1) Cold slice preparation appears to be the cause of the excess spinogenesis. See the following publications that track this phenomenon and how we resolved it:
- Kirov SA, Harris KM (1999) Dendrites are more spiny on mature hippocampal neurons when synapses are inactivated. Nature Neuroscience 2(10):878-883.
- Kirov SA, Sorra KE, Harris KM (1999) Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats. J Neuroscience 19(8):2876-2886.
- Fiala JC, Kirov SA, Feinberg MD, Petrak LJ, Goddard CA, George P and Harris KM (2003) Timing of Neuronal and Glial Ultrastructure Disruption During Brain Slice Preparation and Recovery During Incubation In Vitro. J Comp Neurol. 465(1):90-103.
- Kirov SA, Petrak LJ, Fiala JC, Harris KM (2004) Dendritic spines disappear with chilling but proliferate excessively upon rewarming of mature hippocampus. Neuroscience 127(1):69-80. (Fig. 9 is especially revealing.)
- Bourne JN, Kirov SA, Sorra KE, and Harris KM (2007) Warmer preparation of hippocampal slices prevents synapse proliferation that might obscure LTP-related structural plasticity. Neuropharmacol 52:55-59. (See Fig. 2.)
2) Using warm slice preparations with fast chopping (<5 minutes from decapitation until slice in recovery chamber) then need 3 hours of slice recovery plus >1 hour of test pulses to return spine density to perfusion-fixed levels:
- Bourne JN, Harris KM (2011) Coordination of size and number of excitatory and inhibitory synapses results in a balanced structural plasticity along mature hippocampal CA1 dendrites during LTP. Hippocampus, 21(4):354-73. (Figures 5D, E control - replotted for clarity in Bell et al., listed next.)
- Bell ME, Bourne JN, Chirillo MA, Mendenhall JM, Kuwajima M, Harris KM (2014) Dynamics of nascent and active zone ultrastructure as synapses enlarge during long-term potentiation in mature hippocampus. J Comp Neurol, 522(17):3861-84. (See Fig. 6.)