Sensorimotor function is modulated by the serotonin receptor 1d, a novel marker for gamma motor neurons

Abstract

Gamma motor neurons (MNs), the efferent component of the fusimotor system, regulate muscle spindle sensitivity. Muscle spindle sensory feedback is required for proprioception that includes sensing the relative position of neighboring body parts and appropriately adjust the employed strength in a movement. The lack of a single and specific genetic marker has long hampered functional and developmental studies of gamma MNs. Here we show that the serotonin receptor 1d (5-ht1d) is specifically expressed by gamma MNs and proprioceptive sensory neurons. Using mice expressing GFP driven by the 5-ht1d promotor, we performed whole-cell patch-clamp recordings of 5-ht1d::GFP⁺ and 5-ht1d::GFP⁻ motor neurons from young mice. Hierarchal clustering analysis revealed that gamma MNs have distinct electrophysiological properties intermediate to fast-like and slow-like alpha MNs. Moreover, mice lacking 5-ht1d displayed lower monosynaptic reflex amplitudes suggesting a reduced response to sensory stimulation in motor neurons. Interestingly, adult 5-ht1d knockout mice also displayed improved coordination skills on a beam-walking task, implying that reduced activation of MNs by Ia afferents during provoked movement tasks could reduce undesired exaggerated muscle output. In summary, we show that 5-ht1d is a novel marker for gamma MNs and that the 5-ht1d receptor is important for the ability of proprioceptive circuits to receive and relay accurate sensory information in developing and mature spinal cord motor circuits.

Figure 1. 5-ht1d marks gamma motor neurons and a subset of proprioceptive sensory neurons

(A) In situ hybridization on P11 lumbar spinal cord section showing expression of 5-ht1d in the motor columns. (B) In situ hybridization of adult brain sections showing no detectable expression of 5-ht1d in the facial nucleus (VII; left) while expression can be detected in a few cells in the abducens nucleus (VI; right). Arrow indicates expression in lateral paragigantonuclear nucleus. (C) Double in situ hybridization after development of VAChT alone (left) and after development of 5-ht1d on the same section (middle) shows co-expression of 5-ht1d and VAChT in a subset of motor neurons in P11 lumbar spinal cord. (right) Histogram of soma area estimation of 5-ht1d⁺VAChT⁺ (black bars) and 5-ht1d-VAChT⁺ (white bars) neurons. (D) Expression of 5-ht1d in P11 Egr3^+/+ (top left) and Egr3^−/− (top middle) lumbar spinal cord. Expression of VAChT in P11 Egr3^+/+ (bottom left) and Egr3^−/− (bottom middle) lumbar spinal cord. Quantification of 5-ht1d-expressing cells in lumbar lateral motor column (LMC) shows a significant reduction in Egr3^−/− mice (right; p < 0.0001, Mann-Whitney U-test, mean ± SEM). (E) Expression of GFP (left), parvalbumin (middle) and merged image (right) in lumbar dorsal root ganglia from P2 5-ht1d∷GFP mice.

Figure 2. Electrophysiological properties of 5-ht1d∷GFP⁺ MNs

(A) top: Image of a patched 5-ht1d∷GFP⁺ MN under bright-field light (left), fluorescence (middle) and merged (right). bottom: Cell in (A) filled with biocytin (b1) together with another 5-ht1d∷GFP⁺ (b2) MN and a 5-ht1d∷GFP⁻ (b3) MN. (B) top: Firing properties in response to a +50pA current step showing regular spiking and to a −350pA current step showing a depolarizing sag and rebound spike. bottom: Firing in response to a +350pA, 2s duration, current step shows the initial doublet of spikes followed by regular spiking reaching a steady state frequency at the second half of the trace. inset: the initial doublet at higher magnification. (C) Scatterplot of rheobase current vs. after-hyperpolarization (AHP) decay-time for gamma MNs (black circles), fast alpha MNs (stars) and slow alpha MNs (squares). (D) Example of action potentials from a gamma MN (black line), a fast-like alpha MN (dashed line) and a slow-like alpha MN (grey line) with representative rheobase value, AP half-width and delay to spike.

Figure 3. 5-ht1d is not required for gamma motor neuron survival

(A) In situ hybridization on adult lumbar spinal cord section showed expression of 5-ht1d mRNA in 5-ht1d^+/+ lateral motor columns. No cells positive for 5-ht1d mRNA were found in 5-ht1d^−/− spinal cords. (B) In situ hybridization on adult lumbar spinal cord section of VAChT mRNA present in 5-ht1d^+/+ and 5-ht1d^−/− lateral motor columns (left). Histogram showing frequency distribution of VAChT⁺ motor neuron soma areas in lateral motor column from adult 5-ht1d^+/+ and 5-ht1d^−/− spinal cord section (right).

Figure 4. Serotonin-related genes are expressed by muscle spindles

Images of in situ hybridization experiments of the same anatomical location in serial sections of P2 hind limb muscle tissue stained for the muscle spindle marker Egr3 and the serotonin-related genes Aadc and VMAT2 (top row) and Tph1 (bottom row).

Figure 5. Normal innervation of muscle spindles in 5-ht1d^−/− mice

(A) Confocal images of muscle spindles in the tibialis anterior muscle from P13 5-ht1d^+/+ (left) and 5-ht1d^−/− (right) mice. Sensory axons (VGLUT1⁺TUJ1⁺) and gamma motor axons (VGLUT1⁻TUJ1⁺) were present and distributed similarly in the muscle spindles of control and 5-ht1d^−/− mice . Bungarotoxin (BTX) binds to postsynaptic receptors of motor synapses. (B) Confocal images of motor neuron synapses in muscle spindles visualized with Calbindin (muscle spindle fiber), VAChT (presynaptic motor terminal) and BTX (postsynaptic motor terminal). Quantification of VAChT⁺ BTX⁺ neuromuscular junctions on muscle spindles showed a similar motor innervation between 5-ht1d^−/− mice and control mice (mean ± SEM, two-tailed student's t-test, p = 0.86).

Figure 6. 5-ht1d^−/− mice show reduced amplitude of monosynaptic sensorimotor connections

(A) VGLUT1⁺ contacts on ChAT⁺ motor neurons in adult (P30) thoracic spinal cord of 5-ht1d^+/+ and 5-ht1d^−/− mice (left). (B) Quantification of VGLUT1⁺ contacts on motor neurons (left; 5-ht1d^+/+, n = 57, and 5-ht1d^−/−, n = 51, from 3 animals per genotype; Mann-Whitney U-test, p = 0.33). Bar graph illustrating the ratio of motor neurons with (black) and without (white) detectable VGLUT1 contacts (right, 5-ht1d^+/+, n = 57, and 5-ht1d^−/−, n = 51, from 3 animals per genotype; Chi-squared test, p = 0.17). (C) Schematic of experimental setup for recording responses in ventral root after dorsal root stimulations. (D) Example of averaged responses in L3 ventral roots from a P5 5-ht1d^+/+ (left) and a P3 5-ht1d^−/− (right) mouse. Horizontal arrows indicate monosynaptic and polysynaptic peaks, vertical arrows indicate onset of monosynaptic and polysynaptic episodes. Shaded area indicates part of plot used to estimate polysynaptic area. (E) Graph of amplitude (left) and latency (right) of monosynaptic peak in 5-ht1d^+/+ mice and 5-ht1d^−/− mice (Mean ± SEM, students t-test, p = 0.034 (amplitude), p = 0.17 (latency), n=10 5-ht1d^+/+ mice and n=7 5-ht1d^−/− mice). (F) Graph of amplitude (left), latency (middle) and area (right) of polysynaptic peak in neonatal 5-ht1d^+/+ mice and 5-ht1d^−/− mice (Mean ± SEM, students t-test, p = 0.35 (amplitude), p = 0.88 (latency), p = 0.16 (area) n=10 5-ht1d^+/+mice and n=7 5-ht1d^−/− mice).

Figure 7. Improved motor coordination in 5-ht1d^−/− mice

(A) Graph of the max rotational speed on the rotarod before fall for adult 5-ht1d^+/+ mice and 5-ht1d^−/− mice (Mean ± SEM, 2-way ANOVA, p=0.58, n=10 5-ht1d^+/+mice and n=10 5-ht1d^−/− mice). (B) Hind limb slips when running over a 12 mm beam (left) and time spent crossing the beam (right) of 5-ht1d^+/+ mice and 5-ht1d^−/− mice (Mean ± SEM, students t-test, p = 0.040 (slips), p = 0.99 (time) n=8 5-ht1d^+/+mice and n=7 5-ht1d^−/− mice). (C) Time spent climbing onto a horizontal hanging steel wire with the hind paws in 5-ht1d^+/+ (black) and 5-ht1d^−/− (white) mice (Chi-square test, p = 0.037, n=10 5-ht1d^+/+ mice and n=9 5-ht1d^−/− mice).