In vitro cultured networks can be easily manipulated while maintaining many of the cortical cellular properties
. Therefore, they provide a suitable model to get a first impression of the possible effect of acylated ghrelin in vivo. For proper interpretation of these effects, it is important to mention that the paradigm for the natural establishment of neural circuits is known to proceed in three-stages: 1) early activity-independent wiring to produce a rough map characterized by excessive synaptic connections; 2) subsequent, use-dependent pruning to eliminate inappropriate connections and reinforce the functioning synapses
; and 3) succeeding homeostatic balance with reciprocal influence between the development of neuronal connectivity and intrinsic bioelectrical network activity
At the end of the first phase, when neurons form extensive interconnections, they create functional networks, which exhibit frequent spontaneous action potentials discharges
. The frequency of these bursts of activity usually emerges towards the end of the first week in vitro, and is correlated with the age of the culture
. From then on the activity patterns exhibit periods of elevated firing rates with ongoing repetition of distinctive firing patterns, including network bursts, which evolve gradually in the end of the third week, followed by a drastic shortening of the network bursts after about four weeks of culturing. The burst intensity profiles become quite stable from about 30 days not only in cortical cultures
[56, 57], but also during the early postnatal life, as shown in lightly anesthetized rat neocortex in vivo[58, 59]. The pattern of neuronal activity appears to be strongly dependent upon network interconnectivity
Recently, ghrelin expression was found in dissociated cortical neurons with a clear conditioning- and time-related pattern in the transmitter appearance: very early ghrelin expression at a high level, followed by maturational decrease in the next two weeks of culturing
[27, 28]. This qualitatively mimics the in vivo time course of development of networks, the survival of which requires synapse consolidation and activation during the first two weeks
[61, 62]. In cerebral cortex cultures, the synapse density increases in parallel to the spontaneous activity development from 7 to 21 DIV
. In the initial stages of network development, connectivity is sparse and consequently activity is low. Still, neurons need a certain amount of activity to survive
. The ghrelinergic system might contribute to maintaining a healthy level of activity, possibly through accelerated synaptogenesis. Indeed, our immunostaining clearly illustrates a higher density of synapses in acylated ghrelin treated cultures than in control experiments. Because we used acylated ghrelin, which has been shown to bind only to GHSR-1a
, and most cortical neurons did express GHSR-1a, the synaptogenic effect of ghrelin is most likely mediated by GHSR-1a. Acylated ghrelin increases Ca+2 influx
[44, 66]. E.g. the inositol phosphate signaling pathway, inducing intracellular calcium mobilization
[44, 66, 67] through phospholipase C activation, is specifically associated with the GHSR-1a ligand-dependent activity
[68, 69]. Therefore, we hypothesize that the underlying mechanism for this synaptogenic effect of ghrelin may involve synaptic gene nuclear factors expression, many of which are calcium-dependent
Ghrelin conditioning of the culturing medium resulted in earlier establishment of synaptic contacts and emerging spontaneous neuronal activity as early as 3DIV, which rapidly and continuously increased till 17–18 DIV, followed by a decline till 21–23 DIV. The first activity appeared much earlier in ghrelin treated cultures than in the controls and reached a ”mature” type (i.e. including network bursts) much earlier. In contrast, spontaneous activity of non-treated cultures usually begins toward the end of the first week in vitro and is initially asynchronous
. Thus, chronic ghrelin application had a strong stimulating effect on network activity in developing cultured cells, and to our knowledge, this study demonstrates it for the first time.
Ghrelin probably excites excitatory neurons, as well as inhibitory ones, and therefore, the total activity would increase or decrease, depending on the balance between excitation and inhibition. Approximately 20-25% of the cortical neurons are inhibitory GABAergic
[71, 72], and they usually express a developmental shift in their actions: at young age, GABA depolarizes postsynaptic neurons instead of hyperpolarizing them. This effect takes place during the first 8–9 postnatal days (P) or DIV and does not last beyond day 12
[73, 74]. Ghrelin may directly excite those interneurons, leading to increased network activity in the first two weeks and increased inhibition after the end of the second week
On the other hand, the firing rate of ghr cultures after three weeks in vitro, which is beyond this switch of GABAergic neurons, is still much higher than in ctrl cultures. This suggests that the predominant effect of acylated ghrelin involves the formation of excitatory synapses, thus causing augmented network activity. To validate this, it would be beneficial to determine the effect of ghrelin on postsynaptic density protein 95 (PSD-95) expression, which anchors and organizes postsynaptic neurotransmitter receptors
 and is known to be expressed only at excitatory synapses
[77, 78]. However, this was beyond the scope of the current study.
The decline in firing rate observed in both ghr and ctrl cultures after three weeks, could be ascribed to the phenomenon of synaptic pruning, which is part of the natural formation of neural circuits
, initiated by signaling through NMDA-receptors
. The functional significance of synapse elimination during maturation probably involves adjustment of the excitatory/inhibitory balance on individual neurons and within networks. The main argument in support of this assumption is the specificity of the loss: excitatory synapses are selectively degenerated whereas inhibitory synapses are spared
[80, 81]. This excitatory/inhibitory balance is important for cortical networks to function properly
[82, 83] and the maintenance of this balance requires a form of homeostatic plasticity. In adult cortical networks, it is dynamically regulated to avoid runaway excitation which will bring the network into an epileptic-like state or quiescence, in response to alterations in input strength
[84, 85]. Based on homeostatic considerations, one would expect the higher activity in cultures chronically treated with ghrelin to slow down excitatory synaptogenesis. Still, in acylated ghrelin treated cultures, we found an increased synaptogenesis and elevated firing levels, which suggests that the synaptogenic effect of ghrelin exceeded the homeostatic effect.
The stimulating effect of ghrelin during the initial stages of network development suggests that ghrelin may provide an additional mechanism to maintain healthy activity levels when activity would otherwise be too low, as may occur, for instance, after stroke. In developmental periods of low activity, synapse formation and inter-neuronal communication are both very important for brain development and functioning. Our results suggest that acylated ghrelin may support those processes, at least in our in vitro preparation. This is in agreement with previous studies reporting anti-appoptotic and neuroprotective effects of ghrelin
[19, 86, 87]. It was also supported by our own findings during a difficult culturing period, when most cultures died early. Although most of those cultures became active, the duration of their activity was too short to be included in this study. Still, ghrelin treated cultures survived much longer than control cultures, which were active on average for 8 ± 7 days, while ghrelin treated cultures were active for 18 ± 6 days.
The ghrelin concentrations used in this study are relatively high, compared to physiological levels, which might induce nonspecific effects. However, it has been recently reported that in vitro at 1 μM concentration, 50% of the neurons of area postrema exhibit a response, 48% of the neurons react at 100 nM, 38.7% at 10 nM, and only 20% at 1 nM ghrelin
Though an earlier study showed that concentration did significantly affect synapse densities
, the results from the present experiments indicate no significant effect of different ghrelin concentrations on network activity. It is possible that activity development also depended on concentration, but our sample sizes were not large enough to detect that. However, this study did not focus on a quantitative description of dose dependent activity changes, but aimed to qualitatively describe the effect of acylated ghrelin on network activity.