## 2 Peripheral Ultrasonic Neuromodulation--Technical Specifications
### Metrics for Peripheral Neural Activities  
The assessment of the neuromodulatory effects of US requires a reliable metric of peripheral neural activities, which includes direct and indirect measurements of neural action potentials from peripheral nerves or neurons. In addition, secondary effects of neural activities were also used to indirectly infer the neuromodulatory effects on PNS, including altered organ functions (i.e., bladder contraction, urethral sphincter relaxation), electroencephalogram recordings in the brain, and behavioral signs (e.g., whisker movement, freeze in motion, toe-pinch response). The detection of secondary effects tends to lag US stimulation by hundreds of milliseconds to seconds, a time frame much longer than the direct neuronal effects of milliseconds [38]. This review mostly focuses on the direct assessment of neural action potentials and omits metrics using secondary effects. Nerve axons in the PNS are generally protected by soft connective tissue stacked in multiple layers, in contrast to the neurons and processes in the CNS, protected by bony structures like the skull or the vertebrae. These tightly wrapped tissue layers in the PNS, functioning as electrical insulators, pose a great challenge in recording electrophysiological activities from individual nerve axons, i.e., single-unit recordings [48]; recordings from individual neurons or axons in the CNS are straightforward when the electrodes are placed inside the skull or vertebrae. Consequently, the major metrics to assess the neuromodulatory effects on the PNS are either CAP, as a summation of action potentials from a bulk nerve bundle [47; 49; 50; 51; 52], or evoked muscle forces, as an indicator of motor nerve functions [42; 53; 54]. However, the characteristics of a CAP (peak amplitude, temporal location, and spread) depend on the temporal summation of a population of action potentials from axons with various spatial locations, morphologies, and insulation environment. The CAP characteristics can also be affected by changes in recording conditions, e.g., relative position of the electrode sites and axons, change of access impedance of recording electrodes due to altered moisture conditions, and multiple/chronic use of electrodes [48]. Thus, neither the changes in CAP amplitude nor the assumed changes in conduction delay (Figure 2A) can appropriately be representative of the effects of US neuromodulation. In addition, the signal strength in a CAP record can be misleading, as shown in Figure 2A: the large peak contributed to by fast-conducting A-fibers usually overshadows the small volleys by slow-conducting C-fibers, despite the presence of significantly higher proportions of C-fibers than A-fibers in the PNS [55; 56]. Collectively, CAP appear to be an inappropriate metric for PNS neuromodulation. Further, muscle forces, evoked by US neuromodulation of a nerve, are indirect metrics of nerve activities and limited to the study of motor axons innervating specific muscles. On the other hand, single-unit recording can record action potentials from individual nerve axons (Figure 2B), capable of capturing relatively fine variations of neural responses to mechanical, chemical, and/or thermal stimuli [57; 58; 59]. Single-unit recording relies solely on the temporal information of the spike, which is mainly determined by the action potential transmission and is not affected by experimental artifacts like changes in electrode impedance. Thus, compared with CAP, single-unit recordings are more robust and provide much higher sensitivity to allow the detection of subtle changes of conduction delay in individual axons. However, single-unit recordings are technically challenging and involve microdissection of nerve fiber bundles until action potentials from a single nerve axon are isolated [43; 48].