DOI: 10.5061/dryad.h70rxwdm9
title: Data for: Targeted anatomical and functional identification of antinociceptive and pronociceptive serotonergic neurons that project to the spinal dorsal horn

description: 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.
description: <strong>Quantifcation of cells retrogradely labelled from
 the spinal cord</strong> 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.
 <strong>Whole-cell recordings</strong>
 
 <em>Slice preparation and
 electrophysiology</em> 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
 NaH<sub>2</sub>PO<sub>4</sub>, 25
 NaHCO<sub>3</sub>, 25 glucose, 0.5
 CaCl<sub>2</sub>, 7 MgCl<sub>2</sub>). 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 NaHCO<sub>3</sub>,
 1.25 NaH<sub>2</sub>PO<sub>4</sub>, 2.5 KCl, 5
 HEPES, 14.6 glucose, 2 CaCl<sub>2</sub>, 1
 MgCl<sub>2</sub>), 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 &gt;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 
 <strong>Behavioral/Chemogenetic
 experments</strong> 
 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.
 <em>Hargreaves</em> 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. <em>Cold plantar
 assay</em> 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. 
 <em>von Frey</em> 
 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.
 <em>Rotarod</em> 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 &gt;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.