the brainstem and the cerebellar peduncles using a glass pipette with a ∼20 μm diameter tip (Beierlein et al., 2004). Both sides of the brainstem were injected, primarily corresponding to the position of the vestibular nuclei along the fourth ventricle (Künzle, 1983a) up to the peduncles. However, it was commonly observed that the dye spread to other areas in the brainstem, making it likely that fibres other than vestibulo-cerebellar axons were labeled in this study (Künzle, 1982, 1983b). The brainstem-cerebellum preparation was left to incubate overnight at 5°C in a sealed container with Ringer solution. This ensured adequate time for the dye to fill the mossy fibres. The following day the cerebellum was carefully separated from the brainstem and placed in the recording chamber ventral side up. Fine silver wires were placed transversely over the peduncles and near the caudal tip of the cerebellum for mechanical stabilization. For fluorescence imaging, a Polychrome light source (TILL Photonics, Germany) and a Sensicam (640 × 480 pixels; PCO, Germany) were used together with 63×/0.9 water immersion objective (Zeiss, Germany). This resulted in each pixel covering an area of 0.17 μm on each side at full camera resolution. However, 2 × 2 pixel binning was commonly used, and for high-speed imaging (up to 100 Hz) 4 × 4 or 8 × 8 pixel binning was applied. In some cases a 0.5× lens was added in front of the camera to enlarge the field of view. Axons were readily visible from the ventral surface of the cerebellum through the ependymal cell layer, and could sometimes be followed for up to ∼1 mm. As indicated in Figure 1, rosettes were often widely spaced along the axon. Mossy fibres were identified based on their predominantly parasagittal course and the occasional occurrence of complex rosettes. In contrast, Purkinje cell axons are placed deeper in the cerebellum, just below the Purkinje cell layer, and display few boutons in the granule cell layer (Mugnaini et al., 1974); furthermore, the labelling procedure is commonly expected only to label axons anterogradely (Beierlein et al., 2004). The mossy fibres usually had a diameter of ∼ 1–2 μm, consistent with anatomical data (Mugnaini et al., 1974). In this way we could image activity-related fluorescence signals from presynaptic terminals of myelinated CNS axons in the intact adult cerebellum. Forty mossy fibres were included in this study. Axons were accepted for analysis if they displayed a robust increase in fluorescence when the fibres were activated electrically, and if they could follow repetitive activation at 10–100 Hz. For the intensity of fluorescence excitation used in the present experiments, bleaching was measured as the decrease in fluorescence during a 5-s long illumination without electrical stimulation, and was 0.58% per second of illumination (n = 4, SD ± 0.43%). The data were not corrected for this bleaching. A series of pictures was collected for each stimulation, consisting of a number of reference pictures, followed by pictures accompanied by electrical stimulation. The electrical stimulation was synchronised with picture acquisition. Background fluorescence was measured in a region away from the axon of interest, and for each picture the background was subtracted from the raw fluorescence of the axon under study. Four to six prestimulus pictures were averaged and used as reference. dF/F (%) was calculated as 100 × (Factive − Freference)/Freference. Images were analysed using custom written routines in IDL (ITT Visual Information Solutions); additional data analysis was performed using Origin (Microcal, MA, USA), including analysis of fluorescence decay time constants. Decay time constants were considered to be bi-exponential if the fast and the slow time constants differed by more than a factor of four. The data shown here are for the most part an average of the responses to four identical stimulations. For illustration, pseudocolor dF/F images were spatially smoothed using a 3 × 3 median filter. Mossy fibre complex rosettes often extend substantially in the z-axis. It was therefore rare to find a complex rosette that could be imaged in toto in one focal plane. Therefore, Z-stacks were recorded by stepping the microscope 1 μm in the focus axis by a PC program controlling the Luigs and Neumann microscope z-axis drive. For each focal plane, one or several picture series were acquired during electrical stimulation. For illustration of rosette anatomy, a maximum intensity projection of the z-stack was used as a basis. However, for clarity, additional picture montage was undertaken using data from individual focal planes as indicated in the text.


Presynaptic calcium signalling in cerebellar mossy fibres.

Thomsen LB, Jörntell H, Midtgaard J - Front Neural Circuits (2010)

Anatomical and electrophysiological identification of mossy fibres.  (A) Montage of a mossy fibre trajectory in the whole-cerebellum preparation. The axon was injected with biocytin and reacted with DAB. The cerebellum is viewed from the ventral (ependymal) side. The cerebellar outline is indicated, including the cerebellar peduncles (right). The caudal tip (to the left) has been cut in two with a small sagittal incision to facilitate flattening of the tissue during fixation. The axon was traced using Neurolucida and the position of each identifiable rosette is indicated by red oval (complex rosette) or green circle (simple rosette) superimposed on the cerebellar outline. Below the cerebellar profile is the vertical projection of the Neurolucida tracing to show the dorsal–ventral variations in the mossy fibre trajectory. Colour pictures show representative rosettes. Some of the complex rosettes cannot be resolved in detail due to closely packed branching. All dimensions are measured in the mounted, dehydrated tissue. The 5-μm scale applies to the simple rosettes above and below the scale bar; the 10-μm scale applies to the remaining rosettes. (B) 3-D diagram of Neurolucida mossy fibre tracing from (A). Elevated (ventral) view approximately from the position indicated by “*” in (A). Complex rosettes indicated by red symbols, simple rosettes indicated by green symbols. (C) Montage of bright field image and fluorescence picture of mossy fibre rosette patch recording in a cerebellar slice. Axon injected with Lucifer Yellow. (See also Video in Supplementary Material). (D) I–V curves for axon in (B). Inset below shows rebound depolarization following the largest hyperpolarizing pulse in (D). (E) Local, subthreshold responses (arrowheads) and spike at the beginning of depolarizing pulse in (D). Spike amplitude in (D) and (E) low-pass filtered by recording electrode. (F) Summary graph of I–V responses in (D) at steady state (circles) and at 100 ms after pulse-onset for depolarizing pulses (squares). In the hyperpolarizing direction the peak values are indicated by squares. Data points from additional sweeps are included in (F), omitted for clarity from (D). Resting membrane potential −46 mV (horizontal, dashed line); spike current threshold +100 pA (vertical, dotted line).
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: Anatomical and electrophysiological identification of mossy fibres. (A) Montage of a mossy fibre trajectory in the whole-cerebellum preparation. The axon was injected with biocytin and reacted with DAB. The cerebellum is viewed from the ventral (ependymal) side. The cerebellar outline is indicated, including the cerebellar peduncles (right). The caudal tip (to the left) has been cut in two with a small sagittal incision to facilitate flattening of the tissue during fixation. The axon was traced using Neurolucida and the position of each identifiable rosette is indicated by red oval (complex rosette) or green circle (simple rosette) superimposed on the cerebellar outline. Below the cerebellar profile is the vertical projection of the Neurolucida tracing to show the dorsal–ventral variations in the mossy fibre trajectory. Colour pictures show representative rosettes. Some of the complex rosettes cannot be resolved in detail due to closely packed branching. All dimensions are measured in the mounted, dehydrated tissue. The 5-μm scale applies to the simple rosettes above and below the scale bar; the 10-μm scale applies to the remaining rosettes. (B) 3-D diagram of Neurolucida mossy fibre tracing from (A). Elevated (ventral) view approximately from the position indicated by “*” in (A). Complex rosettes indicated by red symbols, simple rosettes indicated by green symbols. (C) Montage of bright field image and fluorescence picture of mossy fibre rosette patch recording in a cerebellar slice. Axon injected with Lucifer Yellow. (See also Video in Supplementary Material). (D) I–V curves for axon in (B). Inset below shows rebound depolarization following the largest hyperpolarizing pulse in (D). (E) Local, subthreshold responses (arrowheads) and spike at the beginning of depolarizing pulse in (D). Spike amplitude in (D) and (E) low-pass filtered by recording electrode. (F) Summary graph of I–V responses in (D) at steady state (circles) and at 100 ms after pulse-onset for depolarizing pulses (squares). In the hyperpolarizing direction the peak values are indicated by squares. Data points from additional sweeps are included in (F), omitted for clarity from (D). Resting membrane potential −46 mV (horizontal, dashed line); spike current threshold +100 pA (vertical, dotted line).
Mentions: For anterograde labelling of mossy fibres, the calcium indicator Calcium Green-1 dextran (molecular weight 5 kD; Molecular Probes, OR, USA) was dissolved in physiological saline at a concentration of 40 mM. Two microlitres of this solution was pressure-injected into the brainstem and the cerebellar peduncles using a glass pipette with a ∼20 μm diameter tip (Beierlein et al., 2004). Both sides of the brainstem were injected, primarily corresponding to the position of the vestibular nuclei along the fourth ventricle (Künzle, 1983a) up to the peduncles. However, it was commonly observed that the dye spread to other areas in the brainstem, making it likely that fibres other than vestibulo-cerebellar axons were labeled in this study (Künzle, 1982, 1983b). The brainstem-cerebellum preparation was left to incubate overnight at 5°C in a sealed container with Ringer solution. This ensured adequate time for the dye to fill the mossy fibres. The following day the cerebellum was carefully separated from the brainstem and placed in the recording chamber ventral side up. Fine silver wires were placed transversely over the peduncles and near the caudal tip of the cerebellum for mechanical stabilization. For fluorescence imaging, a Polychrome light source (TILL Photonics, Germany) and a Sensicam (640 × 480 pixels; PCO, Germany) were used together with 63×/0.9 water immersion objective (Zeiss, Germany). This resulted in each pixel covering an area of 0.17 μm on each side at full camera resolution. However, 2 × 2 pixel binning was commonly used, and for high-speed imaging (up to 100 Hz) 4 × 4 or 8 × 8 pixel binning was applied. In some cases a 0.5× lens was added in front of the camera to enlarge the field of view. Axons were readily visible from the ventral surface of the cerebellum through the ependymal cell layer, and could sometimes be followed for up to ∼1 mm. As indicated in Figure 1, rosettes were often widely spaced along the axon. Mossy fibres were identified based on their predominantly parasagittal course and the occasional occurrence of complex rosettes. In contrast, Purkinje cell axons are placed deeper in the cerebellum, just below the Purkinje cell layer, and display few boutons in the granule cell layer (Mugnaini et al., 1974); furthermore, the labelling procedure is commonly expected only to label axons anterogradely (Beierlein et al., 2004). The mossy fibres usually had a diameter of ∼ 1–2 μm, consistent with anatomical data (Mugnaini et al., 1974). In this way we could image activity-related fluorescence signals from presynaptic terminals of myelinated CNS axons in the intact adult cerebellum. Forty mossy fibres were included in this study. Axons were accepted for analysis if they displayed a robust increase in fluorescence when the fibres were activated electrically, and if they could follow repetitive activation at 10–100 Hz. For the intensity of fluorescence excitation used in the present experiments, bleaching was measured as the decrease in fluorescence during a 5-s long illumination without electrical stimulation, and was 0.58% per second of illumination (n = 4, SD ± 0.43%). The data were not corrected for this bleaching. A series of pictures was collected for each stimulation, consisting of a number of reference pictures, followed by pictures accompanied by electrical stimulation. The electrical stimulation was synchronised with picture acquisition. Background fluorescence was measured in a region away from the axon of interest, and for each picture the background was subtracted from the raw fluorescence of the axon under study. Four to six prestimulus pictures were averaged and used as reference. dF/F (%) was calculated as 100 × (Factive − Freference)/Freference. Images were analysed using custom written routines in IDL (ITT Visual Information Solutions); additional data analysis was performed using Origin (Microcal, MA, USA), including analysis of fluorescence decay time constants. Decay time constants were considered to be bi-exponential if the fast and the slow time constants differed by more than a factor of four. The data shown here are for the most part an average of the responses to four identical stimulations. For illustration, pseudocolor dF/F images were spatially smoothed using a 3 × 3 median filter. Mossy fibre complex rosettes often extend substantially in the z-axis. It was therefore rare to find a complex rosette that could be imaged in toto in one focal plane. Therefore, Z-stacks were recorded by stepping the microscope 1 μm in the focus axis by a PC program controlling the Luigs and Neumann microscope z-axis drive. For each focal plane, one or several picture series were acquired during electrical stimulation. For illustration of rosette anatomy, a maximum intensity projection of the z-stack was used as a basis. However, for clarity, additional picture montage was undertaken using data from individual focal planes as indicated in the text.

Bottom Line: A paired-pulse depression of the calcium signal lasting more than 1 s affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists.While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells.This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Pharmacology, University of Copenhagen Copenhagen, Denmark.

ABSTRACT
Whole-cell recordings were obtained from mossy fibre terminals in adult turtles in order to characterize the basic membrane properties. Calcium imaging of presynaptic calcium signals was carried out in order to analyse calcium dynamics and presynaptic GABA B inhibition. A tetrodotoxin (TTX)-sensitive fast Na(+) spike faithfully followed repetitive depolarizing pulses with little change in spike duration or amplitude, while a strong outward rectification dominated responses to long-lasting depolarizations. High-threshold calcium spikes were uncovered following addition of potassium channel blockers. Calcium imaging using Calcium-Green dextran revealed a stimulus-evoked all-or-none TTX-sensitive calcium signal in simple and complex rosettes. All compartments of a complex rosette were activated during electrical activation of the mossy fibre, while individual simple and complex rosettes along an axon appeared to be isolated from one another in terms of calcium signalling. CGP55845 application showed that GABA B receptors mediated presynaptic inhibition of the calcium signal over the entire firing frequency range of mossy fibres. A paired-pulse depression of the calcium signal lasting more than 1 s affected burst firing in mossy fibres; this paired-pulse depression was reduced by GABA B antagonists. While our results indicated that a presynaptic rosette electrophysiologically functioned as a unit, topical GABA application showed that calcium signals in the branches of complex rosettes could be modulated locally, suggesting that cerebellar glomeruli may be dynamically sub-compartmentalized due to ongoing inhibition mediated by Golgi cells. This could provide a fine-grained control of mossy fibre-granule cell information transfer and synaptic plasticity within a mossy fibre rosette.

No MeSH data available.


Related in: MedlinePlus