Nanoscale single-cell interfaces allow optical activation of single neurons and sensory-motor modulation in organotypic slices

Nano-technology based tools are allowing neuroscientists to precisely and selectively stimulate and investigate neuronal circuits, with unprecedented accuracy. However, the existing neuron-stimulating interfaces engineered at the nano-scale level are usually facing compromises between being less-invasive or yielding a higher level of specificity. To provide single-cell stimulation, current nano-electronic devices are precise and powerful but exert stressful harm over the targeted neuronal membrane/tissues. On the other hand, most of the optical driven stimulating tool are less invasive, but lack in high spatial resolution, hardly enabling the stimulation of one sole neuron when embedded in a complex three-dimensional tissue. More recent bio-modified nano-structured devices displaying increased bio compatibility, may deliver highly localized stimulations. Nevertheless, the applicability of such tools in physiological environments is limited due to their nature and physical features, limiting as well their translational applications. We were able to deliver single-cell, not invasive, optical stimulation limited to individual and identified neurons, by exploiting TAT-conjugated Silicon-based nano-photodiodes (TAT nPDs) within organotypic spinal cord cultures’ (OSCs) functional micro-circuits. We employed nPDs-mediated stimulation to: • Investigate the use of nPDs as single-cell interface, to optically stimulate individual neurons, exploiting nPDs design via chemical functionalisations • Explore individual neuronal contributions to sensory and pre-motor circuits dynamics and their reciprocal impact • Explore the diverse astrocytic activity patterns in sensory and pre-motor areas of the spinal cord, also in respect to synaptic network changes We showed that near infrared light (NIR) stimulation of single nPD selectively activated individual identified neurons in confined spinal areas of OSCs, tuning circuit-outputs changes. Being the distance between nPD and the cell membrane a limiting factor in the stimulation efficacy, we linked to nPD surface a peptide derived from the viral transactivator of transcription (TAT) of human immunodeficiency virus, to increase nPD adhesion and vicinity to the neuronal membrane. With such an approach we improved the device 3 stimulation efficacy when comparing to our first report (Thalhammer et al., 2022) by employing TAT-nPDs instead of naïve (i.e. not functionalised) nPDs. For example, when TAT-nPD NIR light-mediated stimulation was directed over a single neuron located in superficial layers of the dorsal horn (DH), we induced larger sensory circuit (wind-up) potentiation. We then employed TAT-nPDs to stimulate single pre-motor interneuron in the ventral horn (VH), improving synchronisation of pre-motor outputs, with no increase in neuronal network activity frequency. Hence, neuronal network outputs are specifically modulated by single neuron activation in the dorsal and ventral microcircuits. With nPDs light-stimulation we investigated how the functional connectivity between ventral and dorsal areas in OSCs, translate sensory and motor changes in the adjacent dorsal or ventral areas, in a sort of in-vitro spinal microcircuit sensory-motor integration. The induction of DH sensory enhancement (wind-up) was reflected onto VH as an increase in network synchronization. On the other hand, induction of VH neuronal synchronization via direct VH TAT nPDs-stimulation, induced a mild potentiation of DH sensory circuits probably due to a back-propagating increase in the excitation of the system, entraining unspecifically the entire DH neuronal pathways. We then explored GFAP-positive astrocytes calcium signalling when evoking the different neuronal activity patterns in dorsal and ventral areas. Astrocytes parallel the increase in neuronal synchronization achieved with nPDs-stimulation in VH with an increase in astrocytic calcium transient frequency rate, while in DH neuronal wind-up is not followed by enhanced astrocyte calcium activity. Spinal astrocytes appear to be tuned by the increase in neuronal network synchronization, provided that their basal calcium dynamics be below a certain frequency threshold. In addition, astrocyte CX43-gap junction integrity is needed to regulate basal astrocyte calcium activity, namely to keep it higher in DH or lower in VH. In addition, intact astrocyte syncytium appeared to contribute to the induction of dorsal sensory neuronal plasticity (wind-up).