After Institutional Animal Care and Use Committee approval, male Sprague-Dawley infant (postnatal day 7) and juvenile (postnatal day 28) rats were deeply anesthetized with isoflurane 3% with spontaneous ventilation. The trachea was intubated and animals ventilated using pressure controlled ventilation (Inspira PCV, Harvard Apparatus, Holliston, MA, USA) with humidified oxygen. The ECG was monitored throughout. Anesthetized animals were immobilized with pancuronium bromide and the isoflurane maintained at 2% throughout the study (Tevan Pharmaceutics, North Wales, PA, USA). As illustrated in , a dorsal midline incision was made in trunk skin and L5 DRG and adjacent spinal cord was exposed by laminectomy as previously (Boada et al., 2010
). The tissue was continuously superfused with oxygenated artificial cerebrospinal fluid [aCSF (in mM): 127.0 NaCl, 1.9 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, and 10.0 D-glucose]. The spinal column was secured using custom clamps and the preparation was transferred to a preheated (32-34°C) recording chamber where the superfusate was slowly raised to 37°C (MPRE8, Cell MicroControls, Norfolk, VA, USA). Pool temperature adjacent to the DRG was monitored with a thermocouple (IT-23, Physitemp, Clifton, NJ, USA). Rectal temperature (RET-3, Physitemp) was maintained at 34 ± 1°C with radiant heat.
Schematic diagram of the in vivo rat L5 preparation (lateral view).
DRG soma were impaled with borosilicate microelectrodes (80-250 MΩ) containing 1 M potassium acetate (in some cases also 20% neurobiotin (Vector Laboratories, Burlingame, CA, USA)). Intracellular penetrations with a resting membrane potential of ≤ -35 mV were characterized further. DC output from an Axoclamp 2B (Axon Instruments/Molecular Devices, Sunnyvale, CA, USA) was digitized and analyzed off-line using Spike2 (CED, Cambridge, UK). Sampling rate for intracellular recordings was 21 kHz throughout (MicroPower1401, CED).
After achieving stable cell impalement, the skin was searched with a fine sable-hair brush to locate the peripheral RF. For afferents requiring higher intensities, subsequent searches used increasingly stiffer probes and finally sharp-tipped forceps. Afferents with cutaneous RFs were distinguished from those with deep RFs by displacing skin to ensure that RFs tracked rather than remained stationary. Mechanical thresholds were characterized with calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA). Adaptation rate was frequently evaluated using micromanipulator-based probes; responses to skin stretch and vibratory stimuli were also tested. In all cases, RFs were characterized and measured with the aid of a zoom stereomicroscope.
Active membrane properties of all identified sensory neurons were analyzed including the amplitude and duration of the action potential (AP) and afterhyperpolarization (AHP) of the AP, along with the maximum rates of spike depolarization and repolarization (MRD and MRR); AP and AHP durations were measured at half-amplitude (D50, AHP50) to minimize hyperpolarization-related artifacts. Passive properties were analyzed including membrane resting potential (Em), input resistance (Ri), time constant (Tau), inward rectification, and, where possible, rheobase; all but the latter were determined by injecting incremental hyperpolarizing current pulses (≤ 0.1 nA, 500 ms) through balanced electrodes.
Because intact lumbar DRGs serve multiple nerves, spike latency was obtained by stimulating the RF at the skin surface using a bipolar electrode (0.5 Hz); this was performed following all natural stimulation to prevent potential alterations in RF properties. Because we were interested in latency from terminals, all measurements were obtained using the absolute minimum intensity required to excite neurons consistently without jitter; significantly shorter latencies, seen at traditional (i.e., two- to three-fold threshold) intensities and presumably reflecting spread to more proximal sites along axons. Any neuron with jitter was rejected. Stimuli ranged in duration from 50 to 100 μs; utilization time was not taken into account. Conduction distances were measured for each afferent on termination of the experiment by inserting a pin through the RF (marked with ink at the time of recording) and carefully measuring the distance to the DRG along the closest nerve.
All included cells satisfied the following requirements: resting membrane potential more negative than -30 mV, AP amplitude ≥30 mV and the presence of spike AHP. Passive membrane properties indicative of poor impalement were also reason for exclusion.
2.1 Receptive field (RF) analysis
After establishing the afferent identity, the RF was carefully searched with suprathreshold mechanical stimuli. The force used was equivalent to the group upper limit of their cutoff force (±0.33 mN) for the tactile and for the nociceptive afferents was 9.02 mN unless their threshold was higher in value. The highest force possible without compromise to the cellular impalement was <100.94 mN.
During the glabrous skin RF mapping, different parameters were established: 1) number and location of “spots” with the highest sensibility (lowest threshold responses), 2) absolute RF area (mm2) at threshold intensity, 3) relative RF area (%) normalized to glabrous skin total foot area for age (P7: 69.2 mm2, P28: 355.7 mm2). Specific functional areas (fingers against paw) were both analyzed as independent sectors (P7 fingers (Z1): 27.2 mm2, P7 Paw (Z2): 42.0 mm2; P28 fingers (Z1): 109.4 mm2, P28 Paw (Z2): 246.3 mm2). All values where obtained by diagramming the cellular RF to later establish absolute area. The area measurements were performed using StereoInvestigator 7.0 (MicroBrightField Inc, Williston, VT, USA) that was supported by an Olympus BX51 microscope and a digital camera (Microfire A/R, Optronics, Goleta, CA, USA). The perimeter of the drawing represented the RF mapped (contour) at 4× magnification. Stereoinvestigator automatically performed several contour measurements including the area.
2.2 Data Analysis
All data are presented as mean with standard error of the mean, except for mechanical thresholds (MT) which are presented as medians and ranges. Statistical analysis was performed using SAS 9.2, (Cary, NC, USA). Multivariate statistical analysis was used to predict and categorize neurons. Canonical correlation was utilized to determine the relationship between all of the variables and the fiber subtypes. Canonical variates were generated from the measured variables to predict the highest correlation between variable value and fiber type classification in the P28 animals. The goal was to capture the variance in measurements by redistribution into variates which capture a large share of the variance.
The canonical variates were used to classify the neurons in the P28 animals and determine the sensitivity, specificity and positive and negative predictive value for the classification using the model. The means and standard deviations were determined for all neuronal subtypes determined from the classification of neurons into LTMR, AHTMR and the CHTMR from the canonical correlation. ANOVA was performed to test for differences in LTMR and HTMR within age. Except for MT where Kruskall-Wallis was used. Significant differences between AHTMR and CHTMR were determined using a Bonferroni post-test comparison of means. RF area estimations were compared in the paw and fingers normalized RF area mean values among Tactiles and Nociceptors at each age (P7 and P28) by ANOVA. By convention, P<0.05 was considered significant.