pubmed.ncbi.nlm.nih.gov

Physiological temperatures drive glutamate release onto trigeminal superficial dorsal horn neurons - PubMed

  • ️Wed Jan 01 2014

Physiological temperatures drive glutamate release onto trigeminal superficial dorsal horn neurons

Tally M Largent-Milnes et al. J Neurophysiol. 2014.

Abstract

Trigeminal sensory afferent fibers terminating in nucleus caudalis (Vc) relay sensory information from craniofacial regions to the brain and are known to express transient receptor potential (TRP) ion channels. TRP channels are activated by H(+), thermal, and chemical stimuli. The present study investigated the relationships among the spontaneous release of glutamate, temperature, and TRPV1 localization at synapses in the Vc. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded from Vc neurons (n = 151) in horizontal brain-stem slices obtained from Sprague-Dawley rats. Neurons had basal sEPSC rates that fell into two distinct frequency categories: High (≥10 Hz) or Low (<10 Hz) at 35°C. Of all recorded neurons, those with High basal release rates (67%) at near-physiological temperatures greatly reduced their sEPSC rate when cooled to 30°C without amplitude changes. Such responses persisted during blockade of action potentials indicating that the High rate of glutamate release arises from presynaptic thermal mechanisms. Neurons with Low basal frequencies (33%) showed minor thermal changes in sEPSC rate that were abolished after addition of TTX, suggesting these responses were indirect and required local circuits. Activation of TRPV1 with capsaicin (100 nM) increased miniature EPSC (mEPSC) frequency in 70% of neurons, but half of these neurons had Low basal mEPSC rates and no temperature sensitivity. Our evidence indicates that normal temperatures (35-37°C) drive spontaneous excitatory synaptic activity within superficial Vc by a mechanism independent of presynaptic action potentials. Thus thermally sensitive inputs on superficial Vc neurons may tonically activate these neurons without afferent stimulation.

Keywords: TRPV1; electrophysiology; spontaneous release; temperature; trigeminal nucleus caudalis.

Copyright © 2014 the American Physiological Society.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.

Physiological temperatures drive spontaneous glutamate release in trigeminal nucleus caudalis (Vc). Traces represent basal and temperature responses (30–36°C) in Vc neurons with High (≥10 Hz) spontaneous activity (A) and Low (<10 Hz) spontaneous activity (B). Individual diary plots of spontaneous excitatory postsynaptic current (sEPSC) frequency (black bars) and bath temperature (Temp; red line) over the 1st 15 min of recording are shown in High (C) and Low (D) for cells shown in A and B, respectively. sEPSC rate tracked with increases or decreases in temperature accordingly.

Fig. 2.
Fig. 2.

Temperature acts presynaptically to drive glutamate release. A: neurons segregated into 2 distinct groups: High (red squares, n = 7) with sEPSC rates ≥5 Hz or Low (blue squares, n = 5) with sEPSC rates <5 Hz at 32°C. Significant increases in sEPSC rate were observed at temperatures ≥34°C regardless of basal rate (P < 0.001, ANOVA). B: amplitudes were not different between High and Low neuron groups (P > 0.05, Student's t-test), and temperature did not significantly change event size (P > 0.05, ANOVA). Large symbols represent the mean value (±SE); individual cells are represented by colored lines.

Fig. 3.
Fig. 3.

Temperature-evoked sEPSCs generate postsynaptic action potentials. Representative current-clamp recordings of superficial Vc neurons with sEPSC rates were classified as High, ≥5 Hz (n = 7; A), or Low, <5 Hz (n = 7; B) sEPSC rates at 32°C. As temperature increased from 30 to 36°C, the number of action potentials generated increased regardless of basal sEPSC rate; the magnitude of elicited action potential responses was dependent on sEPSC category as observed at 36°C (left).

Fig. 4.
Fig. 4.

Blockade of voltage-gated Na+ channels in High and Low neurons reveals selective drive of thermally evoked glutamate release. A: the histogram of a representative High neuron where sEPSC frequency tracks closely and is reversible with bath temperature in control solution (left; 0–5 min). Addition of TTX (1 μM) to block voltage-gated Na+ channels isolated activity-independent glutamate release and did not alter the rate of thermally evoked EPSCs (6–10 min). B: miniature EPSC (mEPSC) frequency from High neurons (n = 13) is not significantly different in the control solution (red squares) vs. TTX (red circles; P > 0.05, Student's t-test). Increases in EPSC rate were intact after exposure to TTX, suggesting High neurons were directly activated by temperature. C: a histogram of EPSC frequency and bath temperature for a Low neuron in control solution (sEPSCs, 0–5 min) and in TTX (mEPSCs, 6–10 min). D: for Low neurons, inclusion of TTX (blue circles) in the bath solution significantly attenuated thermally evoked responses but not basal release in control solution (blue squares), suggesting that Low neurons (n = 5) were insensitive to temperature changes from 30 to 36°C (**P ≤ 0.01, ANOVA). Data in B and D are shown as means (±SE).

Fig. 5.
Fig. 5.

Glutamate release in Vc is either temperature-sensitive or -insensitive. A: TTX-isolated, direct inputs to Vc for High (red, n = 36) or Low (blue, n = 18) neurons; mEPSC frequency is temperature-sensitive or -insensitive, respectively. Significant increases in mEPSC rate over the basal frequency at 32°C were observed when the bath was ≥34°C (*P ≤ 0.05, ANOVA) for High inputs. Increasing or decreasing bath temperature to 36 or 30°C, respectively, from 32°C did not significantly change mEPSC rates in Low neurons (blue bar; P > 0.05, ANOVA). Data are represented as means (±SE). B: linear regression analysis of Vc neurons confirmed thermal sensitivity of spontaneous glutamate release from High neurons (red) from Low neurons (blue). Dotted lines represent the 95% confidence interval. C: localization map of recording sites for temperature-sensitive (red circles) and temperature-insensitive (blue triangles) neurons. Thermally defined populations overlapped throughout the Vc. Scale bar = 100 μm. D: transient receptor potential vanilloid type 1 channel (TRPV1) immunoreactivity is restricted to the superficial lamina of Vc in the horizontal slice, paralleling the recording zone in C. Scale bar = 250 μm. C and D are orientated such that rostral is to the left and midline is at the bottom. spV, spinal trigeminal tract; Vi, trigeminal nucleus interpolaris. E: summary pie charts for temperature (left; n = 54)- and capsaicin (CAP; right; n = 26)-responsive neurons in Vc. Color coding is such that: red, temperature-sensitive (Temp+); blue, temperature-insensitive (Temp−); white, CAP-sensitive (CAP+); and yellow, CAP-insensitive (CAP−).

Fig. 6.
Fig. 6.

TRPV1 expression does not correlate to intrinsic rates of release or thermal sensitivity. Representative traces are of superficial Vc mEPSC recordings with High (A; red bars) or Low (B; blue bars) basal release rates in TTX at 32°C (top), at 36°C (middle), and in 100 nM CAP (bottom). Inputs were considered to be CAP-sensitive and, therefore, TRPV1-expressing if application of CAP resulted in a mEPSC rate more than twice that observed in TTX, 32°C. CAP evoked a ≥5-fold increase in mEPSC rate High (C; n = 6). In these same recordings, temperature manipulation directly induced increases or decreases in mEPSC rate when warming or cooling steps, respectively, were applied to High neurons (D). Despite similar responses to CAP application (E), Low neurons with TRPV1 did not have altered rates of glutamate release across 30–36°C (F). CAP (100 nM) was applied at the end of Vc recordings in the presence of TTX (Student's t-test). CAP effects on mEPSCs were analyzed independently from thermally evoked responses (ANOVA). Data in C–F represent the means ± SE. Statistical significant is denoted by *P ≤ 0.05, **P ≤ 0.01.

Fig. 7.
Fig. 7.

Thermal sensitivity persists in neurons lacking TRPV1. Representative traces are of superficial Vc mEPSC recordings insensitive to CAP with High (A; orange bars) or Low (B; green bars) basal release rates in TTX at 32°C (top), at 36°C (middle), and in 100 nM CAP (bottom). Inputs were considered to be CAP-insensitive if application of CAP did not increase the mEPSC rate more than twice that observed in TTX, 32°C. CAP failed to evoke enhanced mEPSC rates in 8 recordings. In TRPV1− recordings with a High mEPSC rate (C; n = 8), temperature changes directly influence mEPSC rate when warming or cooling steps were applied, respectively (D). E and F: rarely, Low neurons were encountered without TRPV1 that did not have altered rates of glutamate release across 30–36°C (F). CAP (100 nM) was applied at the end of Vc recordings in the presence of TTX (Student's t-test). CAP effects on mEPSCs were analyzed independently from thermally evoked responses (ANOVA). Data in C–F represent the means ± SE. Statistical significance was *P ≤ 0.05.

Fig. 8.
Fig. 8.

Vc synaptic populations with respect to temperature-sensitive glutamate release and TRPV1 expression. Summary of the proportion of neurons with mEPSC rates sensitive to modest temperature (Temp+) changes and/or CAP application (TRPV1+) is shown. CAP sensitivity was not predictive of thermal sensitivity such that 4 populations were identified: Temp+/TRPV1+ (red), Temp−/TRPV1+ (blue), Temp+/TRPV1− (orange), and Temp−/TRPV1− (green). CAP (100 nM) was applied at the end of Vc recordings in the presence of TTX, and subsequent effects on mEPSCs were analyzed independently from thermally evoked responses.

Fig. 9.
Fig. 9.

Thermally sensitive miniature glutamate release is enhanced during postnatal development. A: the rate of thermally evoked miniature glutamate release is significantly greater in rats older than 14 days (gray, P14–20 postnatal days, n = 15; black, P21–28, n = 8) compared with P7–13 (white, n = 15) at 36°C (*P < 0.05, 2-way ANOVA). B: in contrast, postnatal development does not change the rate of miniature release onto neurons from thermally insensitive inputs (P7–13, n = 8; P14–20, n = 9; P21–28, n = 4). C: amplitudes of mEPSCs were similar across developmental ages and input types. D: the percentage of Vc cells with either temperature-sensitive (black; Temp+) or -insensitive (white; Temp−) inputs was unchanged across the development age range from P7 to P28. E: responses of mEPSC rates to CAP (100 nM) were similar regardless of age (P7–13, n = 8; P14–20, n = 16; P21+, n = 3; P > 0.05, ANOVA). CAP increased rates in all TRPV1+ neurons (*P < 0.05). All experiments were performed in TTX (1 μM) and gabazine (3 μM). NS, not significant.

Similar articles

Cited by

References

    1. Aicher SA, Hermes SM, Whittier KL, Hegarty DM. Descending projections from the rostral ventromedial medulla (RVM) to trigeminal and spinal dorsal horns are morphologically and neurochemically distinct. J Chem Neuroanat 43: 103–111, 2012 - PMC - PubMed
    1. Anwar IJ, Derbenev AV. TRPV1-dependent regulation of synaptic activity in the mouse dorsal motor nucleus of the vagus nerve. Front Neurosci 7: 238, 2013 - PMC - PubMed
    1. Baccei ML, Bardoni R, Fitzgerald M. Development of nociceptive synaptic inputs to the neonatal rat dorsal horn: glutamate release by capsaicin and menthol. J Physiol 549: 231–242, 2003 - PMC - PubMed
    1. Bae YC, Oh JM, Hwang SJ, Shigenaga Y, Valtschanoff JG. Expression of vanilloid receptor TRPV1 in the rat trigeminal sensory nuclei. J Comp Neurol 478: 62–71, 2004 - PubMed
    1. Beckstead RM, Norgren R. An autoradiographic examination of the central distribution of the trigeminal, facial, glossopharyngeal, and vagal nerves in the monkey. J Comp Neurol 184: 455–472, 1979 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources