Critical Analysis Of The Study On The Relationship Between Retrotrapezoid Neuron (RTN) Groups And Nalcn

Brief Background of the Study

The study conducted by Shi et al. investigates the relationship between retrotrapezoid neuron (RTN) groups and Nalcn to further understand their relationship and underlying mechanisms. Nalcn is a tetrodotoxin (TTX) resistant Na+ leak channel that contributes to the influx of sodium currents in the brain, playing an integral molecular and physiological role in the RTN. The study used shRNA-mediated knockdown to illustrate Nalcn’s contribution to the background Na+ current and excitability in RTN and its role in the activation of RTN via Substance P (SP).

The RTN is an area of the brainstem that regulates pulmonary respiration. Respiratory control involves a central pattern generator (CPG) that uses a tonic excitatory drive through CO2/H+ sensitive neurons in the RTN. The RTN neurons adjust their firing of action potentials in response to changes in CO2/H+ to maintain homeostasis of blood pH. The depletion of Nalcn removes the CO2 response of RTN neurons. These conclusions illustrate the importance and relevance of Nalcn in RTN neuronal respiratory function. Phox2b-expressing RTN neurons are intrinsically chemosensitive. Symptomized by respiratory arrest during sleep, congenital central hypoventilation syndrome (CCHS) is caused by mutations in RTN expression of Phox2b. It manifests itself by reduced CO2 chemosensitivity and a curtailed response to elevated CO2 levels. Prior studies have contextualized an array of diseases associated with Nalcn variants and single human gene mutations affecting breathing and neurodegenerative disorders. However, this study focuses on Nalcn function in these neuronal groups.

The article aims to identify ion channels that might participate in breathing rhythm generation and chemosensitivity, therefore focusing on the inquiry of Nalcn. The research outlines the fundamental function of respiration in physiological processes, and is used in all behavioural states. The investigation of the relationships and underlying mechanisms between nalcn, Phox2b, and neuronal groups of RTN develop an intricate insight on human physiology, and more specifically remedies for diseases including CCHS.

Hypothesis and Research Question

The article does not explicitly state a hypothesis, however it suggests that selective depletion of Nalcn in RTN neurons would result in respiratory dysfunction. The article addresses various scientific questions related to the contribution of Nalcn to endogenous currents in the respiratory neurons, specifically the pH-sensitive RTN neurons. It asks whether Nalcn is required for the activation of RTN neurons by the neuropeptide substance P, as well as the effects of RTN-selective Nalcn depletion. It all ties in to the question, “What are specific physiological roles for Nalcn in control of breathing by RTN respiratory integrator neurons?”Basic Approach Used in the Study Experiments were tested on Phox2b::GFP mice. According to research, Nalcn is expressed in various vertebrates, including humans and mice, and there is 98% sequence similarity between the Nalcn of those two species. The researchers used mice because of this close similarity. Most Phox2b-expressing RTN neurons exhibit an intrinsic chemosensitivity according to the study by Lazarenko et al 2009. This suggests that Phox2b (paired-like homeobox 2b) is a molecular signature of chemosensitive RTN neurons. Reverse-Transcription PCR was performed on RTN neurons. They used multiplex nested single-cell RT-PCR (sc-PCR) or multiplex quantitative sc-PCR (sc-qPCR) to analyze numerous genes in single-celled assays. RT-PCR is a technology in which RNA molecules are converted into their complementary DNA sequences by reverse transcriptases, followed by amplification of cDNA using PCR procedures.

Single-cell analysis preserves information that is lost when measurements are taken by averaging cells together. RT-qPCR is the most widely used technique among methods for targeted analysis. Advantages include single-molecule sensitivity, a short amount of time required to complete measurement, and ease of assay design. Using microfluidic arrays in the final step of the qPCR is useful for single-cell analysis as it increases throughput and reduces costs.

Next, methods of virus production were performed. Two different transduction systems were used to infect the RTN neurons both in vitro and in vivo. Adeno-associated virus 2 (AAV2) was used for slice cultures in vitro and lentivirus for in vivo observation. In the AAV2 production, Nalcn short hairpin RNA shRNA (artificial RNA used to silence specific gene expression) was inserted into complementary AAV targets. In vivo studies consisted of a customized lentiviral targeting vector that was prepared using an artificial promoter designed to target the same shRNA sequence selectively in RTN neurons. Organic cultured slices were prepared from neonatal Phox2b::GFP mice. Three days after preparation, AAV2 was pipetted onto the surface of each slice, and was used 2 to 4 weeks later. In the slices, electrophysiological recordings from GFP-labeled neurons were performed by a fluorescence microscope. The solution was pH-adjustable. Recordings were made using pClamp, a multiclamp amplifier and a Digidata 1440A.

Lentivirus was injected into the RTN of the mice at specific coordinates and depth. There were two sets, a control virus and an Nalcn virus. The pipette electronically injected an exact amount of virus. Mice were examined 4 weeks later in histochemical assays. Breathing measurements were measured in mice that were conscious and unrestrained by plethysmography, the use of devices to determine changes in airway resistance and capacity. There were three incrementing CO2 challenges. Seven minutes at four, six, and eight percent CO2, each step separated by five minutes of pure O2. To analyze responses, the ventilatory flow signals were recorded, then amplified, digitized and analyzed using an EMKA Technology called Iox 2. 7. They measured minute ventilation, tidal volume, and body weight.

For the immunohistochemistry (IHC), mice were transcardially perfused and their brains were transversely cut. DNA fragments were amplified from the brain slices with primes, and sense probes were a negative control. IHC is a sensitive technique for detecting antigens within tissues using antigen-specific antibody. The variations in the initial preparation of tissues, including the amount, age, and pH of fixative, thickness of tissue, and time the tissue was left in fixative, can widely alter the results of IHC.

For cell counts and analysis, serial sections of RTN through the rostrocaudal extent were placed on glass slides and viewed using an epifluorescence microscope. Actual cell numbers were approximately three times higher than counted due to no stereological correction factor. Results of the StudyThe first major finding describes expression of Nalcn at variable levels in chemo-sensitive RTN neurons. RTN was found to be located at the ventral medullary surfaces or neighbouring it. Specifically, Phox2b and VGlut2-expressing RTN neurons express about 95% of Nalcn. Researchers were able to identify in Figure 1C that RTN neurons varied in the pH50 range over which they preserve their discharge. Those with a pH lower than 7. 42 (Type I) expressed less Nalcn and those with a pH higher than 7. 42 (Type II) had a greater expression of Nalcn, as displayed in Figure 1D. The next deduction was that the knockdown of Nalcn in vitro causes decreased basal discharge of RTN neurons. RTN neuronal expression for GFP and mCherry and the co-expression of the two are shown. The subsequent experiments compare Nalcn RTN neurons with controls: acutely dissociated neurons, infected with scrambled and uninfected neurons. RTN neurons expressing Nalcn shRNA had a significant reduction in Nalcn expression, the firing rate in AAV-infected RTN neurons and membrane potentials compared to controls. An important concept was that Nalcn-depleted RTN neurons shifted to more acidified levels causing a lower firing rate. Therefore, Nalcn knockdown hyperpolarizes RTN neurons and decreases neuronal firing. The knockdown of Nalcn decreases a TTX-resistant background Na+current in RTN neurons was found from Figure 3. Holding current, input conductance (Figure 3B) and current density (Figure 3C) were measured. In Nalcn shRNA, all measurements were significantly lower than the controls. Figures 3D/3E/3G consist of similarly formatted graphs (comprised of uninfected, scrambled and Nalcn, respectively). For 3D/3E, there was reduction with inward holding current and decreased input conductance, however with 3G, it was relatively linear meaning a minor effect was shown. Furthermore, current density was measured in Figures 3F (control) and 3H (Nalcn shRNa) concerning the two bath solutions mentioned previously and it had similar results as 3D/3E/3G. It was also discovered that there is a strong association between NMDG-sensitive amplitude and Nalcn expression, but Nalcn knockdown has a lower affinity. Data from Figure 4 conceptualizes that SP activates Nalcn to cause excitation of RTN neurons. The main experiments conducted tested the firing rates of RTN neurons with the application of SP and 5-HT. Figure 4C summarizes the results of the experiment showing that SP has no effect in Nalcn-depleted cells, contrary to its marked increase in the controls. It was also shown that Nalcn expression dissipated in RTN neurons expressing Nalcn shRNA. Additionally, voltage-clamp recordings and its effect of SP on membrane current were also performed. These recordings show that SP-sensitive current and SP-activated conductance weren’t reduced by Nalcn knockdown.

Another experiment they proved was that selective Nalcn in vivo reduced CO2-evoked neuronal activation and ventilatory simulation. In Figure 5C/5D/5E, the yellow arrows illustrate lentiviral infected RTN neurons had reduced expression of Nalcn, while green and white arrows (uninfected RTN and non-RTN neurons) had preserved 90% of Nalcn expression. Figure 5F illustrated that the percentage of cells expressing Nalcn was substantially lower compared to NK1R and VGlut2, indicating the selective knockdown of Nalcn. The expression of cFos can indirectly cause the activation of RTN neurons through the increase of CO2/H+. Imaging expressions were set up again in Figure 6A and viral injections similar to Figure 5 were used again as well. The arrows indicate cFos-immunoreactive neurons with yellow arrows indicating transduced and white arrows as non-transduced. All three sets of images in this figure had similar results displayed in Figure 6B except the transduced neurons injected with Nalcn, resulted in half the number of percentages cFos-labelled cells. Figure 7 illustrates how Nalcn depletion would affect ventilatory responses to raised CO¬¬2. Respiratory flow with 4 different CO2 concentrations following injections with both lentiviruses was performed. The results show that the lentiviral transduction ratio and percentage of pre-injection is lower in Nalcn compared to the control. Before virus injection, the outcome of CO2 on ventilation was similar with those that received injection of either virus. However, after Nalcn depletion, there was a strong reduction due to the subsided CO2-induced increases in frequency and volume illustrated in Figure 7C. The results were consistent with one another in each scenario whether it was the control or Nalcn. Ultimately, selective Nalcn depletion in RTN neurons lower CO2-induced neuronal stimulation. The aforementioned data showcases significant results, characterized by the use of clear tables, graphs and diagrams, promote clarity and understanding of the text. The organization of the data effectively highlights major results from the experiments conducted, while simultaneously addressing the objectivity of the research. The reduced CO2 evoked neuronal activation and ventilatory response to CO2, for example, is explicitly seen in Figure 7c. Most of the data collected is contrasted with control group(s) which create a baseline from which conclusions can be drawn. The firing rate graph showed that the Nalcn-depletion were shifted more to acidified levels and the firing rate was much lower than the controls. The shift is reflective of a lower initial firing rate instead of a difference in intrinsic neuronal pH sensitivity. This is because data collected from the control groups showcases no difference in firing rates (from pH) in control or Nalcn shRNA-expressing neurons. Supplementary information, being a preliminary resource, augments the data being collected. The importance of Nalcn, for instance, is highlighted through past research articles stating that mutations of Nalcn are associated with respiratory dysfunction and the deletion of the channel in postnatal mice leads to disrupted respiration. Introduction of known diseases and their affiliation to Nalcn provides an avenue that not only gauges interest, but conceptually describes the role of the ion channel before delving further into its mechanism on a biochemical level.

Similarly, this paper defines the physiological role of Nalcn in RTN neurons in respiration through CO2 induced activation. From this, the specific function of Nalcn and its mutations are made evident, but it’s not specifically applied to a condition or certain cause. However, through supplementary data from past studies discussing respiratory diseases associated with Nalcn, the application and significance of the research being conducted become clearly defined. The results define the function of Nalcn within the physiological context of respiration. Evidently, the selective depletion of Nalcn and diminished RTN activation in-vivo reduced CO2 evoked neuronal activation and ventilatory response to CO2 by approximately 37%. These results showcase Nalcn physiological function as well as broad scale application of result. Since these results were obtained by the selective depletion of Phox-2b expressing RTN neurons in-vivo, the physiological effects of breathing (i. e. ventilatory responses to CO2) can be more easily assessed and applied to the mentioned respiratory diseases.

Significance of Results

This study presents a cell-specific role for Nalcn (controls CO2 respiratory reflex in RTN neurons) in a defined physiological function (breathing). This study also developed a method to remove Nalcn expression in neurons, effectively removing them from the cell. This method could potentially be applied to other cell types where Nalcn is expressed. This paper has not changed the way of thinking in the field, nor did it refute any existing mechanisms or models. This study supports the notion that “leak” channels such as Nalcn are a mechanism used to maintain RMP. This study demonstrated that depletion of Nalcn decreases RTN neuronal firing rates due to a hyperpolarized cell membrane. This hyperpolarization was shown to be caused by the loss of a TTX-resistant background Na+ current. This current depolarizes the cell membrane, so its removal results in action potentials being less likely to occur in RTN neurons. In addition to the roles listed above, Nalcn also has cell-specific functions, such as excitation of RTN neurons when activated by SP. However, Nalcn is also found in many other types of neurons in other parts of the body. Other experiments could be conducted to see if Nalcn has any other specific roles in other cell-types where it is expressed.

Critique of Study

The article reviewed was published under “The Journal of Neuroscience”. According to the database search engine Web of Science, this article had an impact factor of 5. 97 in 2017 and 6. 517 over the course of five years from 2012 to 2017. This article had a fairly high impact factor of between 5-6 throughout the past five years, meaning that the information is condensed and had been cited multiple times. It assumes that the reader has in-depth knowledge of the topic with an understanding of various experiments and how they are executed. Many abbreviations in this article were not explained or defined which makes it difficult for the reader to interpret the data and understand the methods being applied. A prominent issue with in vitro study that may result in uncredible results is using HEK239T cells to assess knockdown efficiency of each shRNA hairpin as the physiological effects manifested by breathing cannot be assessed. It can be argued that these cells are embryonic and therefore have the potential of differentiating into RTN cells or tissue involved in breathing. However, a complete and thorough assessment requires all confounding variables to be mirrored for credible and accurate results. Applying that principle to this study, in order to identify knockdown efficiency, researchers could integrate the virus construct in RTN cells. By doing so, the purpose of the study is served: changing parameters of which RTN cells exist, and following the corresponding outcome. Another concern lies in the explanation of the conditions in which Nalcn exists. The experiment involving virus production stated that shRNA was utilized for knockdown of Nalcn. It was stated that the shRNA was about 70% effective, but other locations where Nalcn was present were not tested. Considering the location of Nalcn is imperative to determine the effectiveness of Nalcn shRNA.

What can be seen as illogical, however, is RTN neuron type II. This neuron type, as established by the study, is found in alkaline environments (pH>7. 42), and hence will be encompassed by negatively charged ions (OH-); there will be an overall negative charge outside of the RTN, as well as inside. The sodium ions that are meant to leak in, will face difficulty moving down its concentration gradient to facilitate neurotransmission as its cationic nature will encourage it to stay in a more anionic environment. Biology dictates sodium flow down its concentration gradient, while chemistry declares that cations will fundamentally be inherently attracted to anions. This raises confusion as it is not thoroughly addressed through the paper. Lack of clarity is amplified when the paper establishes that RTN neuron type II is more greatly expressed than RTN neuron type I; this fundamental issue, which has not been explored in the paper, has been further regurgitated by declaring that fact. To enhance this study, authors should have explored this contradiction, and establish how type II RTN neurons can exist in basic conditions.