Altered Intrinsic Properties and Inhibitory Connectivity in Aged Parvalbumin-Expressing Dorsal Horn Neurons

Abstract

The incidence of pain symptoms such as allodynia are known to increase with age. Parvalbumin expressing interneurons (PVINs) within the dorsal horn (DH) of the spinal cord play an important role in allodynia whereby their inhibitory connections prevent innocuous touch information from exciting nociceptive pathways. Here we ask whether the functional properties of PVINs are altered by aging, comparing their functional properties in adult (3-7 month) and aged mice (23-28 month). Patch clamp recordings were made from PVINs in laminae IIi-III of parasagittal spinal cord slices. The intrinsic excitability of PVINs changed with age. Specifically, AP discharge shifted from initial bursting to tonic firing, and firing duration during current injection increased. The nature of excitatory synaptic input to PVINs also changed with age with larger but less frequent spontaneous excitatory currents occurring in aged mice, however, the net effect of these differences produced a similar level of overall excitatory drive. Inhibitory drive was also remarkably similar in adult and aged PVINs. Photostimulation of ChR2 expressing PVINs was used to study inhibitory connections between PVINs and unidentified DH neurons and other PVINs. Based on latency and jitter, monosynaptic PVIN to unidentified-cell and PVIN-PVIN connections were compared in adult and aged mice, showing that PVIN to unidentified-cell connection strength increased with age. Fitting single or double exponentials to the decay phase of IPSCs showed there was also a shift from mixed (glycinergic and GABAergic) to GABAergic inhibitory transmission in aged animals. Overall, our data suggest the properties of PVIN neurons in aged animals enhance their output in spinal circuits in a manner that would blunt allodynia and help maintain normal sensory experience during aging.

Keywords: aging; interneuron; mouse; pain; spinal cord.

FIGURE 1

PVINs are more excitable in aged mice. (A) Bar graphs compare the incidence of AP discharge patterns in adult (black bars) and aged PVINs (red bars). TF and IB discharge dominate at both ages but there is a shift from IB to TF discharge in aged mice. Traces show the Initial Bursting and Tonic Firing AP discharge patterns in PVINs during depolarizing current step injections (20 pA step increments, 1 s duration). (B) Current-frequency and current-discharge duration relationships in adult (black dots) and aged (red dots) mice for five depolarizing current steps above rheobase. Note, AP discharge duration is enhanced in the aged population. (C) Overlaid traces of representative AP’s from adult (black) and aged (red) PVINs. (D) Group data comparing AP properties in adult (black) and aged (red) PVINs. None of the six AP properties (rheobase, threshold, AP amplitude, AP halfwidth – APW50, AHP amplitude, and AHP half-width – AHPW50) differed in adult and aged PVINs. *p < 0.05.

FIGURE 2

Excitatory synaptic drive is unchanged in aged PVINs. (A) Traces show continuous sEPSC recordings from adult and aged PVINs. Note, sEPSC amplitude is clearly greater in aged PVINs. (B) Upper overlaid sEPSC traces are captured from the data in (A). The average of these captured events (heavy trace) emphasizes the increased sEPSC amplitude in aged animals. Lower plots show amplitude histograms highlighting a population of right shifted larger sEPSCs in the aged recording. (C) Plots comparing group data for sEPSC properties in PVINs from adult and aged mice. In aged mice, sEPSC amplitude and charge are increased whereas sEPSC frequency is reduced. sEPSC time course (rise and decay time) and synaptic drive (charge per second) are unchanged in aged PVINs (asterisks indicate significant differences, p < 0.05). Each data point represents the mean of at least 20 events.

FIGURE 3

Synaptic inhibition is similar in adult and aged PVINs. (A) Representative traces showing sIPSCs in adult (left, black) and aged (right, red) PVINs. (B) Upper overlaid sIPSC traces captured from records in (A) with averaged responses indicated by heavier traces. Lower plots show amplitude histograms highlighting similar amplitude distributions in adult and aged sIPSC recordings. (C) Plots showing group data for sIPSC properties. Note, the similarly in all sIPSC properties measured in adult and aged mice. Each data point represents the mean of at least 20 events.

FIGURE 4

Aged PVINs increase their postsynaptic output. (A) Traces showing photostimulation-evoked AP discharge in adult and aged PVINs at increasing stimulation frequencies. Photostimulation pulses (1 ms duration) and stimulation protocol are shown in blue. The short horizontal black bars on the 5 Hz discharge traces indicate a membrane potential of −60 mV. Top two plots on the right summarize the reliability of evoked AP discharge at the four photostimulation frequencies for adult and aged PVINs. Bottom graph compares the mean probability for AP discharge at different photostimulation frequencies. (B) Voltage clamp recordings (−70 mV holding potential) showing PVIN photostimulation-evoked inhibitory postsynaptic currents (oIPSCs) generated in unidentified neurons (top traces) and PVINs (bottom traces). Schematics to the left show each photostimulation and recording configuration. Left traces are recorded from adult mice, and middle traces from aged mice (10 consecutive sweeps and average overlayed in each). Right traces show overlaid averages. Note, oIPSC responses are markedly larger at PVIN to unidentified cell connections in aged animals. Group data plots (far right) compare oIPSC amplitude for each recording configuration and show that oIPSC amplitude is increased at PVIN to unidentified cell connections in aged mice. (C) Voltage clamp recordings (−70 mV holding potential) from adult (left) and aged (middle) PVINs on an expanded time scale to show the photocurrent phase of the photostimulation response (highlighted by blue rectangle). Average overlaid photocurrent responses (right trace) show they are similar in recordings from adult and aged animals. Group data (far right plots) compare photocurrent rise time and amplitude in adult and aged PVINs. The similarity in these two measures suggest age related changes in photocurrent properties does not explain the larger oIPSCs observed at PVIN to unidentified cell connections in aged mice. Each data point represents the mean of 10 consecutive traces events. *p < 0.05.