Hi Bill,
Thank you for your reply. Here are some details for my experimental
condition:
1. *Age of the mice/rat*: I used only mice aged from P17-P23.
2. *Presence of blockers for inhibition*: I didn't use any blocker in my
experiments.
3.* Slice preparation*: Mice were anesthetized by isofluorane inhalation and
decapitated. Brains were removed from the skull and immediately immersed in
chilled dissection buffer containing the following (in mM): 87 NaCl, 2.5
KCl, 1.25 NaH2PO4, 25 NaHCO3, 75 sucrose, 10 dextrose, 1.3 ascorbic acid, 7
MgCl2, and 0.5 CaCl2, bubbled with 95% O2/ 5% CO2. Transverse hippocampal
slices (400 μm thick) were prepared using a Vibratome (Leica VT1200S).
Slices were allowed to recover for 45 min in a 35°C submersion chamber
filled with oxygenated artificial cerebrospinal fluid (ACSF) containing the
following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 15 dextrose,
1.3 MgCl2, and 2.5 CaCl2, and then for an additional 1 hr at room
temperature.
4. *Amplitude of the EPSP/stimulus strength*: The evoked maximum value of
fEPSP amplitude varies from 0.4 mV to 1 mV. I choose the stimulus strength
that evokes fEPSP amplitude between 1/2 and 2/3 of the maximum fEPSP value,
and I am not sure if that is still too low to produce any LTD effect. Some
people seem to use the stimulus intensity that can barely evoke a population
spike as the stimulus strength for the LTD induction....Maybe I should try
that?
5. *Brain region*: I place the recording electrode (1.3 MΩ) about 50 um
away from the end of CA1 and 50 um below the pyramidal cell layer , and the
stimulating electrode (*concentric bipolar electrode*) 500 um away from the
recording electrode (and closer to the boundary of CA1-CA3).
6.* Input pathway* : I am stimulating Schaffer Collateral inputs.
7. *Temperature during recording*: It is always around room temperature,
22-25 C.
Thank you again for your time. I 'd very much appreciate any of your
suggestions!
Amy
>>
> Message: 1
> Date: Mon, 15 Aug 2011 08:14:33 -0700 (PDT)
> From: Bill <connelly.bill from gmail.com>
> Subject: [Neuroscience] Re: A question about LTD protocol
> To: neur-sci from net.bio.net> Message-ID:
> <17b1b750-df09-4e72-b3d5-879346bf241c from a10g2000yqn.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>> Hi Amy,
>> Can I have a bit more details?
>> How do you cut the slices?
>> What kind of stimulating electrode do you use?
>> How do you choose the intensity of your stimulus?
>> On Aug 13, 2:06 am, Min-Yu Sun <msun... from student.ucr.edu> wrote:
> > Hi,
> >
> > I meet a problem for using the LTD protocol to induce LTD. I am using a
> > PP-LFS protocol (1Hz, 900 paired-pulses; pulse interval:50 ms, pulse
> > duration: 0.1 ms) to induce LTD on acute hippocampal slices from
> wild-type
> > mice (aged from P17-P23). My amplifier is Axopatch 200B, and the software
> I
> > use is pClamp 10.2. All the experiments are done in room temperature
> (22-25
> > C). The problem is, I can't induce LTD reliably. Sometimes I can induce
> LTD
> > for about 15-20% at 45-60 min after PP-LFS (rise slope of fEPSP is 80-85%
> > compared to baseline level), but sometimes I can't induce LTD at all even
> if
> > I use exactly the same protocol. The rise slope of fEPSP went back to
> around
> > 95-100% (compared to baseline) at 30 min after PP-LFS. I was just
> wondering
> > if there is any experimental condition I might not pay attention to,
> which
> > could extremely affect LTD induction. Does anyone have any thought about
> > this? I 'd very much appreciate for any possible suggestions!
> >
> > Thanks a lot!
> >
> > Amy (Min-Yu)
>>>> ------------------------------
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