Extracellular recording of axonal spikes in the visual cortex

Multichannel extracellular recordings are a major tool to investigate neural populations in vivo, especially in unanaesthetised animals. A significant disadvantage of extracellular recordings though, is that we cannot tell with certainty the type of neurons we are observing. Sure, we have a few tricks up our sleeves, like optical tagging in specific cre-lines, or antidromic activation of selected populations, but in naive recordings, the identity of the recorded neurons is usually inferred by their firing patterns and their extracellular spike waveforms. The most commonly used procedure is to separate fast-spiking interneurons from principal (mainly pyramidal) cells based on their narrower action potentials. This approach works well in the cortex, hippocampus, striatum and amygdala. In some cases, however, extracellular recordings can yield yet another type of waveform, the axonal spikes. The appearance of these units varies from region to region, but is usually characterised by narrow spike waveform (100–150 μs), even narrower than that of the fast-spiking interneurons. In addition, axonal spikes are often positive or triphasic, distinguishing them from respectable somatic extracellular action potentials. For example, terminals of pallidal afferents have been recorded in the thalamus of songbirds (Goldberg & Fee, 2012), entorhinal axons in the alveus (Robbins et al. 2013), thalamic reticular terminals in the ventrobasal thalamus (Barthó et al. 2014), and climbing fibres in the cerebellum (Khaliq & Raman, 2005). In this issue of The Journal of Physiology, Sun et al. (2021) provides a new addition to the list; they record the activity of putative thalamic axon terminals in the cat visual cortex. These units have a positive waveform, high burst ratio, and their receptive fields are mostly non-oriented, suggesting a thalamic origin. Furthermore, while most units cease firing after cortical silencing by muscimol, the putative thalamic axonal spikes’ activity persists. Together, these findings provide an indirect, nevertheless convincing case that the positive spikes originate from the thalamocortical afferents. One may ask, what is the point of recording the axonal spikes instead of the corresponding somatic signals? The main advantage is topography. However meticulous the placing, it is close to impossible to place two electrodes in the exactly corresponding sites of a projection. Another potential it offers is the mapping of functional connectivity in cortical microcircuits, as well as the connection strength across states. There are still a multitude of open questions regarding axonal spikes. For instance, only certain types of axons can be recorded extracellularly, such as thalamocortical axons in this study, but no sign of intracortical excitatory or inhibitory terminals. Similarly, in the thalamus, we see thalamic reticular, but not corticothalamic terminals. This is probably due to the morphology of the axon terminals, but definitely needs to be addressed further with high-resolution silicon probe arrays. In fact, we are not even sure these spikes represent the firing of axon terminals, or passing axons, though indirect evidence suggests that they are indeed terminals. Luckily in this case it does not really matter, as primary sensory cortices are the final destination of first-order thalamus. Finally, how come, that despite the thousands of units clustered in labs throughout the past decades, axonal spikes have been largely unnoticed or ignored? The main reason is that they are often difficult to cluster. In contrast to somatic spikes, they usually appear in a much smaller spatial segment. Since clustering from multisite recordings is based on the different appearance of the same spike on distinct, but spatially close channels, a signal that appears on only one or two channels might easily go unnoticed. Their smaller amplitude (the present paper is an exception), compared with somatic signals, does not help either to separate them from the mess of multiunit activity. Finally, during clustering, one tends to regard rare, unusual waveforms as artefacts, and keep only the reliable ones. These difficulties will abate, as high channel count silicon probes gradually replace wire tetrodes as the weapon of choice; so hopefully more reports of axonal spikes from other brain structures can be expected in the future.