Human iPSC-Derived Neurons with Reliable Synapses and Large Presynaptic Action Potentials.

Understanding the function of the human brain requires determining basic properties of synaptic transmission in human neurons. One of the most fundamental parameters controlling neurotransmitter release is the presynaptic action potential, but its amplitude and duration remain controversial. Presynaptic action potentials have so far been measured with high temporal resolution only in a limited number of vertebrate but not in human neurons. To uncover properties of human presynaptic action potentials, we exploited recently developed tools to generate human glutamatergic neurons by transient expression of Neurogenin 2 (Ngn2) in pluripotent stem cells. During maturation for 3 to 9 weeks of culturing in different established media, the proportion of cells with multiple axon initial segments decreased, while the amount of axonal tau protein and neuronal excitability increased. Super-resolution microscopy revealed the alignment of the pre- and postsynaptic proteins, Bassoon and Homer. Synaptic transmission was surprisingly reliable at frequencies of 20, 50, and 100 Hz. The synchronicity of synaptic transmission during high-frequency transmission increased during 9 weeks of neuronal maturation. To analyze the mechanisms of synchronous high-frequency glutamate release, we developed direct presynaptic patch-clamp recordings from human neurons. The presynaptic action potentials had large overshoots to ∼25 mV and short durations of ∼0.5 ms. Our findings show that Ngn2-induced neurons represent an elegant model system allowing for functional, structural, and molecular analyses of glutamatergic synaptic transmission with high spatiotemporal resolution in human neurons. Furthermore, our data predict that glutamatergic transmission is mediated by large and rapid presynaptic action potentials in the human brain.

Phosphatidylinositol 3,5-bisphosphate facilitates axonal vesicle transport and presynapse assembly.

Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P2 and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.

What calcium channels remember.

A new mechanism involving intermediate gating states of calcium channels explains how analogue postsynaptic potentials influence neurotransmitter release.

Direct whole-cell patch-clamp recordings from small boutons in rodent primary neocortical neuron cultures.

Direct electrical recordings from conventional boutons in the mammalian central nervous system have proven challenging due to their small size. Here, we provide a protocol for direct whole-cell patch-clamp recordings from small presynaptic boutons of primary dissociated cultured neurons of the rodent neocortex. We describe steps to prepare primary neocortical cultures and recording pipettes, followed by identifying boutons and establishing a whole-cell bouton recording. We then provide details on precise pipette capacitance compensation required for high-resolution current-clamp recordings from boutons. For further details on the use and execution of this protocol, please refer to Ritzau-Jost et al.1.

Somatostatin Interneurons Promote Neuronal Synchrony in the Neonatal Hippocampus.

Synchronized activity is a universal characteristic of immature neural circuits that is essential for their developmental refinement and strongly depends on GABAergic neurotransmission. A major subpopulation of GABA-releasing interneurons (INs) expresses somatostatin (SOM) and proved critical for rhythm generation in adulthood. Here, we report a mechanism whereby SOM INs promote neuronal synchrony in the neonatal CA1 region. Combining imaging and electrophysiological approaches, we demonstrate that SOM INs and pyramidal cells (PCs) coactivate during spontaneous activity. Bidirectional optogenetic manipulations reveal excitatory GABAergic outputs to PCs that evoke correlated network events in an NKCC1-dependent manner and contribute to spontaneous synchrony. Using a dynamic systems modeling approach, we show that SOM INs affect network dynamics through a modulation of network instability and amplification threshold. Our study identifies a network function of SOM INs with implications for the activity-dependent construction of developing brain circuits.