Here we have applied, for the first time, optogenetic methods in combination with patch-clamp recordings to analyze the functional integration of stem cell-derived DA neurons grafted into an in vitro PD model. We demonstrate complex bidirectional functional interaction between grafted DA neurons and host cells and extensive intragraft excitatory synaptic connectivity.
Our findings indicate that the organotypic hemisphere cultures used here, having relatively preserved cortico-striatal connections but severed DA input to the striatum, represent a suitable model for preliminary screening of the functional properties of stem cell-derived DA neurons and their integration into host neural circuitries. Most hallmarks of mesencephalic DA neurons in situ
were detected in the stem cell-derived DA cells, including presynaptic D2
. However, the characteristic delayed inward rectifying “sag” was not observed in any of the grafted DA neurons 
. Since grafted stem cell-derived DA neurons lacking “sag” have been shown to ameliorate functional deficits in 6-OHDA lesioned animals 
, the significance of the delayed inward rectifier currents for the therapeutic action of grafted DAergic neurons is unclear. We also found, using perforated patch-clamp recordings in combination with puff application of GABA, that the reversal potential for GABAA
receptor-mediated chloride currents in the DA neurons was near −40 mV. Such depolarized reversal potential of GABAA
receptor-mediated currents seems to be characteristic of endogenous DA neurons 
, but may also indicate partial immaturity 
. To our knowledge, this is the first time that the reversal potential of GABAergic currents has been explored in grafted DA neurons.
Observations in PD patients subjected to intrastriatal transplantation of fetal VM tissue indicate that the grafted DA neurons become functionally integrated into host neural circuitries 
, a process which parallels the time course of clinical improvement. However, due to their ectopic location, intrastriatally grafted DA neurons do not receive “correct” synaptic inputs 
. Exploring the functional synaptic interactions between grafted stem cell-derived DA cells and host neurons in experimental models is not trivial and has been hindered due to technical limitations. Kim et al. 
performed paired electrophysiological recordings from presumed striatal host cells and grafted mouse ES-derived DA neurons. No direct synaptic interactions between these pairs were found 
. Electrical field stimulations within the graft area induced EPSCs in both graft and presumed host neurons 
. The host identity of neurons was inferred solely by their distance from the graft site. In acute slices, field stimulations presumably outside the transplant have been shown to induce both glutamatergic and GABAergic postsynaptic currents in grafted fetal DA neurons 
, as well as in grafted neural stem cell-derived neurons 
. When measuring extracellularly from presumed grafted fetal mesencephalic neurons in vivo
, field stimulations in both the striatum and cortex evoked increased action potential firing in some neurons, while in the majority of grafted neurons such stimulations decreased the action potential firing rate, both suggesting host-to-graft connectivity 
. However, in none of the above studies the source of synaptic inputs to the graft neurons was unequivocally verified to be host-derived.
We demonstrate here that the optogenetic approach makes possible completely selective activation and inhibition of either host or grafted neurons in an unprecedented manner. In our experiments, action potentials induced by light in the ChR2-transduced host striatal neurons did not reveal any direct synaptic connections to the grafted DA cells. Interestingly, silencing of the host striatal, NpHR-transduced neurons by selective light activation increased excitatory synaptic activity in the grafted cells. One possible explanation to this observation is that the grafted neurons receive direct cortical excitatory inputs, which are presynaptically tonically suppressed by inhibitory striatal neuron collaterals, similar to what has been described for cortical innervation of host striatal neurons in vivo 
. Selective silencing of these striatal neurons by optogenetic approach results in a disinhibitory effect, revealed as increased excitatory cortical input to the grafted cells. Optogenetic activation of grafted DA and other neurons resulted in appearance of GABAA
receptor-mediated slow IPSCs only in one out of six recorded host neurons. This connection was most likely polysynaptic since application of glutamatergic receptor antagonists blocked the response. Similar optogenetic activation of grafted neurons always induced increased sEPSC frequency in grafted DA neurons, indicating extensive intragraft excitatory connections.
Taken together, our data indicate that in the in vitro
PD model, there is extensive synaptic interaction between DA neurons and other cell types within the graft and that connections between host and graft are complex, including host striatal presynaptic regulation of host cortical afferents to the grafted DA neurons and polysynaptic graft-to-host connections. Further optogenetic dissection of the functional synaptic interactions between grafted and host neurons will be instrumental in delineating the cellular and synaptic mechanisms underlying behavioral recovery, as well as those which may give rise to adverse effects 
following DA cell transplantation in patients with Parkinson's disease.