The results of this IHC study indicate that the Piezo2 protein is expressed in pyramidal neurons of the NC and HC, Purkinje cells of the cerebellar cortex and mitral cells of the OB. These results are consistent with earlier and more recent studies of Piezo2 expression rodent and human brains [26, 27, 34, 35, 40, 41]. Although Piezo2 is well established as a pressure sensitive channel in peripheral neurons [5, 17-25] further functional studies, using patch clamp [43] and/or single cell Ca2+ imaging [44] are required to confirm that Piezo2 is functionally active in these different neuron types. Interestingly, the first patch clamp study demonstrating single pressure-activated single channel currents in NC and HC pyramidal neurons in mouse brain slices, indicated a low frequency channel activity in the absence of applied pressure that increased in frequency with increasing steady state pressure [43]. Furthermore, recording from a CA3 pyramidal neuron (see Figure 8 of ref. 43) demonstrated that relatively brief (~ 1 s) negative pressure pulses of >25 mm Hg were able to stimulate repetitive firing of action potentials at increasingly higher frequencies (e.g., 3 Hz with a 27 mm Hg pulse and 24 Hz with a 31 mm Hg pulse). In the future, it will be important to extend these studies to positive pressure pulses which have been reported to selectively activate Piezo2 channels [e.g., see 45].
It is also worth considering the nature (e.g., amplitude and time course) of the exogenous and endogenous pressure that neurons might experience within the brain that might be capable of activating Piezo2 channels. The finding that large exogenous pressures, simulating blast forces, alter Piezo2 channel expression in the NC and HC [26, 27], particularly in response to repetitive blasts, indicates the potential for a neuropathological role of Piezo2 in traumatic brain injury (TBI). Moreover, one might expect that abnormal activation of Piezo2 in these brain regions could contribute to the acute and often reversible deficits in motor, sensory and cognitive functions associated with concussion and mild TBI. However, TBI is just one of several neuropathological conditions, including hydrocephalus, cerebral hemorrhage and brain tumors, known to elevate the normally low (< 10 mm Hg) baseline intracranial pressure (ICP) to higher levels (> 25 mm Hg), all with serious clinical consequences [46-49]. Given positive pressures pulses as low as ≤ 5 mm Hg can activate Piezo2 channels [45], it would not be surprising that pressures 5-fold higher would induce abnormal Piezo2 activities and contribute to deficits in brain function. A special case is normal pressure hydrocephalus (NPH) in which enlarging ventricles compress the brain parenchyma [50]. Interestingly, the dementia associated the NPH is often misdiagnosed as Alzheimer’s disease (AD), but can be distinguished by ventriculoperitoneal shunts that reduce ICP, and often rapidly reverses this form of dementia [50].
In terms of evolution and natural selection, there would seem little selective advantage for brain neurons to express Piezo2, especially if the channel only functions in a neuropathological role, with no benefit for improved normal brain performance. Therefore, a more relevant and interesting question is whether smaller changes in ICP (≤ 10 mm Hg) that occur in the healthy brain might also produce changes in Piezo2 activity and thereby somehow enhance normal brain function. Although ICP in healthy subjects is maintained at low baseline values of 0-10 mm Hg [46, 49] ICP also undergoes metronomic-like, pulsatile changes involving rapid (~200 ms) pulses with each heartbeat [46-48], as well as much slower (5-10 s) pulses associated with breathing [51-53]. Moreover, specific volitional breathing practices ___involving slow inspiration/expiration cycles and/or diaphragmatic vs thoracic breathing ___ performed to improve attention or reduce stress/anxiety, cause even larger pulsatile changes in ICP [53]. This raises the intriguing possibility that the presence of Piezo2 might allow specific neurons to transduce these pulsatile ICP changes, thereby entraining the rhythmic neural network oscillations previously measured with EEG and shown to underlie specific brain/behavioral states [54-56]. In this case, it may be that the pulsatile fluctuations in ICP started out as epiphenomena [1] representing the unavoidable cerebral responses to breathing and cardiac rhythms. However, over time selective pressure would tend to favor Piezo2 expression, if that conferred on neurons a new mechanism for synchronizing neural network communication, especially within the much larger brains of humans.
Direct experimental evidence for the actual idea that breathing can induce entrainment of neural networks goes back 80 years with the discovery by Edgar Adrian in 1942 that nasal breathing in rodents causes rhythmic firing of mitral neurons within the OB, as well as neurons within the piriform cortex [57]. Many subsequent studies have verified and extended his discovery, showing that nasal breathing, not only entrains OB oscillations at the rodent’s breathing frequency (0.5- 5 Hz), but also modulates the amplitude of higher frequency oscillations (80 -120 Hz) in the OB as well as other downstream brain regions including the HC and NC [30-32] . Furthermore, these neural network oscillations have been associated with specific changes in rodent behaviors including whisking, memory formation and emotional (fear) responses [58-67]. Perhaps most significantly, many of the observations related to respiration-locked oscillations seen in rodents, have now been confirmed in humans, using either intracranial EEG recording from epileptic patients or high density EEG recording from healthy subjects [55, 56, 68, 69].
Three different, non-mutually exclusive mechanisms may explain respiration entrained neural network oscillations [30-32, 70-72]. The first mechanism, referred to as olfactory re-afferent discharge (ORD) proposes that nasal airflow during nasal breathing activates mechanosensitive primary olfactory neurons (PON) within the nasal epithelium turn activating via their direct synaptic connections, mitral cells within the OB [72, see Fig. 7]. In this mechanism, the OB acts as a “global clock” for other brain regions, synchronizing via its synaptic connections, neural networks across widespread brain regions, including the HC and NC [31, 32]. Evidence supporting this mechanism, is that tracheotomy or ablation of the nasal epithelium (or removal of the OB) in rodents, reduces the synchronized activity in the OB and/or in downstream brain regions [57, 58, 73, 74]. Moreover, in both humans and rodents, air pressure pulses delivered to the nostrils can restore and/or alter the oscillation frequency [63, 68].
The second mechanism referred to as “respiratory corollary discharge” (RCD) depends upon efferent copy discharges from neurons in the brain stem nuclei that regulate breathing [75-77, Fig. 7]. Support for this mechanism includes the finding that although tracheotomy or ablation of the nasal epithelium reduces the neural oscillations in rodents, they do not completely block them [58, 77]. Moreover, in humans the oscillations persist during mouth breathing and therefore in the absence of any nasal airflow) [69, 77]. Further evidence for the RCD mechanism is that neurons in the brain stem respiratory nuclei form connections with neurons in the locus coeruleus that via their projections may alter activity in widely spaced neural networks throughout the brain [76].
The third mechanism, and the least investigated, we refer to as intrinsic resonance discharge (IRD). In this case, it is the intrinsic properties of the neural networks involving either intrinsic neuronal membrane properties or synaptic micro-circuitry, which dictates that they fire or resonate at a frequency close to the respiratory frequency [56]. The evidence also for the IRD mechanism is again that respiration-entrained oscillations are retained during mouth breathing or in the absence of nasal airflow or functional PONs, [58, 69, 77]. Moreover, IRD may in part account for a somewhat puzzling feature of the ORD mechanism, which relates to the mechanosensitivity of the odor sensitive GPCR in the PONs. Evidence indicates that their mechanosensitivity depends upon a second messenger pathway, and consequently display long latencies of ~300 ms in response to pressure pulses [72]. Furthermore, because of GPCR adaptation, their maximum frequency response to repetitive pressure pulse stimuli has been measured at only 0.5 Hz [72], which is significantly slower than the rodent’s normal breathing frequency and entrainment frequency induced in the mitral cells of the OB (2-6 Hz). Although the convergence of many PON inputs to the mitral cells may still generate rhythmic activity that follows respiration, even when only a fraction of the PONs responding with each respiratory cycle [see 72], an alternative or reinforcing mechanism could be that the mitral cells themselves possess an intrinsic resonance to the breathing cycle. Specifically, we hypothesize that Piezo2 in mitral cells, as well as neurons in the NC and HC, confer an intrinsic resonance that further reinforces their entrainment by the breathing via their transduction of pulsatile ICP changes (Fig. 7). Because stretch activated channels, including Piezo channels, are characterized by their fast gating [78] and ability to accurately track relative high frequency (e.g., ³ 5 Hz) pressure pulses [79, 80] they could confer resonance for breathing-associated, as well as the even higher frequency cardiac-associated, ICP pulses [46-48, 81-83]. Moreover, the expression of Piezo2 in cerebellar PC may also explain the sniffing dependent activation of the human cerebellum [84] and the highly correlated slow (~ 1 Hz) oscillations seen between mouse PCs [85].
An intrinsic Piezo2/ICP resonance mechanism may also developed a more important role for human breathing-induced entrainment because of additional selective pressures. In particular, in humans nasal breathing is not obligatory as in rodents. Also in humans, olfaction has become a relatively unimportant sensory function, adding only little advantage for survival, which is unlike in rodents where olfaction, sniffing and whisking are tightly linked and critical for their normal behavior and survival. Moreover, humans are the only species capable of volitionally switching breathing patterns (e.g., to forced inspiration/expiration cycles) in order to promote changes in mood, cognition and emotions. Humans are also unique in that they consciously modulate through their breathing, both their heartbeat and emotions [81, 82]. Interestingly, on this point a study of single unit recording from epileptic patients indicated a higher proportion of hippocampal and amygdala neurons show entrainment with the cardiac cycle compared with the respiratory cycle [83].
Regardless of the exact mechanism, a key issue for all mechanisms is their reliability and degree of synchronization that they can induce within widely separated neural networks. This synchronization has special implications for the unity of experience, as it relates to perception, motor control and memory retrieval, especially in the much larger human brain. In the case of the first two mechanisms, the overall synchronization will depend upon signaling delays within multi-synaptic, axonal transmission pathways. In comparison, the Piezo2/ICP pulse dependent IRD mechanism will only be rate limited by the speed of ICP pulse transmission throughout the brain, powered by the perennial actions of the respiratory and cardiac pumps. In the ideal case when Pascal’s law applies, namely a rigid, fluid-filled and closed container, a pressure pulse will spread throughout the container almost instantaneously at the speed of sound (i.e., ~1500 m/s) [46]. However, this may represent an upper estimate since the brain contained within the skull is actually a semi-closed system that involves cerebral venous outflow as well as arterial inflow [45-47]. Plus, CSF and blood tend to circulate between the brain and spinal cord, particularly during inspiration and expiration [51-53]. In addition, the brain parenchyma, ventricles, and vasculature are themselves compliant rather than rigid, which will also tend to dissipate and slow the spread of ICP changes. In one notable study [86], aimed at measuring ICP transmission in the human brain, two ICP sensors, placed 5 cm apart in the lateral ventricle and brain parenchyma, measured a phase shift of 10 ms [86]. This would predict a pulse velocity of only 5 m/s. (i.e., which compares with a predicted 50 ms phase shift for a velocity of 1,500 m/s). However, there are two caveats regarding this lower estimate. First, the ICP pulse does not have a single origin but arises throughout the arterial tree [46]. Second, the pressure pulsations may occur within the dense microvascular embedded in the parenchyma so that no individual neuron is more than 100 mm from a pulsating vessel [87]. Both effects would tend to minimize delays and thereby synchronize the ICP pulse transmission to all neurons within neural networks throughout the brain.
Limitations and future studies
The results of any IHC study are dependent on the specificity/selectivity of the Ab as determined by various Ab validation criteria including genetic, orthogonal and independently generated Abs [40]3. Although now four independently generated anti-PIEZO2 Abs including ours, raised against different regions of the PIEZO2 protein [26, 27, 34, 40 Table1] indicate Piezo2 expression in the rodent and human brain. Nevertheless, further verification with techniques independent of antibodies are required. For example, by the development and use of highly sensitive GFP- tagged Piezo2 transgenic mice [e.g., 88, GENSAT]. Given Piezo1 is also expressed in rodent and human brain neurons [2] and that both Piezo1 and Piezo2 are essential for pressure sensation in peripheral baroreceptors [24] it will also be important to establish the expression patterns and the specific roles of both Piezo channels in the different brain neurons (i.e., by using also a combination GFP-tagged Piezo transgenic mouse). The functional expression of pressure-activated channels in the specific neuron types in mouse brain slices and ideally in situ in the brains of living/breathing animals needs to be verified and these studies should be combined with conditional Piezo knock out mice in order to establish the channel’s molecular identity.
Footnote 3: https://www.proteinatlas.org/about/assays+annotation#ihk
Perhaps the biggest challenge in terms of testing the intrinsic resonance hypothesis will be to demonstrate that Piezo channels, actually participate in the entrainment of neural network oscillations. One possible approach may be to use the conditional Piezo knock out mice and compare respiratory entrained neural network oscillations with those reported previously for wild type mice [30-32, 59-61]. It would also be very interesting to study human patients that show a specific loss of Piezo2 function [89], to determine how their measured EEG and behavioral responses to specific breathing protocols, compare with reported responses of normal human subjects [54-56, 68, 69].
Finally, we would like to end where we began with a more compete version of the quote from Hutcheson and Yaron [1]. “A question therefore arises: are resonances used by neurons or are they simply epiphenomena?” A broad answer to this question is that, in nature, epiphenomena seldom remain epiphenomena for long; they are the raw material for evolutionary advances. It would be surprising to find that the brain has not found a use for a set of mechanisms capable of tuning neurons to specific frequencies, particularly in light of the prevalence of robust brain rhythms.” Here, Hutcheson and Yarom are referring to, at the time, the relatively unexplored role of the various EEG recorded electrical brain rhythms [90]. However, it may be the metronomic-like and perennial ICP pulses, transduced by pressure sensitive channels, prove even more pivotal in tuning and synchronizing the brain to the fundamental frequencies of life.