Characterization hiPSC-derived neural progenitor cells and neurons to investigate the role of NOS1AP isoforms in human neuron dendritogenesis.

Abnormal dendritic arbor development has been implicated in a number of neurodevelopmental disorders, such as autism and Rett syndrome, and the neuropsychiatric disorder schizophrenia. Postmortem brain samples from subjects with schizophrenia show elevated levels of NOS1AP in the dorsolateral prefrontal cortex, a region of the brain associated with cognitive function. We previously reported that the long isoform of NOS1AP (NOS1AP-L), but not the short isoform (NOS1AP-S), negatively regulates dendrite branching in rat hippocampal neurons. To investigate the role that NOS1AP isoforms play in human dendritic arbor development, we adapted methods to generate human neural progenitor cells and neurons using induced pluripotent stem cell (iPSC) technology. We found that increased protein levels of either NOS1AP-L or NOS1AP-S decrease dendrite branching in human neurons at the developmental time point when primary and secondary branching actively occurs. Next, we tested whether pharmacological agents can decrease the expression of NOS1AP isoforms. Treatment of human iPSC-derived neurons with d-serine, but not clozapine, haloperidol, fluphenazine, or GLYX-13, results in a reduction in endogenous NOS1AP-L, but not NOS1AP-S, protein expression; however, d-serine treatment does not reverse decreases in dendrite number mediated by overexpression of NOS1AP isoforms. In summary, we demonstrate how an in vitro model of human neuronal development can help in understanding the etiology of schizophrenia and can also be used as a platform to screen drugs for patients.

Neurotransmitter Release Can Be Stabilized by a Mechanism That Prevents Voltage Changes Near the End of Action Potentials from Affecting Calcium Currents.

At chemical synapses, presynaptic action potentials (APs) activate voltage-gated calcium channels, allowing calcium to enter and trigger neurotransmitter release. The duration, peak amplitude, and shape of the AP falling phase alter calcium entry, which can affect neurotransmitter release significantly. In many neurons, APs do not immediately return to the resting potential, but instead exhibit a period of depolarization or hyperpolarization referred to as an afterpotential. We hypothesized that presynaptic afterpotentials should alter neurotransmitter release by affecting the electrical driving force for calcium entry and calcium channel gating. In support of this, presynaptic calcium entry is affected by afterpotentials after standard instant voltage jumps. Here, we used the mouse calyx of Held synapse, which allows simultaneous presynaptic and postsynaptic patch-clamp recording, to show that the postsynaptic response is affected significantly by presynaptic afterpotentials after voltage jumps. We therefore tested the effects of presynaptic afterpotentials using simultaneous presynaptic and postsynaptic recordings and AP waveforms or real APs. Surprisingly, presynaptic afterpotentials after AP stimuli did not alter calcium channel responses or neurotransmitter release appreciably. We show that the AP repolarization time course causes afterpotential-induced changes in calcium driving force and changes in calcium channel gating to effectively cancel each other out. This mechanism, in which electrical driving force is balanced by channel gating, prevents changes in calcium influx from occurring at the end of the AP and therefore acts to stabilize synaptic transmission. In addition, this mechanism can act to stabilize neurotransmitter release when the presynaptic resting potential changes. SIGNIFICANCE STATEMENT: The shape of presynaptic action potentials (APs), particularly the falling phase, affects calcium entry and small changes in calcium influx can produce large changes in postsynaptic responses. We hypothesized that afterpotentials, which often follow APs, affect calcium entry and neurotransmitter release. We tested this in calyx of Held nerve terminals, which allow simultaneous recording of presynaptic calcium currents and postsynaptic responses. Surprisingly, presynaptic afterpotentials did not alter calcium current or neurotransmitter release. We show that the AP falling phase causes afterpotential-induced changes in electrical driving force and calcium channel gating to cancel each other out. This mechanism regulates calcium entry at the end of APs and therefore stabilizes synaptic transmission. This also stabilizes responses when the presynaptic resting potential changes.

Presynaptic Calcium Channel Open Probability and Changes in Calcium Influx Throughout the Action Potential Determined Using AP-Waveforms.

Action potentials arriving at a nerve terminal activate voltage-gated calcium channels and set the electrical driving force for calcium entry which affects the amount and duration of neurotransmitter release. During propagation, the duration, amplitude, and shape of action potentials often changes. This affects calcium entry, and can cause large changes in neurotransmitter release. Here, we have used a series of amplitude and area matched stimuli to examine how the shape and amplitude of a stimulus affect calcium influx at a presynaptic nerve terminal in the mammalian brain. We identify fundamental differences in calcium entry following calcium channel activation by a standard voltage jump vs. an action potential-like stimulation. We also tested a series of action potential-like stimuli with the same amplitude, duration, and stimulus area, but differing in their rise and decay times. We find that a stimulus that matches the rise and decay times of a physiological action potential produces a calcium channel response that is optimized over a range of peak amplitudes. Next, we determined the relative number of calcium channels that are active at different times during an action potential, which is important in the context of how local calcium domains trigger neurotransmitter release. We find the depolarizing phase of an AP-like stimulus only opens ~20% of the maximum number of calcium channels that can be activated. Channels continue to activate during the falling phase of the action potential, with peak calcium channel activation occurring near 0 mV. Although less than 25% of calcium channels are active at the end of the action potential, these calcium channels will generate a larger local calcium concentration that will increase the release probability for nearby vesicles. Determining the change in open probability of presynaptic calcium channels, and taking into account how local calcium concentration also changes throughout the action potential are both necessary to fully understand how the action potential triggers neurotransmitter release.

Opposing Influence of Sensory and Motor Cortical Input on Striatal Circuitry and Choice Behavior.

The striatum is the main input nucleus of the basal ganglia and is a key site of sensorimotor integration. While the striatum receives extensive excitatory afferents from the cerebral cortex, the influence of different cortical areas on striatal circuitry and behavior is unknown. Here, we find that corticostriatal inputs from whisker-related primary somatosensory (S1) and motor (M1) cortex differentially innervate projection neurons and interneurons in the dorsal striatum and exert opposing effects on sensory-guided behavior. Optogenetic stimulation of S1-corticostriatal afferents in ex vivo recordings produced larger postsynaptic potentials in striatal parvalbumin (PV)-expressing interneurons than D1- or D2-expressing spiny projection neurons (SPNs), an effect not observed for M1-corticostriatal afferents. Critically, in vivo optogenetic stimulation of S1-corticostriatal afferents produced task-specific behavioral inhibition, which was bidirectionally modulated by striatal PV interneurons. Optogenetic stimulation of M1 afferents produced the opposite behavioral effect. Thus, our results suggest opposing roles for sensory and motor cortex in behavioral choice via distinct influences on striatal circuitry.

The origin of quantal size variation: vesicular glutamate concentration plays a significant role.

Fusion of a single vesicle induces a quantal response, which is critical in determining synaptic strength. Quantal size varies at most synapses. Its underlying mechanisms are not well understood. Here, we examined five sources of variation: vesicular glutamate concentration ([Glu]v), vesicle volume, ultrafast fusion pore closure, the postsynaptic receptor, and the location between release and the postsynaptic receptor cluster at glutamatergic, calyx of Held synapses. By averaging 2.66 million fusion events from 459 synapses, we resolved the capacitance jump evoked by single vesicle fusion. This capacitance jump, an indicator of vesicle volume, was independent of the amplitude of the miniature EPSC (mEPSC) recorded simultaneously at the same synapses. Thus, vesicle volume is not the main source of mEPSC variation. The capacitance jump was not followed by submillisecond endocytosis, excluding ultrafast endocytosis as a source of variation. Larger mEPSCs were increased to a lesser extent by presynaptic glutamate dialysis, and reduced to a lesser extent by gamma-DGG (gamma-D-glutamylglycine), a competitive AMPA receptor blocker, suggesting that a higher glutamate concentration in the synaptic cleft contributes to the large size of mEPSCs. Larger mEPSCs were not accompanied by briefer rise times, inconsistent with the prediction by, and thus arguing against, the scenario that larger mEPSCs are caused by a shorter distance between the release site and the postsynaptic receptor cluster. In summary, the different amplitudes of mEPSCs were mainly attributable to release of vesicles having similar volumes, but different glutamate amounts, suggesting that [Glu]v is a main source of quantal size variation.

Active presynaptic ribosomes in the mammalian brain, and altered transmitter release after protein synthesis inhibition.

Presynaptic neuronal activity requires the localization of thousands of proteins that are typically synthesized in the soma and transported to nerve terminals. Local translation for some dendritic proteins occurs, but local translation in mammalian presynaptic nerve terminals is difficult to demonstrate. Here, we show an essential ribosomal component, 5.8S rRNA, at a glutamatergic nerve terminal in the mammalian brain. We also show active translation in nerve terminals, in situ, in brain slices demonstrating ongoing presynaptic protein synthesis in the mammalian brain. Shortly after inhibiting translation, the presynaptic terminal exhibits increased spontaneous release, an increased paired pulse ratio, an increased vesicle replenishment rate during stimulation trains, and a reduced initial probability of release. The rise and decay rates of postsynaptic responses were not affected. We conclude that ongoing protein synthesis can limit excessive vesicle release which reduces the vesicle replenishment rate, thus conserving the energy required for maintaining synaptic transmission.

Generation and transplantation of reprogrammed human neurons in the brain using 3D microtopographic scaffolds.

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries, but reprogrammed neurons are dissociated and spatially disorganized during transplantation, rendering poor cell survival, functionality and engraftment in vivo. Here, we present the design of three-dimensional (3D) microtopographic scaffolds, using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming, neural network establishment and support neuronal engraftment into the brain. Scaffold-supported, reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices, showing an ∼ 3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼ 38-fold at the injection site relative to injected isolated cells, and allowed delivery of multiple neuronal subtypes. Thus, 3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance.

Small voltage changes at nerve terminals travel up axons to affect action potential initiation.

Nerve terminals are generally considered to be the destination points for electrical signals, which propagate unidirectionally from the soma to nerve terminals. We found that small hyperpolarizations or depolarizations ( approximately 10 mV) generated under physiological conditions in rat nerve terminals backpropagated up the axon ( approximately 400-800 microm) and changed the threshold for initiating action potentials and thus firing patterns. These results suggest a mechanism for information processing in neurons and neuronal circuits.

Methods for patch clamp capacitance recordings from the calyx.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.