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Synaptic transmission in vertebrate neuromuscular junctions forms the basis of this study. The communication between neurons in the nervous system occurs largely due to neurotransmitter release at the synapses. Messages on the various significant nervous system functions are coordinated through the synaptic junctions and the release of neurotransmitters. Ryanodine receptors are found in the somata of the Purkinje cells, basket cells and pre-synaptic terminals of specific synapses and terminals of basket cells. Calcium in the extracellular fluid triggers the neurotransmitter release. Now it is understood that pre-synaptic stores could participate in this same function. Ryanodine-sensitive stores of calcium also play a role.

The quantum is the amount of spontaneous signals occurring in the absence of pre-synaptic action potentials and is equivalent to the release of one neurotransmitter vesicle (Katz, 1969). The action potentials are called miniature currents. For central synapses in the brain, large miniature currents are believed to arise from the release of many neurotransmitter or presynaptic vesicles and in the range of several quanta (Bekkers, 1994). Yoshida’s study (1994) revealed that these multivesicular miniature events could actually be tetrodotoxin-resistant action potentials in the pre-synaptic terminals.

Other researchers have tried to explain the phenomenon from another angle, using the presence of intracellular calcium stores in the pre-synaptic terminals. Nakanishi et al localized inositol triphosphate receptors in the neural tissue of the developing and adult mouse brain (1991). These were immunolocalised in the pre-synaptic terminals of the deep cerebellar nuclei and the retina of the eyes. Narita’s studies (1998, 2000) revealed the action of ryanodine-sensitive calcium stores at the frog neuromuscular junctions.

It was discovered that agents which influence the ryanodine-sensitive Calcium stores also increased the intracellular Calcium in the pre-synaptic cells and regulated acetyl choline release during high frequency stimulation. Mothet et al (1998) studied the action potentials at the pre-synaptic terminals of the buccal ganglia in Aplysia. They indicated that ryanodine inhibited while the pre-synaptic injection of Cyclic ADP Ribose augmented the action potential evoked release of acetyl choline at synapses. Studies also showed that caffeine with or without ryanodine modifies Calcium stores at the pre-synaptic terminals in autonomic ganglia (Peng, 1996; Smith et al, 1996) and in photoreceptors (Krizaj, 1999). Studies on hippocampal pyramidal cells have shown that

Caffeine or thapsigargin influences the frequency of miniature IPSCs.

Making an assumption, from prior studies described above, that spontaneous Calcium release from pre-synaptic Calcium stores may provide the synchronisation mechanism that causes multivesicular miniature IPSCs and the fact that such a hypothesis has not been tested systematically previously, the authors have taken up this topic for their study on cerebellar interneuron Purkinje cells.

This Study

 Methods

Experiments were conducted on the sagittal cerebellar slices of decapitated rats aged 10-14 days of age. During the experimental recordings, the slices were perfused in saline containing prescribed concentrations of NaCl, KCl, Na H2PO4, NaHCO3, CaCl2, MgCl2 and glucose with 95:5 mixture of oxygen and carbon dioxide. Experiments were done at room temperature.

For tight-seal whole-cell recordings, pipettes filled with a solution of appropriate concentrations of CsCl, MgCl2, HEPESCs, BAPTA-Cs (Molecular Probes, Eugene, Oregon), CaCl2, Na-GTP and Na-ATP and of pH 7.3 were used. Capacitance cancellation and series resistance compensation had been done. Kynurenic acid had been added to the extracellular solution to block the inotropic gluatamate receptors. TTX was present in the solution for all recordings.

The calcium free solutions were prepared by leaving out Calcium and adding EGTA Na.

Membrane potential was maintained at -60mV and the current was filtered at

1.5-2 kHz. Sampling was done continuously with brief interruptions. Detection and analysis were done using the IGOR-Pro programming environment. In experiments

needing a Calcium channel blocker, cytochrome was added to the external solution.

The toxin was prepared while the ryanodine was purchased.

Testing the Calcium

The Calcium in the basket cells were tested using the Two-photon laser scanning

Fluorescence microscopy. For studying the action potential-evoked calcium increases, bicuculline was added to the external solution and the calcium sensitive probe Oregon

Green was put into the pipettes. Scans were done and pulses were applied at the end of each 8th scan. This was repeated every minute in external solution which contained saline

in order to get a baseline. The external solution was then changed to the solution containing ryanodine and recording proceeded for another 15 minutes. Another set of recordings were done with external solution not having calcium but having EGTA Na.

The internal solution also had EGTA and Cs instead of K as the main cation.

The responses in Spontaneous Calcium transients also were recorded using molecular probes in the external solution. A pseudo line scan was also done. For immunocytochemistry, a rabbit polyclonal anti-serum was raised to the 16 amino-acids found in all mammals. A C terminal cysteine enabled conjugation to haemocyanin. The conjugated peptide was used to immunize rabbits. Then the ELISA confirmed the specificity. The sarcoplasmic and endoplasmic reticulum microsomes derived from the

skeletal muscle, cardiac tissue, whole brain and cerebellum were used for immunoblot analysis.

Effects of external Calcium concentration.

Large amplitude miniature IPSCs in cerebellar Purkinje cells were found sensitive to extracellular Calcium. With calcium free solution, the mIPSC frequency fell suddenly to half the control level. Continued exposure to the low level of external calcium caused the frequency of mIPSC to continue declining but at a slower rate. On washing after this, the frequency recovered and reached its initial level. The amplitude of the mIPSCs on the other hand showed a steady decline all through and no recovery on washing.

With high levels of Calcium, the frequency of the mIPSCs increased rapidly and significantly. The change in amplitude varied from no response to a minimal increase. The inference was that Calcium strongly influenced mIPSCs in the Purkinje cells though frequency and amplitude were differently affected. The rapid change in frequency was interpreted as the reaction of intracellular Calcium to external Calcium changes. The slow change in amplitude was considered due to the extracellular influence on the pre-synaptic stores. The prolonged extracellular calcium removal could have caused selective elimination of large amplitude miniature IPSCs.

Repeating with a calcium-free solution, many large amplitude miniature IPSCs were seen again. Then there was a sudden drop and then the amplitudes reduced to become concurrent with the control and the IPSCs were also less. The reduction seen when external Calcium was removed was not due to post-synaptic modifications.

On returning to the calcium-rich solution, a slight recovery of both amplitude and frequency occurred. Paired Student’s t-tests indicate significant changes in mean amplitude and frequency between mIPSCs recorded during a 3-min control period and after 15–18 min in Calcium-free external solution. 6 sham experiments were also conducted by keeping the slices in Calcium containing external solution all throughout and these showed no obvious change. The time course of decay of the IPSCs was slower in calcium-free external solution when compared to depolarization-induced calcium transients.

Effects of elevated intracellular Calcium

Elevated intracellular Calcium in the Purkinje cells caused a speedy frequency reduction and a slow increase in amplitude. This sudden fall could not be explained by the intracellular calcium as BAPTA buffered the Calcium in the cells and could not have caused the IPSCs.

Effect of the axons in large amplitude miniature IPSCs

The immediate slow changes in the amplitude of IPSCs on withdrawal of external calcium for 3 minutes could not be attributed to the delayed removal of external calcium

from pre-synaptic release sites. The effect was also not due to local Calcium influx caused by the TTX insensitive axonal depolarization. For the latter test, external Cd,

 a non-selective channel blocker, was used. It reduced the action potential-evoked pre-synaptic Calcium transients seen in the axons and pre-synaptic terminals of cerebellar interneurons. The slow changes in amplitude were therefore not connected to rundown or

altered post-synaptic receptors or delayed extracellular calcium removal. The only remaining explanation was that multivesicular release under the pre-synaptic calcium stores could have caused the changes in amplitude. The lack of recovery after external Calcium restoration could be due to the slow store refilling of intracellular Calcium

when action potentials and subsequent calcium influx are blocked.

Rise Time Kinetics

The multi-vesicular release also could not completely explain the slow changes in amplitude. Rise time of IPSCs as a function of amplitude was studied after extended external calcium removal. In 6 of the 8 cells tested, the rise time was heterogenous in nature where two subpopulations were concerned. Slower decay kinetics was also noted.

A faster rise time was seen in the proximal dendrites and soma. Faster IPSCs were more sensitive to external calcium removal than slow ones and these IPSCs arose at somatic synapses. The origin being multivesicular, synchronisation time must have been in the range of submillisecond. Some IPSCs had slower rise time and decays and were less sensitive to external calcium removal. These IPSCs could be arising from dendrites and could be having synchronisation of 1-4ms.

Ryanodine receptors

The two photon laser illumination was used to focus on the ryanodine receptors stained with a high-affinity Calcium-sensitive dye, Oregon Green BAPTA-1. Transient rises of Calcium in the stained ryanodine receptors in response to short trains of action potentials were measured. The fluorescence rises were noted.

Ryanodine-sensitive Calcium stores are associated with the large amplitude mIPSCs. In the experiment Ryanodine in large concentrations of 100 μM blocked the receptors. The response recorded showed that Ryanodine reduced the mean amplitude and frequency of mIPSCs simultaneously. The responses to muscimol were not affected by ryanodine and the researchers assumed that ryanodine had no post synaptic effect.

Axonal spots with calcium stores were identified in pre-synaptic terminals by recording responses to short action potentials. The intracellular Calcium fluctuations in response to differing concentrations of external calcium to which small concentrations of ryanodine were added was checked. Repeated scanning in the presence of TTX was done. Spontaneous calcium transients were noted before and after addition of ryanodine. Bright spots of fluorescence were noted at the pre-synaptic terminals. This signified the presence and increase of Calcium at the pre-synaptic terminals.

Experiments were then done with small concentrations of Ryanodine 10 μM. Large amplitude mIPSCs were recorded. High frequency bursts and amplitudes of the responses were greatly increased. The bursts could reflect the response at multivesicular and monovesicular sites. The histogram comparing the amplitudes in the control and after ryanodine was added showed a significant difference. The spontaneous Calcium transients occur at basket cell axons and their frequency was increased by small concentrations of ryanodine.


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