Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy

Abstract

We report on an 'unbiased' molecular characterization of individual, adult neurons, active in a central, anterior hypothalamic neuronal circuit, by establishing cDNA libraries from each individual, electrophysiologically identified warm sensitive neuron (WSN). The cDNA libraries were analyzed by Affymetrix microarray. The presence and frequency of cDNAs were confirmed and enhanced with Illumina sequencing of each single cell cDNA library. cDNAs encoding the GABA biosynthetic enzyme Gad1 and of adrenomedullin, galanin, prodynorphin, somatostatin, and tachykinin were found in the WSNs. The functional cellular and in vivo studies on dozens of the more than 500 neurotransmitters, hormone receptors and ion channels, whose cDNA was identified and sequence confirmed, suggest little or no discrepancy between the transcriptional and functional data in WSNs; whenever agonists were available for a receptor whose cDNA was identified, a functional response was found. Sequencing single neuron libraries permitted identification of rarely expressed receptors like the insulin receptor, adiponectin receptor 2 and of receptor heterodimers; information that is lost when pooling cells leads to dilution of signals and mixing signals. Despite the common electrophysiological phenotype and uniform Gad1 expression, WSN transcriptomes show heterogeneity, suggesting strong epigenetic influence on the transcriptome. Our study suggests that it is well-worth interrogating the cDNA libraries of single neurons by sequencing and chipping.

Figure 1

Figure 1A. Warm sensitive neurons in the Preoptic area that were subject of single cell transcriptomics study by chipping and sequencing. Thermoregulatory network is depicted showing the central integrative role of temperature sensitive GABAergic projection neurons (highlighted in red) in activation of thermogenesis in Brown Adipose Tissue, respiratory rate and cardiac output. It depicts data from multiple retrograde tracing studies that show that the inhibitory warm sensitive neurons project to DMH and or rRPA., and that the skin temperature and deep body temperature is affecting the activity of these neurons through Glutamatergic afferents while the local temperature is belived to exert its effect through circulation communicated heat affects. POA: Preoptic Area, DMH: Dorsomedial Hypothalamus, rRPA: rostral Raphe Pallidus, DRG: Dorsal Root Ganglion, BAT: Brown Adipose Tissue, LPB: Lateral parabrachial nucleus. Adapted from Nakamura, K. and S. F. Morrison (2008) and modified with our data. Figure 1B. Flow chart of experimental design for single cell transcriptomics-based identification and in vitro and in vivo validation of the functional secretome and receptor repertoire of individual warm sensitive neurons. The numbers in red show the number of transcripts identified in individual neuron cDNA libraries by chipping using the Affymetrix mouse microarray, in the warm sensitive neurons, the number of cDNAs confirmed by NextGen sequencing that produces reads of 48–100 nucleotides, and accepting reads that are fully matching, in the functional validation experiments involved agonists that were tested electrophysiologically on spontaneously active warm sensitive neurons in slice (at least on ten to fifteen such neurons), 31of these agonist for recptors whose cDNA we identified, were found able to affect the behavior of the cells, i.e., have functional receptors expressed on warm sensitive cells, and the remaining ones may not havefunctional receptors expressed or the receptors do not affect the tested electrophysiological properties, or the number of cells tested was too low to find a cell with a functional receptor expressed, 21 recptor agonists were injected into the POA as cDNA encoding their receptor, or channel was found, 18 of these produced hypo- or hyperthermic response at the 0.01 to 10 nanomoles dose.

Figure 1

Figure 1A. Warm sensitive neurons in the Preoptic area that were subject of single cell transcriptomics study by chipping and sequencing. Thermoregulatory network is depicted showing the central integrative role of temperature sensitive GABAergic projection neurons (highlighted in red) in activation of thermogenesis in Brown Adipose Tissue, respiratory rate and cardiac output. It depicts data from multiple retrograde tracing studies that show that the inhibitory warm sensitive neurons project to DMH and or rRPA., and that the skin temperature and deep body temperature is affecting the activity of these neurons through Glutamatergic afferents while the local temperature is belived to exert its effect through circulation communicated heat affects. POA: Preoptic Area, DMH: Dorsomedial Hypothalamus, rRPA: rostral Raphe Pallidus, DRG: Dorsal Root Ganglion, BAT: Brown Adipose Tissue, LPB: Lateral parabrachial nucleus. Adapted from Nakamura, K. and S. F. Morrison (2008) and modified with our data. Figure 1B. Flow chart of experimental design for single cell transcriptomics-based identification and in vitro and in vivo validation of the functional secretome and receptor repertoire of individual warm sensitive neurons. The numbers in red show the number of transcripts identified in individual neuron cDNA libraries by chipping using the Affymetrix mouse microarray, in the warm sensitive neurons, the number of cDNAs confirmed by NextGen sequencing that produces reads of 48–100 nucleotides, and accepting reads that are fully matching, in the functional validation experiments involved agonists that were tested electrophysiologically on spontaneously active warm sensitive neurons in slice (at least on ten to fifteen such neurons), 31of these agonist for recptors whose cDNA we identified, were found able to affect the behavior of the cells, i.e., have functional receptors expressed on warm sensitive cells, and the remaining ones may not havefunctional receptors expressed or the receptors do not affect the tested electrophysiological properties, or the number of cells tested was too low to find a cell with a functional receptor expressed, 21 recptor agonists were injected into the POA as cDNA encoding their receptor, or channel was found, 18 of these produced hypo- or hyperthermic response at the 0.01 to 10 nanomoles dose.

Figure 2. Warm sensitive neurons (n=8) included into the transcriptomics study are all GABAergic

A. Electrophysiological characterization of warm sensitive neurons: as the temperature of the bath in which the Preoptic area tissue slice is suspended increases, the firing rate of warm sensitive neurons increases. B. Firing rate and thermal coefficients of warm sensitive neurons whose transcriptome was examined by microarray analysis of their cDNA library. C. PCR confirmation of GAD1 in 4 out of 8 WS cells, it should however be noted that Affymetrix chipping and NextGen sequencing, both, showed that 8 of 8 of the warm sensitive neurons expressed the GAD1 cDNA.

Figure 3. The experimental paradigm: Identification and functional validation of a Prototypic molecular component of the warm sensitive neuron receptor repertoire; the pyrogen receptor, IL-1R1, and its downstream signaling components

Earlier electrophysiological and binding studies have shown IL-1R1 expression in the POA. These data thus serve as confirmation of the chipping, PCR, sequencing, electrophysiological and CBT measurement based functional identification strategy for receptors on warm sensitive neurons. A. Many components of the Interleukin 1 signaling pathway were identified by microarray analysis and NextGen sequencing of cDNA libraries from warm sensitive neurons. Transcripts found to be expressed with a significant p value of detection are shaded grey. B. Application of IL-1β (0.1nM) hyperpolarizes the cell and inhibits the spontaneous firing rate of POA/AH warm sensitive neurons. C. POA injection of IL-1β (150ng/0.5ul) elicited increases in core body temperature in wild type (wild type of what?) treated mice relative to vehicle treated mice. D. These changes in body temperature are not due to differences in motor activity of the IL-1β treated mice relative to the vehicle treated control mice.

Figure 4

Figure 4A. Cluster analysis of warm sensitive neurons. The warm sensitivity of individual hypothalamic neurons was assessed electrophysiologically and the cytoplasmic contents of the cell harvested by aspiration. During the third round of aRNA amplification, the amplified single cell transcriptome was labeled with biotin for use as a probe for microarray analysis. The probes were analyzed on Illumina long oligonucleotide arrays and the resultant mRNA hybridization intensities quantified and the results were analyzed by GeneSpring (Agilent) analysis and displayed as a heatmap of normalized intensities. This analysis shows that the warm sensitive neurons do cluster together, but with some variation. Explain WS1–WS7 Figure 4B. Go - diagram on functional class distribution of the transcripts that were confirmed by NextGene sequencing of the cDNA libraries of 2 warm sensitive neurons, in separate experiments. This pie chart shows the fraction of extent cellular mRNAs that are present in GO designated functional classes. Approximately 8% of the cellular mRNA encodes receptors: ie cell membrane receptors, nuclear recptors.

Figure 4

Figure 4A. Cluster analysis of warm sensitive neurons. The warm sensitivity of individual hypothalamic neurons was assessed electrophysiologically and the cytoplasmic contents of the cell harvested by aspiration. During the third round of aRNA amplification, the amplified single cell transcriptome was labeled with biotin for use as a probe for microarray analysis. The probes were analyzed on Illumina long oligonucleotide arrays and the resultant mRNA hybridization intensities quantified and the results were analyzed by GeneSpring (Agilent) analysis and displayed as a heatmap of normalized intensities. This analysis shows that the warm sensitive neurons do cluster together, but with some variation. Explain WS1–WS7 Figure 4B. Go - diagram on functional class distribution of the transcripts that were confirmed by NextGene sequencing of the cDNA libraries of 2 warm sensitive neurons, in separate experiments. This pie chart shows the fraction of extent cellular mRNAs that are present in GO designated functional classes. Approximately 8% of the cellular mRNA encodes receptors: ie cell membrane receptors, nuclear recptors.

Figure 5. Abundance distribution of G-protein coupled receptor cDNAs in warm sensitive neurons

The Y-axis represents the read counts for the mRNAs encoding predicted G-protein coupled receptors that are present in these cells. The X-axis is the different genes that are expressed in these cells. The read counts at the far right of the graph appear to be 0 but are rather 1 or 2 which shows up as 0 on a Log(N) scale. There are receptors at very high levels such as Frizzled Homolog 3 (1858 reads) and the NPY receptor, Y1 (774 reads) and several others represented by only 2 or 3 read counts such as the orphan G-protein coupled receptor 133 or melanocortin 5 receptor. The large difference in RNA read counts for this class of molecules suggests a uniqueness to the receptor encoding repertoire of these cells and by analogy a functional uniqueness. The presence of (nearly) 35 receptor mRNAs at 1–2 read counts suggests that these mRNAs are either expressed at low levels or expressed in a subregion of the cell such as the dendrite where low levels of expression could have dramatic effects. Alternatively, the presence of these mRNAs shows that these genes are, epigenetically, able to be transcribed and with the proper stimulation may produce transcribed to higher abundances. In this model the low expressers may be an indicator of the additional epigenetic capacity of these warm sensitive neurons to respond to stimuli. Excluded from this analysis are the olfactory receptors.

Figure 6. Olfactory receptors in warm sensitive neurons

These circle graphs show the distribution of predicted olfactory receptors in individual warm sensitive neurons as reflected in RNAseq read counts. One cell is represented by the outer circle and the other by the inner circle. The read counts for a particular receptor are represented by the arc around the circle that each colored box circumscribes. The range in expression of the genes in these cells of this class of receptors is ranging from 1 (in both cells) to 71 (warm sensitive neuron 1) and 541 (warm sensitive neuron 2). There are greater than 30 types of olfactory receptors expressed per cell with half of them expressed at greater than 2 read counts. The colors that are shared between the circles do not represent the same gene. None of the olfactory receptors found in one warm sensitive neuron were found in the other neuron suggesting exclusivity to the pattern of olfactory receptor expression in hypothalamic neurons.

Figure 7. Functional validation in vitro and in vivo of the receptor transcripts identified in single warm sensitive neuron cDNA libraries: Hypothermic and hyperthermic effects can be exerted through receptors of warm sensitive neurons identified by single cell transcriptomics and validated by use of the recptor agonist injected into the POA and tested on the firing rate of WSN in POA slice

Bombesin acting at BSR3 receptor activates firing of the warm sensitive neurons and causes hypothermia upon POA injection, while Prolactin inhibits firing of warm sensitive neurons and upon injection into the POA causes hyperthermia, respectively.

Figure 8. Receptor repertoire and secretome of warm sensitive neurons in POA identified by microarray and confirmed by NextGen sequencing of individual neuron cDNA libraries

The mRNA was harvested from the soma, and it provides no information about where in the cell, dendrite, axon terminal, soma the translated proteins will be functional in these GABAergic peptidergic projection neurons. The figure depicts only such signal substances and receptors whose cDNA have been found by both microarray and confirmed by sequencing and in many cases by functional tests using recptor agonists.