Motivated by the work of Potter, we first reasoned that neuron networks that develop in vitro from embryonic neurons were deprived of their natural stimuli during development. We hypothesized that chronic stimulation would train the network for more spontaneous communication within such an in vitro hippocampal system. The results clearly show an increase in spike frequency within bursts that depended on the duration of chronic stimulation over the course of culture time, as well as a 50% increase in active electrodes with chronic stimulation. The bursts per minute also increased with chronic stimulation, although not monotonically.
Most paradoxical in our results is the increase in several measures with 1 hr/day of stimulation and a reduction at 3 hr/day. It is tempting to compare these results to findings in the literature for denser, unstimulated cultures in which bursting activity gradually develops, firing rates increase, and intense superbursts occur (Wagenaar et al., 2006
). By following burst dynamics over 6 weeks of development, van Pelt et al. (2004)
found the temporal onset and offset of the bursts first broadened to a peak at 3 weeks, then sharpened with shorter overall lengths. These may be analogous to the changes evident in and in the statistics in . The argument is that the stimulation paradigms have accelerated the natural phases of increasing then decreasing and sharpened bursts, with higher spike rates within the burst. Still, it is only speculation that our stimulation paradigms, chosen for convenience, may have accentuated the cultures’ development.
We hypothesized a simple concept of greater induction or strengthening of spontaneous activity from stimuli delivered adjacent to the recording electrode. Instead, we observed no simple relationship between chronic stimulation for 0, 1 and 3 hr/day and spike rate on the scale of minutes. However, examination of shows clearly a direct relationship between chronic stimulation and spike frequency within a burst. These somewhat discrepant results are reconciled by observing that show a non-linear relationship of burst duration with chronic stimulation. These results indicate that chronic stimulation increases spike rate within a burst, but not necessarily overall burst rate. Higher spike rate within bursts may indicate stronger information transfer evoked by chronic stimulation.
The chronic stimulation protocol and measures of responses certainly have limitations. We did not optimize the stimulation train, or the number of hours/day or days of chronic stimulation; these parameters were chosen for convenience. We also wanted to avoid over-stimulation and neuron death. It is likely that the spike frequency within bursts has not reached the maximum with 3 hr/day stimulation. The percent active electrodes could surely be increased by increasing the density of plated neurons or by the addition of extra astroglia (Boehler et al., 2007
). Although we stimulated with a paired-pulse stimulus, other stimuli at higher frequency are likely to affect the burst characteristics (Wagenaar et al., 2005
In the absence of external stimulation, cultured networks develop spontaneous activity in vitro (Thomas et al., 1972
; Gross, 1979
; Pine, 1980
). But the nature of this activity may be different from in vivo. In vitro networks have a smaller than expected proportion of excitatory connections (Vogt et al., 2005
) and a high fraction of inhibitory GABAergic synapses (Liu et al., 2000
; Robain et al., 1987
; Rozenberg et al., 1989
). The decline of these GABAergic synapses and the rise of glutamatergic transmission in vivo may require the external stimulation that neurons receive in the brain. Conversely, synaptic development and transmission is compromised if external stimulation is blocked during development in vivo (Turrigiano and Nelson, 2004
). Therefore, the synaptic scaling of excitatory and inhibitory receptors is likely to be different in neuronal networks in vitro with or without external stimuli during development. In comparison to spike rate measures also made at 3 weeks in rat neurons, but in cortical neurons in Neurobasal/B27, Chiappalone et al. (2006)
found a spontaneous spike rate of 2.2 Hz that compares well with our unstimulated rate of 2.6 Hz. Their work and ours both found a burst rate of 5 bursts/min. However, their threshold for spike detection of 7× S.D. was much less than ours of 11.
Finally, these results raise interesting questions about the nature of brain network communication and bursts. Since burst activity represents 90% of the total activity that we observe under all conditions, can we reasonably conclude that information coding occurs in bursts and less so in individual or smaller groups of action potentials? Others have observed these high levels of bursting activity and used short term stimulation to modulate patterns of activity (Maeda et al., 1995
; Maeda et al., 1998
; Stegenga et al., 2008). Certainly bursting activity occurs during development in vivo (Kim and McCormick, 1998
; Leinekugel et al., 2002
) and is different from epileptiform activity. Our finding that the frequency within a burst of spontaneous activity increases with prior chronic stimulation suggests that the prior stimulation increased the synaptic drive of the network in order to produce faster firing rates or lowered inhibitory connections. To establish this conjecture, we will need to compare synaptic densities, resting membrane potentials and inhibitory to excitatory inputs in the unstimulated and chronically stimulated networks. Also, a resting membrane potential that is more depolarized with chronic stimulation (closer to threshold) could explain higher spontaneous spike rates within bursts from a higher ratio of excitatory to inhibitory inputs (Li et al., 1998
; Kim and McCormick, 1998
In conclusion, chronic stimulation of planar hippocampal networks on MEAs during the course of culture results in more spontaneous activity than unstimulated cultures, with higher spike rates within bursts. Thus, chronic stimulation appears to change the dynamics of the network so that proximity to stimulus is no longer the most important determinant of spike rate.