Isolectin B(4)-positive and -negative nociceptors are functionally distinct

Abstract

Small-diameter sensory neurons that are primarily nociceptors can be divided neurochemically into two populations: isolectin B(4) (IB(4))-positive nonpeptidergic neurons, and IB(4)-negative peptidergic neurons. It has been shown that IB(4)-positive neurons depend on glial-derived neurotrophic factor (GDNF), whereas IB(4)-negative neurons depend on NGF for survival during postnatal development (Molliver et al., 1997). Furthermore, these two populations of nociceptors terminate in distinct regions of the superficial spinal cord. To date, however, no evidence exists that indicates whether these two groups of nociceptors have distinct functional roles in the process of nociception (Snider and McMahon, 1998). To search for functional differences, we performed whole-cell voltage and current-clamp recordings on acutely isolated adult mouse dorsal root ganglion neurons that were labeled with fluorescent IB(4). We found that IB(4)-positive neurons have longer-duration action potentials, higher densities of TTX-resistant sodium currents, and smaller noxious heat-activated currents than IB(4)-negative neurons. Furthermore, we show that NGF, but not GDNF, directly increases the number of neurons that respond to noxious heat. The different electrophysiological properties expressed by IB(4)-positive and -negative small neurons, including their different heat sensitivities, indicates that they may relay distinct aspects of noxious stimuli both acutely and after injury in vivo.

Fig. 1.

A, Left, Fluorescent and phase-contrast images of field of sensory neurons fixed after labeling with IB₄ -FITC. Right, Distribution of IB₄-positive and -negative sensory neurons by soma diameter. Dorsal root ganglion neurons from all spinal levels were isolated, placed in culture, and then fixed and stained 24 hr later (n = 4 mice, 530 neurons). B,Left, Profile of a somatic action potential (AP) before (top) and after (bottom) addition of IB₄-FITC. This neuron was IB₄ positive. Note that binding of IB₄ did not alter the shape or duration of the AP. Right, In six IB₄-positive neurons, IB₄ binding did not alter the duration of the somatic AP.

Fig. 2.

A, Examples of APs in IB₄-positive and -negative neurons with (top) and without (bottom) inflections on the falling phase. Insets show the first derivative of each spike and illustrate that the APs in the top have an inflection, whereas those in bottom do not. Note that IB₄-positive neurons have longer duration APs than IB₄-negative neurons. B, Mean duration of AP measured at 75% of spike amplitude. The asteriskindicates that the duration of APs in IB₄-positive neurons is significantly longer than in IB₄-negative neurons (p < 0.05; t test).

Fig. 3.

A, Examples of whole-cell voltage-clamp recordings under conditions selective for Na⁺ currents in an IB₄-negative and an IB₄-positive neuron. TTX-resistant Na⁺current was recorded in the presence of 1 μm TTX, and TTX-sensitive current was acquired by electronically subtracting the TTX-resistant current from the Total Na⁺current. The overshoot at one test-pulse in the calculated TTX-sensitive component of the IB₄-positive neuron was caused by variability in the voltage clamp and electronically subtracting the TTX-resistant current from the total current.B, Mean TTX-sensitive Na⁺ current density for all neurons at different test potentials. Peak TTX-sensitive currents were divided by total cell capacitance to give the current density. C, Mean TTX-resistant Na⁺ current density for all neurons. TTX-resistant currents were recorded in the presence of 1 μm TTX, and peak currents were corrected for cell capacitance.

Fig. 4.

A, Examples of whole-cell voltage-clamp recordings from three different small-diameter neurons and their responses to a ramp of heated buffer applied to cell membrane (24–49°C in 10 ± 1 sec). Neuron that did not respond to heat (left) was IB₄ negative, neuron with small inward current (middle) was IB₄ positive, and neuron with large inward current (right) was IB₄ negative. B, Left, Percentage of IB₄-positive and -negative neurons that responded to a noxious heat stimulus. Right, Magnitude of heat-evoked currents. The asterisk indicates that currents in IB₄-negative neurons were significantly larger than those in IB₄-positive neurons (p < 0.05; χ² test).C, Cultures of neurons were treated with no growth factor (Control), NGF (100 ng/ml), or GDNF (50 ng/ml) for 24 hr and then tested for responses to noxious heat. Values in parenthesesindicate the number of responding cells per number of cells tested for each group. Single asterisk indicates that percentage of responding cells in NGF-treated IB₄-negative neurons is significantly different from that of IB₄-negative control neurons. Double asterisk indicates that percentage of responding cells in NGF-treated IB₄-positive neurons is significantly different from that of IB₄-positive control neurons. D, Mean inward currents evoked in neurons treated with no growth factor, NGF, or GDNF for 24 hr. Values inparentheses indicate the number of responding cells in each group.

Fig. 5.

Putative roles of IB₄-positive and -negative neurons in nociceptive function.