Abstract
Neurons located in dorsal root ganglia (DRG) are crucial for transmitting peripheral sensations such as proprioception, touch, temperature, and nociception to the spinal cord before propagating these signals to higher brain structures. To date, difficulty in identifying modality-specific DRG neurons has limited our ability to study specific populations in detail. As the calcium-binding protein parvalbumin (PV) is a neurochemical marker for proprioceptive DRG cells we used a transgenic mouse line expressing green fluorescent protein (GFP) in PV positive DRGs, to study the functional and molecular properties of putative proprioceptive neurons. Immunolabeled DRGs showed a 100% overlap between GFP positive (GFP+) and PV positive cells, confirming the PVeGFP mouse accurately labeled PV neurons. Targeted patch-clamp recording from isolated GFP+ and GFP negative (GFP−) neurons showed the passive membrane properties of the two groups were similar, however, their active properties differed markedly. All GFP+ neurons fired a single spike in response to sustained current injection and their action potentials (APs) had faster rise times, lower thresholds and shorter half widths. A hyperpolarization-activated current (Ih) was observed in all GFP+ neurons but was infrequently noted in the GFP− population (100% vs. 11%). For GFP+ neurons, Ih activation rates varied markedly, suggesting differences in the underlying hyperpolarization-activated cyclic nucleotide-gated channel (HCN) subunit expression responsible for the current kinetics. Furthermore, quantitative polymerase chain reaction (qPCR) showed the HCN subunits 2, 1, and 4 mRNA (in that order) was more abundant in GFP+ neurons, while HCN 3 was more highly expressed in GFP− neurons. Likewise, immunolabeling confirmed HCN 1, 2, and 4 protein expression in GFP+ neurons. In summary, certain functional properties of GFP+ and GFP− cells differ markedly, providing evidence for modality-specific signaling between the two groups. However, the GFP+ DRG population demonstrates considerable internal heterogeneity when hyperpolarization-activated cyclic nucleotide-gated channel (HCN channel) properties and subunit expression are considered. We propose this heterogeneity reflects the existence of different peripheral receptors such as tendon organs, muscle spindles or mechanoreceptors in the putative proprioceptive neuron population.
Highlights
Our experience of somatosensory stimuli such as proprioception, pain, and touch, results from the detection of stimuli in the periphery by specialized receptors before the propagation of these signals along primary afferents
Input resistance was lower in green fluorescent protein (GFP)+ cells when compared with GFP− cells (152.18 ± 18.34 vs. 388.63 ± 68.79 MΩ, p = 0.01, n = 35 and 32, respectively)
This study used targeted patch-clamp electrophysiology and molecular analysis of hyperpolarization-activated cyclic nucleotidegated (HCN) channel subtypes to compare the properties of putative proprioceptive neurons (GFP+) with a group of non-proprioceptive ‘‘other’’ sensory populations (GFP−)
Summary
Our experience of somatosensory stimuli such as proprioception, pain, and touch, results from the detection of stimuli in the periphery by specialized receptors before the propagation of these signals along primary afferents. Primary afferents are the peripheral axons of pseudo-unipolar sensory neurons, whose cell bodies are clustered in dorsal root ganglia (DRG). Several studies have shown that Ih is present in both large (presumably proprioceptive) and small (nociceptive) diameter neuronal populations Their specific electrophysiological properties, are known to differ (Doan and Kunze, 1999; Gao et al, 2012). This suggests sensory neurons of different modalities express a unique pattern of hyperpolarization activated cyclic nucleotide gated (HCN) channels that form tetramers and can be comprised of four distinct subunits, HCN1-4. We undertook quantitative polymerase chain reaction (qPCR) and immunolabeling analysis to compare the expression of HCN subunits 1–4
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