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a needle. If the dura is not pierced, it will prevent smooth
insertion of the cannula in the brain and skew the injection.
(j) Lower the cannula to touch the brain, and start injection by
infusion mode on the pump (0.5
L/min) while lowering the
cannula slowly (1 mm/min) to the fi nal site of injection. The
Z (depth) coordinate to reach the CA1 area in a 20-22-day-
old rat is 2.6 mm depth (from height at which the cannula
touches the brain). As soon as the cannula is at destination,
change the fl ow rate to 0.1
μ
μ
L/min. Inject at total of 0.5
μ
L
of viral solution per site of injection.
(k) Remove cannula slowly (0.5 mm/min).
(l) If both hemispheres are to be injected, repeat the procedure
from point h to l.
(m) Remove rat from frame and close skin with suture. For post-
operative pain, inject the rat subcutaneously with buprenor-
phine solution (0.03 mg/kg).
In this section, I provide guidelines for preparation of hippocampal
slices and electrophysiological analysis of synaptic function of
infected neurons. A detailed description of this type of electro-
physiology protocol is beyond the scope of this chapter and can be
found elsewhere [ 25 ].
(a) 24 h after in vivo infection, prepare transverse hippocampal
slices (or coronal slices containing the hippocampus) from
brains of infected animals. For slicing, we use a sucrose-based
ice-cold solution containing (in mM): 2.5 KCl, 1.25
NaH 2 PO 4 , 10 MgSO 4 , 0.5 CaCl 2 , 26 NaHCO 3 , 234 Sucrose,
and 11 Glucose (saturated with 95 % O 2 and 5 % CO 2 ). Two-
hundred and fi fty micrometers slices are produced using a
vibratome. Cutting thicker slices will decrease the number of
infected slices you can record from as the viruses do not always
spread very far within the hippocampus.
(b) After dissection, slices are kept in regular oxygenated (satu-
rated with 95 % O2 and 5 % CO2) artifi cial cerebrospinal fl uid
(ACSF) made of (in mM): 119 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 ,
1.3 MgSO 4 , 2.5 CaCl 2 , 26 NaHCO 3 , and 11 Glucose at a
temperature of 36 °C for 1 h, and then at room temperature.
(c) For recording, slices are placed in recording chamber of elec-
trophysiology setup and are submerged in oxygenated ACSF
at a temperature of 30-32 °C. For accurate analysis of
excitatory synaptic function, 100
3.3 Electro-
physiological Analysis
M Picrotoxin should be
added to the ACSF to block GABAergic transmission. Infected
GFP-expressing neurons are visualized by epi-fl uorescence.
(d) Infected neurons and neighbor uninfected neurons are patch
clamped using 4-7 M
μ
glass electrodes fi lled with an patch
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