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Data for: Targeted anatomical and functional identification of antinociceptive and pronociceptive serotonergic neurons that project to the spinal dorsal horn


Ganley, Robert (2023), Data for: Targeted anatomical and functional identification of antinociceptive and pronociceptive serotonergic neurons that project to the spinal dorsal horn, Dryad, Dataset,


Spinally-projecting serotonergic neurons play a key role in controlling pain sensitivity and can either increase or decrease nociception depending on physiological context. It is currently unknown how serotonergic neurons mediate these opposing effects. Utilizing virus-based strategies, we identified two anatomically separated populations of serotonergic hindbrain neurons located in the lateral paragigantocellularis (LPGi) and the medial hindbrain, which respectively innervate the superficial and deep spinal dorsal horn and have contrasting effects on pain perception. Our tracing experiments revealed an unexpected high selectivity of serotonergic neurons of the LPGi for transduction with spinally injected AAV2retro vectors while medial hindbrain serotonergic neurons were largely resistant to AAV2retro transduction. Taking advantage of this selectivity, we employed intersectional chemogenetic approaches to demonstrate that activation of the LPGi serotonergic projections decreases thermal sensitivity, whereas activation of medial serotonergic neurons increases sensitivity to mechanical von Frey stimulation. Together these results suggest that there are functionally distinct classes of serotonergic hindbrain neurons that differ in their anatomical location in the hindbrain, their postsynaptic targets in the spinal cord, and impact on nociceptive sensitivity. At least the LPGi neurons give rise to rather global and bilateral projections throughout the rostrocaudal extent of the spinal cord suggesting that they contribute to widespread systemic pain control.


Quantifcation of cells retrogradely labelled from the spinal cord

Animals were injected with AAVs at least 2 weeks before animals were perfusion fixed. Alternatively animals were injected with modified rabies virus at either 5 or 7 days before perfusion. Fixed hindbrains were sectioned at 60 micrometers, which were immunostained with primary antibodies raised against the antigens of interest. FIJI with cellcounter plugin were used to manually count cells, which were recorded and processed in Microsoft Excel. See methods in manuscript for further details.

Whole-cell recordings

Slice preparation and electrophysiology

Hindbrain slices were prepared from TPH2::Cre animals that had received a bilateral injection of AAV9.flex.tdTomato into the ventral hindbrain. Animals were aged 3-6 weeks at the time of injection and were prepared for electrophysiological recordings 1-2 weeks later. Animals were decapitated and the brain was rapidly dissected and placed in ice cold oxygenated dissection solution (containing in mM (65 NaCl, 105 sucrose, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, 0.5 CaCl2, 7 MgCl2). The hindbrain was isolated, glued to a block of 2% agarose and installed in a slicing chamber. Transverse slices of hindbrain were cut at 250 μm on a vibrating blade microtome (D.S.K microsclicer DTK1000), which were allowed to recover for at least 30 min in oxygenated aCSF at 34°C prior to recording, containing (in mM) 120 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 5 HEPES, 14.6 glucose, 2 CaCl2, 1 MgCl2), pH 7.35-7.40, osmolarity 305-315 mOsm.

During recording, slices were perfused with aCSF at a flow rate of 2-3 ml/min. Targeted recordings were taken from tdTomato-expressing neurons using glass microelectrodes filled with a K-gluconate internal solution (containing 130 K-Gluconate, 5 NaCl, 1 EGTA, 10 HEPES, 5 Mg-ATP, 0.5 Na-GTP, 2 biocytin). Whole-cell recordings were acquired using a HEKA EPC10 amplifier with Patchmaster software at a sampling frequency of 20 kHz (HEKA Elektronik). A biophysical characterization of passive and active membrane properties was performed in current and voltage clamp modes, and the access resistance was monitored between recordings using a 10 mV voltage step protocol. Data were excluded if the access resistance changed >30% during recording.

The relative position of the recorded/labelled neurons in each slice was noted, and slices were fixed overnight in 4% PFA at 4°C at the end of each experiment. Slices were immunoreacted with primary antibodies against tdTOM and TPH2, which were revealed the next day with secondary antibodies conjugated to Cy3 or Alexa 647. Biocytin was revealed with Avidin-Alexa A488 and the position of the filled neurons was assigned to the NRM or the LPGi, which could be determined based on the pattern of TPH2 immunoreactivity in the slice. Cells located outside of these two regions were not analyzed further

Behavioral/Chemogenetic experments

For specific DREADD-mediated activation of descending serotonergic pathways, an intersectional approach was used for labelling the lateral hindbrain neurons whereas direct labelling was used to label the medial neurons. TPH2::Cre mice received an intraspinal injection of AAV2retro.flex.FLPo.BFP, and one week later received bilateral injections into the ventral hindbrain with AAV1.FRT.hM3Dq.mCherry. Behavioral tests were performed after 10 days incubation time to allow the expression of the viral transgene. Before experiments mice were acclimatized to the behavioral setup for one hour. For the Hargreaves plantar, cold plantar, electronic von Frey, and Rotarod assays, six measurements were taken for each time point and an average of these was reported. All measurements were taken from both hindlimbs of all animals. Alternatively, serotonergic neurons in the medial hindbrain were labelled by injection of AAV8.hsyn.flex.hM3Dq into the NRM (injection coordinates -6, 0, 5.9) and the same behavioral tests were performed with the same experimental design.


Sensitivity to heat stimuli was assessed with the Hargreaves plantar assay (IITC). Mice were placed on a pre-heated transparent platform set to a temperature of 30°C, and the withdrawal latencies were recorded using a timed infrared heat source. A resting intensity of 5% and an active intensity of 20% was used for stimulations, and a maximum cutoff time of 32 s was set to avoid tissue damage.

Cold plantar assay

Mice were placed on a 5 mm borosilicate glass platform and were stimulated from beneath with dry ice pellets. The time taken to withdraw the paw was measured using a stopclock and a maximum stimulation time of 20 s was used to avoid tissue damage.

von Frey

Von Frey thresholds were measured using an electronic von Frey algesiometer (IITC). Animals were adapted on a mesh surface, and the plantar surface of each paw was stimulated with a bendable plastic filament attached to a pressure sensitive probe. Pressure was applied to the plantar surface in a linear manner until the animal withdrew its paw, and the maximum pressure (the pressure at which the animal withdrew) was displayed on the device.


Sensorimotor coordination was evaluated using an accelerating rotarod, and the time taken for animals to fall from the rotating barrel was recorded. The barrel rotated from 4-40 rpm over a period of 300 s, and increased speed constantly throughout each experiment. Values were discarded if the animal jumped from the barrel, and if the animal jumped in >50% of trials for a given time point these data were discarded from the experiment. Two training sessions were given for all animals prior to the experiment being started to ensure a stable performance in the absence of treatment.