Grae Davis gave a Brains and Behavior talk entitled, "Homeostatic Control of Neural Function: From Phenomenology to Molecular Design".
He talked about homeostasis of the transmitter release that compensates reduced sensitivity or motoneuronal innervation of the postsynaptic muscle. If postsynaptic GluR was blocked by a toxin, the frequency of mEPSC increases and the size of EPSP recovers in 10 min. This was shown also by some mutants.
This recovery of EPSP amplitude is activity-independent. It seems like that the muscle is sensing the tonic release of transmitter from the presynaptic terminals, and release some retrograde messenger that regulates the transmitter release.
One candidate was called ephrins and Eph receptor.
Tuesday, December 9, 2008
Thursday, December 4, 2008
Thompson and Watson (2005) Melibe swim paper
Sint1 cell bodies are located on the medial dorsal surface just caudal to the prominent tentacular lobe that rises from the center of the pleural ganglion. 30-50 micron.
Sint1 branches in the pleural ganglion neuropil near the base of the optic lobe and projects to the ipsilateral pedal ganglion via the dorsal pleuralpedal connective, where it forms a series of arborizations.
A single sint2 is found near the dorsal midline of each pedal ganglion.
LY staining shows that sint2 branches in the pedal ganglion neuropil and sends a major process to the opposite pedal
ganglion via the circumesophageal, pedal-pedal connective.
sint1 can cause phase advance/delay.
There is reciprocal inhibition between left and right sint1 neurons, via direct IPSP.
sint1 innervates motoneurons.
sint1 and sint2 are electrically coupled.
Mutual inhibition between sint2 neurons.
Mutual inhibition between sint2 and the contralateral sint1
Activity in Sint1 and Sint2 dissociates during other locomotor behaviors.
the CPG for swimming is formed dynamically, when activity in the sint1 and sint2 cell pairs becomes bound together. When this does not occur, the same interneurons appear to function independently during the performance of other behaviors that involve the same or similar musculature, such as turning.
Sint1 branches in the pleural ganglion neuropil near the base of the optic lobe and projects to the ipsilateral pedal ganglion via the dorsal pleuralpedal connective, where it forms a series of arborizations.
A single sint2 is found near the dorsal midline of each pedal ganglion.
LY staining shows that sint2 branches in the pedal ganglion neuropil and sends a major process to the opposite pedal
ganglion via the circumesophageal, pedal-pedal connective.
sint1 can cause phase advance/delay.
There is reciprocal inhibition between left and right sint1 neurons, via direct IPSP.
sint1 innervates motoneurons.
sint1 and sint2 are electrically coupled.
Mutual inhibition between sint2 neurons.
Mutual inhibition between sint2 and the contralateral sint1
Activity in Sint1 and Sint2 dissociates during other locomotor behaviors.
the CPG for swimming is formed dynamically, when activity in the sint1 and sint2 cell pairs becomes bound together. When this does not occur, the same interneurons appear to function independently during the performance of other behaviors that involve the same or similar musculature, such as turning.
Monday, November 24, 2008
Jing's CC9&10 paper (2008)
They generated rat antibody for 5-HT.
Briefly, the antigen was prepared by coupling 2 mg serotonin oxalate to 10 mg BSA (Sigma-Aldrich) in 1 ml of 50 mM NaH2PO4, pH 7.2, using 100 microL 16% paraformaldehyde (EMS). Followingovernight incubation at 4°C, coupled antigen was purified from the reaction using a Microcon-30 spinning at 13,800 x g for 30 min at 4°C. After washing the retentate four times with 0.4 ml of 50 mM NaH2PO4, it was resuspended in 0.5 ml of the same buffer and transferred to a new tube...
Tissues were fixed in 4% paraformaldehyde, 0.2% picric acid, 25% sucrose, and 0.1MNaH2PO4, pH 7.6, for either 3 h at room temperature or overnight at 4°C.
Washing buffer (WB; 2% Triton X-100, 1% BSA, 154 mM NaCl, 50 mM EDTA, 0.01% thimerosal, and 10mM Na2HPO4, pH 7.4).
Both the dorsal and the ventral surfaces of the cerebral ganglion were desheathed. The cerebral ganglion was then twisted at the commissure to make it possible to access CC9/10 on the dorsal surface and contralateral CBIs on the ventral surface.
Frequent IPSPs are observed simultaneously in both CC9/10 cells (Fig. 2B). Activation of one CC9/10 can induce polysynaptic inhibition in the other CC9/10 (Fig. 2C1).
CC9/10 responds to noxious stimulus by tonic firing.
CC9/10 firing evokes locomotion.
The locomotion can be seen very well from PPCN (para-pedal commissural nerve).
The frequency of the locomotory rhythm is different depending on whether you stimulated P9 or AT4 nerve.
The cycle frequency of locomotor activity reflects the firing frequency of CC9/10 activity.
CC9 and CC10 may act as locomotion initiators.
One function of CC9/10 is to provide excitation to serotonergic modulatory neurons of the pedal ganglion.
It enhances the induction of the locomotory response.
CC9/10 weakly excites contralateral MCC and CBI-2. MCC>CBI-2
CC9/10 increase excitability (?) of MCC, but this could be just because of depolarization.
CC9–10 are broadly activated and their responses are not site-specific.
Aplysia locomotes, and then eats.
The Journal of Neuroscience, November 19, 2008 • 28(47):12349 –12361
Neural Analog of Arousal: Persistent Conditional Activation of a Feeding Modulator by Serotonergic Initiators of Locomotion
Jian Jing, Ferdinand S. Vilim, Elizabeth C. Cropper, and Klaudiusz R. Weiss
Briefly, the antigen was prepared by coupling 2 mg serotonin oxalate to 10 mg BSA (Sigma-Aldrich) in 1 ml of 50 mM NaH2PO4, pH 7.2, using 100 microL 16% paraformaldehyde (EMS). Followingovernight incubation at 4°C, coupled antigen was purified from the reaction using a Microcon-30 spinning at 13,800 x g for 30 min at 4°C. After washing the retentate four times with 0.4 ml of 50 mM NaH2PO4, it was resuspended in 0.5 ml of the same buffer and transferred to a new tube...
Tissues were fixed in 4% paraformaldehyde, 0.2% picric acid, 25% sucrose, and 0.1MNaH2PO4, pH 7.6, for either 3 h at room temperature or overnight at 4°C.
Washing buffer (WB; 2% Triton X-100, 1% BSA, 154 mM NaCl, 50 mM EDTA, 0.01% thimerosal, and 10mM Na2HPO4, pH 7.4).
Both the dorsal and the ventral surfaces of the cerebral ganglion were desheathed. The cerebral ganglion was then twisted at the commissure to make it possible to access CC9/10 on the dorsal surface and contralateral CBIs on the ventral surface.
Frequent IPSPs are observed simultaneously in both CC9/10 cells (Fig. 2B). Activation of one CC9/10 can induce polysynaptic inhibition in the other CC9/10 (Fig. 2C1).
CC9/10 responds to noxious stimulus by tonic firing.
CC9/10 firing evokes locomotion.
The locomotion can be seen very well from PPCN (para-pedal commissural nerve).
The frequency of the locomotory rhythm is different depending on whether you stimulated P9 or AT4 nerve.
The cycle frequency of locomotor activity reflects the firing frequency of CC9/10 activity.
CC9 and CC10 may act as locomotion initiators.
One function of CC9/10 is to provide excitation to serotonergic modulatory neurons of the pedal ganglion.
It enhances the induction of the locomotory response.
CC9/10 weakly excites contralateral MCC and CBI-2. MCC>CBI-2
CC9/10 increase excitability (?) of MCC, but this could be just because of depolarization.
CC9–10 are broadly activated and their responses are not site-specific.
Aplysia locomotes, and then eats.
The Journal of Neuroscience, November 19, 2008 • 28(47):12349 –12361
Neural Analog of Arousal: Persistent Conditional Activation of a Feeding Modulator by Serotonergic Initiators of Locomotion
Jian Jing, Ferdinand S. Vilim, Elizabeth C. Cropper, and Klaudiusz R. Weiss
Xue-Ying Jiang and Tom Abrams' paper (1998)
The first EPSP and the second EPSP in a paired pulse are evoked at distinct loci.
Paired-pulse ratio decreases with repeated testing.
Paired-pulse ratio depends on the size of the initial EPSP size.
The second EPSP size does not do much in determining the PPR.
The decrease in PPR is independent from synaptic depression.
The decrease of PPR occurs even without the depression of the 1st EPSP.
The decrease of PPR develops much faster than the depression of the 1st EPSP.
The PPR is not affected by the depression of the 1st EPSP.
Depresssion reduces EPSP2 along with EPSP1
Does synaptic depression change large synapses into small synapses?
No, they are different.
The paired pulse ratio is independent from the initial synaptic strength.
Use-dependent plasticity of paired-pulse facilitation provides evidence that a separate set of release sites contributes to EPSP2
A dramatic reduction in the 2nd EPSP after a single paired-pulse trial, when there was minimal change in the first.
Post-tetanic potentiation persists through repeated testing
Facilitation is not simply a modest form of PTP.
Interaction of serotonin-induced facilitation with the paired-pulse ratio
Serotonin-induced facilitation persisted only in the 1st EPSP. The 2nd EPSP shows a transient increase.
Contribution of Ca2+ and phosphorylation to paired-pulse facilitation
Facilitation is Ca dependent, but not phosphatase-dependent.
Paired-pulse ratio decreases with repeated testing.
Paired-pulse ratio depends on the size of the initial EPSP size.
The second EPSP size does not do much in determining the PPR.
The decrease in PPR is independent from synaptic depression.
The decrease of PPR occurs even without the depression of the 1st EPSP.
The decrease of PPR develops much faster than the depression of the 1st EPSP.
The PPR is not affected by the depression of the 1st EPSP.
Depresssion reduces EPSP2 along with EPSP1
Does synaptic depression change large synapses into small synapses?
No, they are different.
The paired pulse ratio is independent from the initial synaptic strength.
Use-dependent plasticity of paired-pulse facilitation provides evidence that a separate set of release sites contributes to EPSP2
A dramatic reduction in the 2nd EPSP after a single paired-pulse trial, when there was minimal change in the first.
Post-tetanic potentiation persists through repeated testing
Facilitation is not simply a modest form of PTP.
Interaction of serotonin-induced facilitation with the paired-pulse ratio
Serotonin-induced facilitation persisted only in the 1st EPSP. The 2nd EPSP shows a transient increase.
Contribution of Ca2+ and phosphorylation to paired-pulse facilitation
Facilitation is Ca dependent, but not phosphatase-dependent.
Shakiryanova's vesicle mobility paper (2007)
It has been postulated that vesicle mobility is increased to enhance release of transmitters, its mechanism was unknown.
Ryanodine receptor mediated Ca2+-induced Ca2+ release from ER is necessary for the sustained DCV mobilization.
This RyR-receptor mediated release is short-lived, and triggers activation of CaMKII as a downstream activator.
ryanodine (high purity) and other chemicals were purchased from Calbiochem.
4-bromo-clacimycin is a Ca2+ ionophore.
cytosolic calcium sensor, Cameleon 2.1.
CaMKII activity can be blocked by a membrane-permiable version of AIP-II (Antennapedia peptide fused).
veratridine, a sodium channel opener (that results in an increase of intracellular Ca2+).
Ryanodine receptor mediated Ca2+-induced Ca2+ release from ER is necessary for the sustained DCV mobilization.
This RyR-receptor mediated release is short-lived, and triggers activation of CaMKII as a downstream activator.
ryanodine (high purity) and other chemicals were purchased from Calbiochem.
4-bromo-clacimycin is a Ca2+ ionophore.
cytosolic calcium sensor, Cameleon 2.1.
CaMKII activity can be blocked by a membrane-permiable version of AIP-II (Antennapedia peptide fused).
veratridine, a sodium channel opener (that results in an increase of intracellular Ca2+).
Two serotonin blockers in Aplysia
cinanserin, methiothepin, cyproheptadine, spiperone.
10 mM 5-HT stock solutions were made in 0.1 M acetic acid solutions and stored at 4℃.
methiothepine blocked AC-coupled 5-HT receptors.
spiperone (100microM) blocks PKC-dependent modulatory effects of 5-HT, which is the facilitation from the depressed synapse.
spiperone is a selective antagonist of PLC-coupled 5-HT receptors in Aplysia.
Staurosporine is the PKC blocker.
Serotonin Receptor Antagonists Discriminate Between PKA- and PKC-Mediated Plasticity in Aplysia Sensory Neurons
Bogdan Dumitriu, Jonathan E. Cohen, Qin Wan, Andreea M. Negroiu and Thomas W. Abrams
J Neurophysiol 95: 2713-2720, 2006. First published October 19, 2005; doi:10.1152/jn.00642.2005
10 mM 5-HT stock solutions were made in 0.1 M acetic acid solutions and stored at 4℃.
methiothepine blocked AC-coupled 5-HT receptors.
spiperone (100microM) blocks PKC-dependent modulatory effects of 5-HT, which is the facilitation from the depressed synapse.
spiperone is a selective antagonist of PLC-coupled 5-HT receptors in Aplysia.
Staurosporine is the PKC blocker.
Serotonin Receptor Antagonists Discriminate Between PKA- and PKC-Mediated Plasticity in Aplysia Sensory Neurons
Bogdan Dumitriu, Jonathan E. Cohen, Qin Wan, Andreea M. Negroiu and Thomas W. Abrams
J Neurophysiol 95: 2713-2720, 2006. First published October 19, 2005; doi:10.1152/jn.00642.2005
Thursday, November 20, 2008
Quantal release of transmitter
The amount of quantal release is determined by both the number of available synaptic vesicles as well as by the probability that an available vesicle is released.
EPSCs can be described as the product of the number of available vesicles (n), release probability of each quanta (Pr) and size of the quantal unit q (mEPSC size):
EPSC = n·Pr·q
If N is the number of release sites (= maximum number of available vesicles), and Pa is the number of available vesicles.
n = N·Pa
EPSC = N·Pa·Po·q
This is the release-site model (Vere-Jones, 1966), assuming that the number of release site is limited.
The value q can be determined by recording mEPSC, sustaining presynaptic neuron depolarized, or stimulating the presynaptic neuron in low-Ca saline.
N can be estimated by giving a depleting stimulus. N is dependent on active zone constituents and mechanisms of vesicle recycling.
EPSCs can be described as the product of the number of available vesicles (n), release probability of each quanta (Pr) and size of the quantal unit q (mEPSC size):
EPSC = n·Pr·q
If N is the number of release sites (= maximum number of available vesicles), and Pa is the number of available vesicles.
n = N·Pa
EPSC = N·Pa·Po·q
This is the release-site model (Vere-Jones, 1966), assuming that the number of release site is limited.
The value q can be determined by recording mEPSC, sustaining presynaptic neuron depolarized, or stimulating the presynaptic neuron in low-Ca saline.
N can be estimated by giving a depleting stimulus. N is dependent on active zone constituents and mechanisms of vesicle recycling.
Wednesday, September 17, 2008
A ground wire
There was an argument about the ground wire used in electrophysiological recordings.
Of course, the surface area of the ground wire should be as large as possible, because 1) it will minimize the junctional resistance between the electrode and the saline, and 2) it will last longer.
The ground wire works as a current sink. It sucks up all current you apply through the microelectrodes, including those generated by zapping and by the capacitance compensation. A thicker wire should last longer . The ground becomes more stable with increased surface area because lower current density on surface would cause less voltage drop.
The AgC2 layer is very thin on the purchased silver pellets. It will go away soon after a heavy usage in current- and voltage-clamping. One has to chloride it frequently. However, if the AgCl2 layer became too thick, then it often often flakes off by a shock, or accidentally touching it with forceps. This is why I don't like those purchased pellets. Thier prices are ridiculous ly high. That's the other reason why I hate it.
Of course, the surface area of the ground wire should be as large as possible, because 1) it will minimize the junctional resistance between the electrode and the saline, and 2) it will last longer.
The ground wire works as a current sink. It sucks up all current you apply through the microelectrodes, including those generated by zapping and by the capacitance compensation. A thicker wire should last longer . The ground becomes more stable with increased surface area because lower current density on surface would cause less voltage drop.
The AgC2 layer is very thin on the purchased silver pellets. It will go away soon after a heavy usage in current- and voltage-clamping. One has to chloride it frequently. However, if the AgCl2 layer became too thick, then it often often flakes off by a shock, or accidentally touching it with forceps. This is why I don't like those purchased pellets. Thier prices are ridiculous ly high. That's the other reason why I hate it.
Friday, September 12, 2008
Sarah Palin and Hillary Clinton
There are two kinds of people in this world.
One kind of people would try to see the truth. They can wipe off their expectation with what they saw, heard, found or learned. If I speak to these people, they would look at my eyes and listen to me and try to understand what I said.
The other kind of people look at what they expect. They don't try to learn but just categorize things into a few things that they already knew. If they face something or people that they cannot categorize, they just trash it. Their eyes are not seeing the world but looking at what they have in their mind.
If I spoke to these people, they would judge me by seeing how I look different and where I am from, before understanding what I said. They think they are smarter than I am even though the situation is quite opposite. They are proud of not understanding things and not knowing things. They are proud of their stupidity.
I think this kind of people start warfare.
Living in this country as an alien, I become very good at telling these two kinds of people by just seeing them, hearing them speak.
Hillary Clinton, she is very smart. I know she would listen to me if I talk to her.
Sarah Palin, she won't. I know that. I can tell.
One kind of people would try to see the truth. They can wipe off their expectation with what they saw, heard, found or learned. If I speak to these people, they would look at my eyes and listen to me and try to understand what I said.
The other kind of people look at what they expect. They don't try to learn but just categorize things into a few things that they already knew. If they face something or people that they cannot categorize, they just trash it. Their eyes are not seeing the world but looking at what they have in their mind.
If I spoke to these people, they would judge me by seeing how I look different and where I am from, before understanding what I said. They think they are smarter than I am even though the situation is quite opposite. They are proud of not understanding things and not knowing things. They are proud of their stupidity.
I think this kind of people start warfare.
Living in this country as an alien, I become very good at telling these two kinds of people by just seeing them, hearing them speak.
Hillary Clinton, she is very smart. I know she would listen to me if I talk to her.
Sarah Palin, she won't. I know that. I can tell.
Monday, June 23, 2008
Pleurobranchaea
A monster came to our lab.
Pleurobranchaea californica.
Dr. Gillette said this is a super-Tritonia.
I gave it a piece of salt, but did not induce the escape swim.
Pleurobranchaea californica.
Dr. Gillette said this is a super-Tritonia.
I gave it a piece of salt, but did not induce the escape swim.
Monday, June 9, 2008
Tank 080609
Tanks look great.
Slugs ate seapens well. No death reported over the weekend.
Tank D had no water in upper level, because the bypass valve was left open. The water condition was fine, though. I cleaned the upper level and adjusted the valve.
15 Tritonia in A, 15 Tritonia in B, ? Aplysia in C, and 5 Tritonia in E.
Slugs ate seapens well. No death reported over the weekend.
Tank D had no water in upper level, because the bypass valve was left open. The water condition was fine, though. I cleaned the upper level and adjusted the valve.
15 Tritonia in A, 15 Tritonia in B, ? Aplysia in C, and 5 Tritonia in E.
Friday, June 6, 2008
Tank 080606
There was the temperature alert from Tank D this morning.
I went down to check it and found that the bottom floor where motor and cooling units are sitting was all wet. There must be some water leak from somewhere.
Tank D often has this this leakage problem. I could not found detect where it is. No cracks, no loose connection.
Today, I found it.
This tube pass water from the upper tank to the lower tank.
I went down to check it and found that the bottom floor where motor and cooling units are sitting was all wet. There must be some water leak from somewhere.
Tank D often has this this leakage problem. I could not found detect where it is. No cracks, no loose connection.
Today, I found it.
This tube pass water from the upper tank to the lower tank.The nozzle of this connection was loose. It was spraying water on the acrylic board.
I must have hit the cover when I opened or closed this cover for cleaning. I cleaned them all three times this week.
I must have hit the cover when I opened or closed this cover for cleaning. I cleaned them all three times this week.
Wednesday, May 21, 2008
Mysterious synapses
There are so many things that are still unknown in Tritonia brain.
Especially, the synaptic connectivities of neurons are so mysterious and puzzling.
One typical example is the synapse from VSI-B to VSI-follower cell (VSIF).
VSI-B excites VSIF from both sides, ipsilaterally and contralaterally though PdN6, but it is totally unknown where they connect. Is it direct synapse, or indirect, or mediated another electrically-coupled cell?
(from 4/29 lab meeting)
Today, I found another mystery in the synaptic output of DSI.
DSI was thought to project its axon to the contralateral PdG.
What I found was...
Especially, the synaptic connectivities of neurons are so mysterious and puzzling.
One typical example is the synapse from VSI-B to VSI-follower cell (VSIF).
VSI-B excites VSIF from both sides, ipsilaterally and contralaterally though PdN6, but it is totally unknown where they connect. Is it direct synapse, or indirect, or mediated another electrically-coupled cell?
(from 4/29 lab meeting)
Today, I found another mystery in the synaptic output of DSI.
DSI was thought to project its axon to the contralateral PdG.
What I found was...
Thursday, April 3, 2008
Patch-clamp amplifier vs. intracellular amplifier
People often ask me what the differences are between Axopatch and Axoclamp (both are made by Axon Instruments).
The answer is simple: Axopatch is a highly-sensitive I-V converter (ammeter). Axoclamp is a highly-stable voltmeter.
Axopatch or so-called patch-clamp amplifiers are designed for recording the membrane current, but it can also measure the membrane potential under the "current clamp mode".
On the other hand, Axoclamp is an amplifier designed for recording voltage with high-resistance microelectrodes. You can also measure membrane current by using "voltage-clamp mode." One may think that the continuous single-electrode voltage clamp mode in Axoclamp sounds very similar or almost identical to the whole-cell patch clamp by Axopatch.
Having either one being able do both voltage and current recordings, why do we need both?
The headstage of Axopatch contains a highly sensitive I-V converter, so that it can measure current with better s/n ratio. It can indirectly measure the voltage by calculating from the current being passed and membrane input resistance, but very susceptible to errors due to changes in the electrode resistance. This is why the patch pipette has to be big, with a resistance as low as possible.
Ironically, the large electrode tip makes he recorded potential less accurate, because of the junctional current. Junctional current is produced by the difference between the electrode solution and the cytoplasmic fluid. This current can be minimized by adjusting the ionic content of the electrode solution and reducing the size of the electrode tip (but this will increase the electrode resistance).
The headstage of Axoclamp contains the "voltage follower" circuit. This voltage follower circuit is the "magic" that makes it possible to record voltage with a sharp mega-ohm electrode. A voltage follower produce virtually infinite input resistance, which makes the electrode resistance almost negligible. Thus, the voltage recorded by Axoclamp is more accurate and relieble than Axopatch.
The current measured under voltage clamp mode in Axoclamp is actually the current generated artificially in the feedback circuit. Such indirect measurement is bad in s/n ratio and slower than Axopatch.
The answer is simple: Axopatch is a highly-sensitive I-V converter (ammeter). Axoclamp is a highly-stable voltmeter.
Axopatch or so-called patch-clamp amplifiers are designed for recording the membrane current, but it can also measure the membrane potential under the "current clamp mode".
On the other hand, Axoclamp is an amplifier designed for recording voltage with high-resistance microelectrodes. You can also measure membrane current by using "voltage-clamp mode." One may think that the continuous single-electrode voltage clamp mode in Axoclamp sounds very similar or almost identical to the whole-cell patch clamp by Axopatch.
Having either one being able do both voltage and current recordings, why do we need both?
The headstage of Axopatch contains a highly sensitive I-V converter, so that it can measure current with better s/n ratio. It can indirectly measure the voltage by calculating from the current being passed and membrane input resistance, but very susceptible to errors due to changes in the electrode resistance. This is why the patch pipette has to be big, with a resistance as low as possible.
Ironically, the large electrode tip makes he recorded potential less accurate, because of the junctional current. Junctional current is produced by the difference between the electrode solution and the cytoplasmic fluid. This current can be minimized by adjusting the ionic content of the electrode solution and reducing the size of the electrode tip (but this will increase the electrode resistance).
The headstage of Axoclamp contains the "voltage follower" circuit. This voltage follower circuit is the "magic" that makes it possible to record voltage with a sharp mega-ohm electrode. A voltage follower produce virtually infinite input resistance, which makes the electrode resistance almost negligible. Thus, the voltage recorded by Axoclamp is more accurate and relieble than Axopatch.
The current measured under voltage clamp mode in Axoclamp is actually the current generated artificially in the feedback circuit. Such indirect measurement is bad in s/n ratio and slower than Axopatch.
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