Metabolic Stability of Hippocampal Slice Preparations During Prolonged Incubation

Abstract

Hippocampal slices were prepared under three conditions: (1) in medium containing glucose and oxygen at 4 degrees C; (2) as in (1), but at 37 degrees C; (3) in medium devoid of glucose and oxygen at 37 degrees C. The rates of recovery to roughly steady-state levels and through 8 h of incubation were monitored for energy metabolite levels and related parameters. In vitro stable values are compared with in situ hippocampal levels. Regardless of the conditions under which slices were prepared, metabolite levels required up to 3 h to stabilize, and these levels were maintained or improved through 8 h of incubation. Further, the maximal concentrations of metabolites were independent of the conditions of slice preparation. Total adenylates and total creatine levels reached 55% of those in vivo. Lactate decreased from the decapitation-induced high levels, but stabilized at concentrations about twice those in rapidly frozen brain. Cyclic AMP and cyclic GMP exhibited peak levels at 30 min of incubation, and cyclic GMP remained elevated for 3 h. Although all three methods of slice preparation resulted in similar metabolite profiles on incubation, the initial decreases in high energy phosphates were delayed by chilling. Most striking, the slices prepared in the absence of glucose and oxygen exhibited much smaller orthodromic evoked potentials in the dentate gyrus. The presence of glucose and oxygen during preparation of the slices appears to be critical to the electrophysiological response of the tissue.

N-methyl-D-aspartate receptor activation increases cAMP levels and voltage-gated Ca2+ channel activity in area CA1 of hippocampus.

“…Slices were allowed to adjust to the recording chamber for 1.5-2 hr to allow cAMP levels to stabilize (15). Slices were removed from the chamber and rapidly frozen either after the test pulses (control) or 1 min after tetanic stimulation (tetanus).…”

Intracellular ATP Influences Synaptic Plasticity in Area CA1 of Rat Hippocampus via Metabolism to Adenosine and Activity-Dependent Activation of Adenosine A1Receptors

“…However, it is worth noting that other metabolites, such as cGMP and cAMP, lactate, or phosphocreatine, also require 1-3 h to achieve a steady state (Whittingham et al, 1984b). Likewise, the phosphorylation status of proteins involved in synaptic plasticity, such as GluA1, ERK2, and MEK1/2, changes during the first 3 h of incubation (Ho et al, 2004), whereas a recovery period of 4 h has been suggested for achieving stable long-term recordings of LTP in brain slices (Sajikumar and Real-time measurement of adenosine release during LTP induction reveals significantly higher adenosine release in slices treated for 2 h in 1 mM Rib and 50 M Ade.…”

“…Indeed, a substantially compromised energetic state of brain slices at the time of cutting has been demonstrated (Fredholm et al, 1984;Whittingham et al, 1984b), with high energy phosphate levels (ATP, phosphocreatine) in brain slices being as much as 50% lower than their in situ values (Thomas, 1957;Whittingham et al, 1984a;Schurr and Rigor, 1989). Accordingly, basal conditions in hippocampal slices have been described as reflecting a post-ischemic recovery state (Hossmann, 2008).…”

Timing of neuronal and glial ultrastructure disruption during brain slice preparation and recovery in vitro

“…Slices can exhibit increased numbers of synapses (Wenzel et al, 1994;Johnson and Ouimet, 2002) and a complete loss of dendritic microtubules (Burgoyne et al, 1982). In addition to these persistent changes, a transient drop in glycogen and ATP occurs during the initial stages of incubation (McIlwain and Tresize, 1956;Whittingham et al, 1984;Lipton, 1988;Feig and Lipton, 1990). This is accompanied by a loss of synaptic transmission that gradually recovers during the first hour in vitro (Schurr et al, 1984;.…”