Author response:
Public Reviews:
Reviewer #1 (Public review):
The paper by Chen et al describes the role of neuronal themo-TRPV3 channels in the firing of cortical neurons at a fever temperature range. The authors began by demonstrating that exposure to infrared light increasing ambient temperature causes body temperature to rise to a fever level above 38{degree sign}C. Subsequently, they showed that at the fever temperature of 39{degree sign}C, the spike threshold (ST) increased in both populations (P12-14 and P7-8) of cortical excitatory pyramidal neurons (PNs). However, the spike number only decreased in P7-8 PNs, while it remained stable in P12-14 PNs at 39 degrees centigrade. In addition, the fever temperature also reduced the late peak postsynaptic potential (PSP) in P12-14 PNs. The authors further characterized the firing properties of cortical P12-14 PNs, identifying two types: STAY PNs that retained spiking at 30{degree sign}C, 36{degree sign}C, and 39{degree sign}C, and STOP PNs that stopped spiking upon temperature change. They further extended their analysis and characterization to striatal medium spiny neurons (MSNs) and found that STAY MSNs and PNs shared the same ST temperature sensitivity. Using small molecule tools, they further identified that themo-TRPV3 currents in cortical PNs increased in response to temperature elevation, but not TRPV4 currents. The authors concluded that during fever, neuronal firing stability is largely maintained by sensory STAY PNs and MSNs that express functional TRPV3 channels. Overall, this study is well designed and executed with substantial controls, some interesting findings, and quality of data. Here are some specific comments:
(1) Could the authors discuss, or is there any evidence of, changes in TRPV3 expression levels in the brain during the postnatal 1-4 week age range in mice?
To our knowledge, no published studies have documented changes in TRPV3 expression levels in the brain during the 1st to 4th postnatal weeks in mice. Research on TRPV3 expression in the mouse brain has primarily involved RT-PCR analysis of RNA from dissociated tissue in adult mice (Jang et al., 2012; Kumar et al., 2018), largely due to the scarcity of effective antibodies for brain tissue sections at the time of publication. Furthermore, the Allen Brain Atlas lacks data on TRPV3 expression in the developing or postnatal brain. To address this gap, we plan to examine TRPV3 expression at P7-8, P12-13, and P20-23 as part of our manuscript revision.
(2) Are there any differential differences in TRPV3 expression patterns that could explain the different firing properties in response to fever temperature between the STAY- and STOP neurons?
This is an excellent question and one we plan to explore in the future by developing reporter mice or viral tools to monitor the activity of cells with endogenous TRPV3 expression. To our knowledge, these tools do not currently exist. Creating them will be challenging, as it requires identifying promoters that accurately reflect endogenous TRPV3 expression. We have not yet quantified TRPV3 expression in STOP and STAY neurons; however, our analysis of evoked spiking activity at 30, 36, and 39°C suggests that TRPV3 expression may mark a population of pyramidal neurons that tend to STAY spiking as temperatures increase. To investigate this further, we are considering patch-seq for TRPV3 expression on recorded neurons. This is a complex experiment, as it requires recording activity at three different temperatures and subsequently collecting the cell contents. While success is not guaranteed, we are committed to attempting these experiments as part of our revisions.
(3) TRPV3 and TRPV4 can co-assemble to form heterotetrameric channels with distinct functional properties. Do STOP neurons exhibit any firing behaviors that could be attributed to the variable TRPV3/4 assembly ratio?
There is some evidence that TRPV3 and TRPV4 proteins can physically associate in HEK293 cells and native skin tissues (Hu et al., 2022). TRPV3 and TRPV4 are both expressed in the cortex (Kumar et al., 2018), but it remains unclear whether they are co-expressed and co-assembled to form heteromeric channels in cortical excitatory pyramidal neurons. Examination of the I-V curve from HEK cells co-expressing TRPV3/4 heteromeric channels shows enhanced current at negative membrane potentials (Hu et al., 2022).
Currently, we cannot characterize cells as STOP or STAY and measure TRPV3 or TRPV4 currents simultaneously, as this would require different experimental setups and internal solutions. Additionally, the protocol involves a sequence of recordings at 30, 36, and 39°C, followed by cooling back to 30°C and re-heating to each temperature. Cells undergoing such a protocol will likely not survive till the end.
In our recordings of TRPV3 currents—which likely include both STOP and STAY cells—we do not observe a significant current at negative voltages, suggesting that TRPV3/4 heteromeric channels may either be absent or underrepresented, at least at a 1:1 ratio. However, the possibility that TRPV3/4 heteromeric channels could define the STOP cell population is intriguing and plausible.
(4) In Figure 7, have the authors observed an increase of TRPV3 currents in MSNs in response to temperature elevation?
We have not recorded TRPV3 currents in MSNs in response to elevated temperatures.
(5) Is there any evidence of a relationship between TRPV3 expression levels in D2+ MSNs and degeneration of dopamine-producing neurons?
This is an interesting question, though it falls outside our current research focus in the lab. A PubMed search yields no results connecting the terms TRPV3, MSNs, and degeneration. However, gain-of-function mutations in TRPV4 channel activity have been implicated in motor neuron degeneration (Sullivan et al., 2024) and axon degeneration (Woolums et al., 2020). Similarly, TRPV1 activation has been linked to developmental axon degeneration (Johnstone et al., 2019), while TRPV3 blockade has shown neuroprotective effects in models of cerebral ischemia/reperfusion injury in mice (Chen et al., 2022).
The link between TRPV activation and cell degeneration, however, may not be straightforward. For instance, TRPV1 loss has been shown to accelerate stress-induced degradation of axonal transport from retinal ganglion cells to the superior colliculus and to cause degeneration of axons in the optic nerve (Ward et al., 2014). Meanwhile, TRPV1 activation by capsaicin preserves the survival and function of nigrostriatal dopamine neurons in the MPTP mouse model of Parkinson's disease (Chung et al., 2017).
(6) Does fever range temperature alter the expressions of other neuronal Kv channels known to regulate the firing threshold?
This is an active line of investigation in our lab. The results of ongoing experiments will provide further insight into this question.
Reviewer #2 (Public review):
Summary:
The authors study the excitability of layer 2/3 pyramidal neurons in response to layer four stimulation at temperatures ranging from 30 to 39 Celsius in P7-8, P12-P14, and P22-P24 animals. They also measure brain temperature and spiking in vivo in response to externally applied heat. Some pyramidal neurons continue to fire action potentials in response to stimulation at 39 C and are called stay neurons. Stay neurons have unique properties aided by TRPV3 channel expression.
Strengths:
The authors use various techniques and assemble large amounts of data.
Weaknesses:
(1) No hyperthermia-induced seizures were recorded in the study.
The goal of this manuscript is to uncover the age-related physiological changes that enable the brain to retain function at fever temperatures. These changes may potentially explain why most children do not experience febrile seizures or why, in the rare cases when they do occur, the most prominent window of susceptibility is between 2-5 years of age (Shinnar and O’Dell, 2004), as this may coincide with the window during which these developmental changes are normally occurring. While it is possible that impairments in these mechanisms could result in febrile seizures, another possibility is that neural activity may fall below the level required to maintain normal function.
(2) Febrile seizures in humans are age-specific, extending from 6 months to 6 years. While translating to rodents is challenging, according to published literature (see Baram), rodents aged P11-16 experience seizures upon exposure to hyperthermia. The rationale for publishing data on P7-8 and P22-24 animals, which are outside this age window, must be clearly explained to address a potential weakness in the study.
This manuscript focuses on identifying the age-related physiological changes that enable the brain to retain function at fever temperatures. To this end, we examine two age periods flanking the putative window of susceptibility (P12-14), specifically an earlier timepoint (P7-8) and a later timepoint (P20-23). The inclusion of these time points also serves as a negative control, allowing us to determine whether the changes we observe in the proposed window of susceptibility are unique to this period. We believe that including these windows ensures a thorough and objective scientific approach.
(3) Authors evoked responses from layer 4 and recorded postsynaptic potentials, which then caused action potentials in layer 2/3 neurons in the current clamp. The post-synaptic potentials are exquisitely temperature-sensitive, as the authors demonstrate in Figures 3 B and 7D. Note markedly altered decay of synaptic potentials with rising temperature in these traces. The altered decays will likely change the activation and inactivation of voltage-gated ion channels, adjusting the action potential threshold.
In Figure 4B, we surmised that the temperature-induced reductions in inhibition and the subsequent loss of the late PSP primarily contribute to the altered decay of the synaptic potentials.
(4) The data weakly supports the claim that the E-I balance is unchanged at higher temperatures. Synaptic transmission is exquisitely temperature-sensitive due to the many proteins and enzymes involved. A comprehensive analysis of spontaneous synaptic current amplitude, decay, and frequency is crucial to fully understand the effects of temperature on synaptic transmission.
Thank you for the opportunity to provide clarification. It was not stated, nor did we intend to imply, that in general, E-I balance is unchanged at higher temperatures. Please see the excerpt from the manuscript below. The statements specifically referred to observations made for experiments conducted during the P20-26 age range for cortical pyramidal neurons. We have a parallel line of investigation exploring the differential susceptibility of E-I balance based on age and temperature. Additionally, our measurements focus on evoked activity, rather than spontaneous activity, as these events are more likely linked to the physiological changes underlying behavior in the sensory cortex.
“As both excitatory and inhibitory PNs that stay spiking increase their firing rates (Figure 5B) and considering that some neurons within the network are inactive throughout or stop spiking, it is plausible that these events are calibrated such that despite temperature increases, the excitatory to inhibitory (E-I) balance within the circuit may remain relatively unchanged. Indeed, recordings of L4-evoked excitatory and inhibitory postsynaptic currents (respectively EPSCs and IPSCs) in wildtype L2/3 excitatory PNs in S1 cortex, where inhibition is largely mediated by the parvalbumin positive (PV) interneurons, showed that E-I balance (defined as E/E+I, the ratio of the excitatory current to the total current) remained unchanged as temperature increased from 36 to 39°C (Figure 5E).”
(5) It is unclear how the temperature sensitivity of medium spiny neurons is relevant to febrile seizures. Furthermore, the most relevant neurons are hippocampal neurons since the best evidence from human and rodent studies is that febrile seizures involve the hippocampus.
Thank you for the opportunity to clarify. Our goal was not to establish a link between medium spiny neuron (MSN) function and febrile seizures. The manuscript's focus is on identifying age-related physiological changes that enable supragranular cortical cells in the brain to retain function at fever temperatures. MSNs were selected for mechanistic comparison in this study because they represent a non-pyramidal, non-excitatory neuronal subtype, allowing us to assess whether the physiological changes observed in L2/3 excitatory pyramidal neurons are unique to these cells.
(6) TRP3V3 data would be convincing if the knockout animals did not have febrile seizures.
Could you kindly provide the reference indicating that TRPV3 KO mice have seizures? Unfortunately, we were unable to locate this reference. It is important to distinguish febrile seizures, which occur within the range of physiological body temperatures (~ 38 to 40°C), from seizures resulting from heat stroke, a severe form of hyperthermia occuring when body temperature exceeds 40.0 °C. Mechanistically, these may represent different phenomena, as the latter is typically associated with widespread protein denaturation and cell death, whereas febrile seizures are usually non-lethal. Additionally, TRPV3 is located on chromosome 17p13.2, a region not currently associated with seizure susceptibility.
Reviewer #3 (Public review):
Summary:
This important study combines in vitro and in vivo recording to determine how the firing of cortical and striatal neurons changes during a fever range temperature rise (37-40 oC). The authors found that certain neurons will start, stop, or maintain firing during these body temperature changes. The authors further suggested that the TRPV3 channel plays a role in maintaining cortical activity during fever.
Strengths:
The topic of how the firing pattern of neurons changes during fever is unique and interesting. The authors carefully used in vitro electrophysiology assays to study this interesting topic.
Weaknesses:
(1) In vivo recording is a strength of this study. However, data from in vivo recording is only shown in Figures 5A,B. This reviewer suggests the authors further expand on the analysis of the in vivo Neuropixels recording. For example, to show single spike waveforms and raster plots to provide more information on the recording. The authors can also separate the recording based on brain regions (cortex vs striatum) using the depth of the probe as a landmark to study the specific firing of cortical neurons and striatal neurons. It is also possible to use published parameters to separate the recording based on spike waveform to identify regular principal neurons vs fast-spiking interneurons. Since the authors studied E/I balance in brain slices, it would be very interesting to see whether the "E/I balance" based on the firing of excitatory neurons vs fast-spiking interneurons might be changed or not in the in vivo condition.
As requested, in the revised manuscript, we will include examples of single spike waveforms and raster plots for the in vivo recordings. Please note that all recordings were conducted in the cortex, not the striatum. To clarify, we used published parameters to separate the recordings based on spike waveform, which allowed us to identify regular principal neurons and fast-spiking interneurons. The paragraph below from the methods section describes this procedure.
“ Following manual curation, based on their spike waveform duration, the selected single units (n= 633) were separated into putative inhibitory interneurons and excitatory principal cells (Barthóet al., 2004). The spike duration was calculated as the time difference between the trough and the subsequent waveform peak of the mean filtered (300 – 6000 Hz bandpassed) spike waveform. Durations of extracellularly recorded spikes showed a bimodal distribution (Hartigan’s dip test; p < 0.001) characteristic of the neocortex with shorter durations corresponding to putative interneurons (narrow spikes) and longer durations to putative principal cells (wide spikes). Next, k-means clustering was used to separate the single units into these two groups, which resulted in 140 interneurons (spike duration < 0.6 ms) and 493 principal cells (spike duration > 0.6 ms), corresponding to a typical 22% - 78% (interneuron – principal) cell ratio”.
In vivo patching to record extracellular and inhibitory responses at 36°C and then waiting 10 minutes to record again at 39°C would be an extremely challenging experiment. Due to the high difficulty and expected very low yield, these experiments will not be pursued for the revision studies.
(2) The author should propose a potential mechanism for how TRPV3 helps to maintain cortical activity during fever. Would calcium influx-mediated change of membrane potential be the possible reason? Making a summary figure to put all the findings into perspective and propose a possible mechanism would also be appreciated.
Thank you for your helpful suggestions. In response to your recommendation, we will include a summary figure detailing the hypothesis currently described in the discussion section of the manuscript. The excerpt from the discussion is included below.
“Although, TRPV3 channels are cation-nonselective, they exhibit high permeability to Ca2+ (Ca²⁺ > Na⁺ ≈ K⁺ ≈ Cs⁺) with permeability ratios (relative to Na+) of 12.1, 0.9, 0.9, 0.9 (Xu et al., 2002). Opening of TRPV3 channels activates a nonselective cationic conductance and elevates membrane depolarization, which can increase the likelihood of generating action potentials. Indeed, our observations of a loss of the temperature-induced increases in the PSP with TRPV3 blockade are consistent with a reduction in membrane depolarization. In S1 cortical circuits at P12-14, STAY PNs appear to rely on a temperature-dependent activity mechanism, where depolarization levels (mediated by higher excitatory input and lower inhibitory input) are scaled to match the cell’s ST. Thus, an inability to increase PSPs with temperature elevations prevents PNs from reaching ST, so they cease spiking.”
(3) The author studied P7-8, P12-14, and P20-26 mice. How do these ages correspond to the human ages? it would be nice to provide a comparison to help the reader understand the context better.
Ideally, the mouse-human age comparison would depend on the specific process being studied. Please note that these periods are described in the introduction of the manuscript. The relevant excerpt is included below. Let us know if you need any additional modifications to this description.
“Using wildtype mice across three postnatal developmental periods—postnatal day (P)7-8 (neonatal/early), P12-14 (infancy/mid), and P20-26 (juvenile/late)—we investigated the electrophysiological properties, ex vivo and in vivo, that enable excitatory pyramidal neurons (PNs) neurons in mouse primary somatosensory (S1) cortex to remain active during temperature increases from 30°C (standard in electrophysiology studies) to 36°C (physiological temperature), and then to 39°C (fever-range).”
