Characterisation of cold-selective lamina I spinal projection neurons

Reviewer #3 (Public review):

Summary:

Razlan and colleagues provide a detailed anatomical characterization of lamina I projection neurons in the mouse spinal cord that are densely innervated by primary afferents activated by cooling of the skin. The authors, building on their previous anatomical work, validate a Trpm8-Flp mouse line, show synaptic contacts between Trpm8⁺ boutons and projection neurons at the ultrastructural level, and demonstrate at the physiological level that these neurons specifically respond to cooling stimuli. Next, by taking advantage of their previous transcriptomic analysis of ALS neurons, they identify calbindin as a marker for cold-activated lamina I projection neurons and map their ascending projections to the rostral lateral parabrachial area, caudal periaqueductal gray, and ventral posterolateral thalamus, well-known thermosensory and thermoregulatory centers. Altogether, these findings provide strong anatomical and functional evidence for a direct line of transmission from Trpm8⁺ sensory afferents through Calb1⁺ lamina I neurons to key supraspinal centers controlling perception of cold and thermoregulatory responses.

Strengths:

The combination of mouse genetics, electron microscopy, ex vivo physiology, and viral tracing provides convincing evidence for a direct cold pathway. The work validates the Trpm8-Flp line by extensive anatomical and molecular characterization. Integration with previous transcriptomic and anatomical data neatly links the cold-selective lamina I neurons to a molecularly defined cluster of ALS neurons, strengthening the bridge between molecular identity, anatomy, and physiological function.

Weaknesses:

While anatomical evidence for direct synaptic connectivity between Trpm8+ afferents and lamina I projection neurons is compelling, a physiological demonstration of strict monosynaptic transmission is not shown. The conclusion that these inputs are exclusively monosynaptic should be toned down. Similarly, the statement that "Lamina I ALS neurons that are surrounded by Trpm8 afferents are cold-selective" should also be toned down as only a few neurons have been tested and it cannot be excluded that other neurons with similar characteristics may be polymodal.

Reviewer #1 (Public review):

Summary:

Spinal projection neurons in the anterolateral tract transmit diverse somatosensory signals to the brain, including touch, temperature, itch, and pain. This group of spinal projection neurons is heterogeneous in their molecular identities, projection targets in the brain, and response properties. While most anterolateral tract projection neurons are multimodal (responding to more than one somatosensory modality), it has been shown that cold-selective projection neurons exist in lamina I of the spinal cord dorsal horn. Using a combination of anatomical and physiological approaches, the authors discovered that the cold-selective lamina I projection neurons are heavily innervated by Trpm8+ sensory neuron axons, with calb1+ spinal projection neurons primarily capturing these cold-selective lamina I projection neurons. These neurons project to specific brain targets, including the PBNrel and cPAG. This study adds to the ongoing effort in the field to identify and characterize spinal projection neuron subtypes, their physiology, and functions.

Strengths:

(1) The combination of anatomical and physiological analyses is powerful and offers a comprehensive understanding of the cold-selective lamina I projection neurons in the spinal cord dorsal horn. For example, the authors used detailed anatomical methods, including EM imaging of Trpm8+ axon terminals contacting the Phox2a+ lamina I projection neurons. Additionally, they recorded stimulus-evoked activity in Trpm8-recipient neurons, carefully selected by visual confirmation of tdTomato and GFP juxtaposition, which is technically challenging.

(2) This study identifies, for the first time, a molecular marker (calb1) that labels cold-selective lamina I projection neurons. Although calb1+ projection neurons are not entirely specific to cold-selective neurons, using an intersectional strategy combined with other genes enriched in this ALS group or cold-induced FosTRAP may further enhance specificity in the future.

(3) This study shows that cold-selective lamina I projection neurons specifically innervate certain brain targets of the anterolateral tract, including the NTS, PBNrel, and cPAG. This connectivity provides insights into the role of these neurons in cold sensation, which will be an exciting area for future research.

Weaknesses:

(1) The sample size for the ex vivo electrophysiology is small. Given the difficulty and complexity of the preparation, this is understandable. However, a larger sample size would have strengthened the authors' conclusions.

(2) The authors used tdTomato expression to identify brain targets innervated by these cold-selective lamina I projection neurons. Since tdTomato is a soluble fluorescent protein that fills the entire cell, using synaptophysin reporters (e.g., synaptophysin-GFP) would have been more convincing in revealing the synaptic targets of these projection neurons.

(3) The summary cartoon shown in Figure 7 can be misleading because this study did not determine whether these cold-selective lamina I projection neurons have collateral branches to multiple brain targets or if there are anatomical subtypes that may project exclusively to specific targets. For example, a recent study (Ding et al., Neuron, 2025) demonstrated that there are PBN-projecting spinal neurons that do not project to other rostral brain areas. Furthermore, based on the authors' bulk labeling experiments, the three main brain targets are NTS, PBNrel, and cPAG. The VPL projection is very sparse and almost negligible.

Reviewer #2 (Public review):

Summary:

In this study, the authors took advantage of a semi-intact ex vivo somatosensory preparation that includes hindlimb skin to characterize the response of projection neurons in the dorsal horn of the spinal cord to peripheral stimulation, including cold thermal stimuli. The main aim was to characterize the connectivity between peripheral afferents expressing the cold-sensing receptor TRPM8 and a set of genetically tagged neurons of the anterolateral system (ALS). These ALS neurons expressed high levels of the calcium-binding protein calbindin 1.

In addition, combining different viral tracing methods, the authors could identify the anatomical targets of this specific subset of projection neurons within the brainstem and diencephalon.

Strengths:

The use of a relatively new (seldom used previously) transgenic line to label TRPM8-expressing afferents, combined with the genetic characterization of a previously identified subset of projection neurons, adds a specificity to the characterization. The transgenic line appears to capture well the subpopulation of Trpm8-expressing neurons

In addition, the use of electron microscopy techniques makes the interpretation of the structural contacts more compelling.

The writing is clear, and the presentation of findings follows a logical flow.

Overall, this study provides solid, novel information about the brain circuits involved in cold thermosensation.

Weaknesses:

In the characterization of recorded neurons in close contact or in the absence of this contact with TRPM8 afferents, the number of recorded neurons is relatively low. In addition, the strength of thermal stimuli is not very well controlled, preventing a more precise characterization of the connectivity.

The authors could provide some sense of the effort needed to record from the 6 cold-activated neurons described. How many preparations were needed, etc?

Reviewer #3 (Public review):

Summary:

Razlan and colleagues provide a detailed anatomical characterization of lamina I projection neurons in the mouse spinal cord that are densely innervated by primary afferents activated by cooling of the skin. The authors, building on their previous anatomical work, validate a Trpm8-Flp mouse line, show synaptic contacts between Trpm8⁺ boutons and projection neurons at the ultrastructural level, and demonstrate at the physiological level that these neurons specifically respond to cooling stimuli. Next, by taking advantage of their previous transcriptomic analysis of ALS neurons, they identify calbindin as a marker for cold-activated lamina I projection neurons and map their ascending projections to the rostral lateral parabrachial area, caudal periaqueductal gray, and ventral posterolateral thalamus, well-known thermosensory and thermoregulatory centers. Altogether, these findings provide strong anatomical and functional evidence for a direct line of transmission from Trpm8⁺ sensory afferents through Calb1⁺ lamina I neurons to key supraspinal centers controlling perception of cold and thermoregulatory responses.

Strengths:

The combination of mouse genetics, electron microscopy, ex vivo physiology, and viral tracing provides convincing evidence for a direct cold pathway. The work validates the Trpm8-Flp line by extensive anatomical and molecular characterization. Integration with previous transcriptomic and anatomical data neatly links the cold-selective lamina I neurons to a molecularly defined cluster of ALS neurons, strengthening the bridge between molecular identity, anatomy, and physiological function.

Weaknesses:

While anatomical evidence for direct synaptic connectivity between Trpm8+ afferents and lamina I projection neurons is compelling, a physiological demonstration of strict monosynaptic transmission is not shown. The conclusion that these inputs are exclusively monosynaptic should be toned down. Similarly, the statement that "Lamina I ALS neurons that are surrounded by Trpm8 afferents are cold-selective" should also be toned down as only a few neurons have been tested and it cannot be excluded that other neurons with similar characteristics may be polymodal.