Rat CSF: Measured Concentrations

Rat CSF: Measured Concentrations

Rat CSF: measured concentrations

156 mM Na+2.2 mM Ca2+

126 mM Cl-2.8 mM lactate

2.8 mM K+pH 7.35

3.6 mM Glucose302 mOsmol

Extracellular solutions:

•Glucose (10 – 26 mM): difficult to obtain healthy acute slices with physiological concentrations

•Antioxidants (sodium ascorbate – 0.4 – 1.0 mM): reduce oxidative damage to mitochondria and membrane – unclear if ATP crosses the membrane

•ATP (pyruvate (2 mM), phosphocreatine, creatine phosphokinase, ATP): add to offset rapid depletion of ATP that occurs during slice preparation

•Li – can be exchanged for Na+, permeant through NMDA receptors but does not support Na+-Ca2+ exchanger

•MnCl2 – can be added in exchange for Ca to block Ca channels

•Osmolarity and Charge Balance: can substitute NaCl with choline chloride or sucrose to maintain osmolarity and charge

[D.O. – X] * [0.342] * V = g of sucrose to add

where:

D.O. = Desired Osmolarity

X = Osmolarity of Solution

V = Volume in liters

g = grams

Patch Pipette Solutions: – dilute cytosolic contents, ~270-300 mOsmol; slightly hypo-osmotic is better

•CsMeSO3, Cs gluconate, CsF, Cs acetate, CsCl – Cs will block most K+ channels, including SK channels; usually used to amplify EPSCs/IPSCs

•F- will interfere with G-protein coupled responses

•QX-314/Lidocaine – blocks Na+ currents (and blocks GABAB receptors)

•GTP – conserves G-protein mediated responses

•EGTA – (good for calcium, shows selectivity versus other divalents) used to prepare precise [Ca2+]i, BAPTA faster chelator so it is used test for Ca++ dependent processes (4-20 mM usual; harder to get good seal; need to backfill)

•K-gluconate, KCl, K-methylsulfate, K aspartate – common forms of K+ (can cause large liquid junction potentials)

•K-gluconate shows little run-down or changes in spontaneous activity for up to 5 hr, blocks Ca2+ activated K+ channels (See Velumian et al., 1997, Pflugers Archiv v433, p343)

•K-methylsulfate – good for studies on Ca2+-activated K+ currents

•Rundown caused by changes in [Ca2+]i and phosphates that affect voltage- and ligand-gated channels – ATP Regenerative Solution – 4 mM K2ATP, 20 mM K2-creatine phosphate, 50 U/ml phosphocreatine kinase, 6 mM MgCl2

•N-methylglucamine – impermeant cation that can substitute for K+ to eliminate voltage-dependent K+ currents

•Mg, HEPES – common, usually 1-2 mM, 5-10 mM, respectively

•Protease inhibitors – leupeptin (100 M) or calpain inhibitor 1 (50 M); not that common

•Backfilling – utilized when reagents of high molecular weight; calcium chelators (or proteins/enzymes/antibiotics) are in the pipette – allows for gigaohm seal and then diffusion of other substances

•Labeling – back-filled 0.5% biocytin or Alexa dyes (eg. 488 nm, 594 nm) for visualizing processes

•MK-801-NMDA channel blocker-add to internal to block NMDA receptors in patched cell (1-2 mM)

•Filter internal through a 0.2 m filter; keep frozen until use, one use per vial

References:

  1. Patch-Clamp Analysis:Advanced Techniques. Walz, Boulton, and Baker (Eds.). Humana Press.
  2. Kay, A.R. (1992) An intracellular medium formulary. J Neurosci Methods. 44(2-3), pp 91-100.