The bipolar recording electrode is placed percutaneously into the belly o f either the biceps fem oris or sem itendonosus m uscle Single shot epidural injections can be

made after a simple dissection over the lower lumbar vertebrae avoiding the need for laminectomy. After am plification (xlO K ) and filtering (< lH z , > lK H z) the raw signal is digitised (2KHz Maclab A D C ) and stored electronically. Data was analysed using M aclab Chart software.

2 .8 .5 EM G recording

EMG recording was commenced no sooner than 30 minutes after reducing the inspired halothane concentration from the surgical levels (2%) to age-related recording levels. For 21, 10 and 3 day old rats these were 0.7%, 0.9% and 1.0% respectively. These levels represent equivalent anaesthetic depths (equi-MAC values) and are fully described in Chapter 3. This was to allow time for equilibration to steady state-alveolar halothane concentration and hence a stable plane of anaesthesia.

Bipolar EMG electrodes (Ainsworks, London) comprising stainless steel needles (0.33mm external diameter) with a central insulated copper wire core were place percutaneously into the belly of the biceps femoris muscle. The total cross- sectional area of the recording needle was at least 0.45mm^ ,while that of the inner copper core was at least O.Olmm^. Raw signals were amplified using a headstage amplifier (NLIOO, Neurolog, Digitimer) with an input resistance of lO^ohms. Preamplification (xlOOO) and filters (low pass: IHz; high pass: IKhz; notch: 50Hz) were used (NL104, NL125) before display of the EMG on a digital storage

oscilloscope (Hameg HM205). The signal was fed to an analog-to-digital signal converter for further data analysis (Maclab/4s). EMG recordings from the hamstring muscles in response to mechanical (von Frey hairs) and electrical stimulation of the hindpaw were made when the animals were under steady state anaesthesia.

Von Frey filaments were chosen as a stimulus as they are practical, simple and could be used in parallel behavioural experiments on awake, freely moving animals. Electrical stimulation was chosen as it allows synchronous activation of afferent fibres in a fashion that guarantees C- fibre activation. Measures of reflex latency are also made possible using this stimulus modality. Heat was not chosen as a stimulus because the heat threshold for eliciting the withdrawal reflex in halothane anaesthetised animals would almost certainly be above that for causing significant tissue damage (i.e. tissue bums).

Von Frey hairs of graded gram weight were pressed onto the plantar surface of the hindpaw for 1 sec. A 2 minute rest interval was observed before repeated

stimulation if reflex withdrawal was elicited. Up to three von Frey hairs above threshold were sequentially applied. The range of mechanical stimulus strengths used was regularly between 5 and 50 gm weight. The calibration of the hairs is shown in Diagram 9.

Electrical stimulation was applied via percutaneous electrodes placed at the medial and lateral borders of the paw. Controlled-current stimuli (Stimulus Isolator, Digitimer) were applied as a train of 15 square wave pulses of 2ms duration at a frequency of lOOHz. A range of 1.5mA to 10mA was used and up to three electrical stimuli above threshold were recorded.

Reflex muscle activity was defined to occcur when the EMG signal increased at least three times above baseline noise (S:N ratio 1:3). This level of activity always resulted in visible limb contraction. All experiments were terminated using an overdose of intra-peritoneal thiopentone. v o n F re y h a ir c a lib r a tio n

a)

C a lib r a tio n o v e r tim e

d ) 17 13 75-, 2 5 - 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Week No. Diagram 9. von Frey hair calibration

von Frey hairs were used as a mechanical stimulus to elicit a withdrawal reflex response. Calibration was completed regularly using an electronic bench top weighing scale. A calibration scale for the full set of filaments is shown in a). The two insets show details for the lower range (b) and the range used in these experiments (c). The stress developed in nylon filaments in compression is known to be influenced by temperature and humidity. Three selected calibration values in (d) are plotted over time. A seasonal variation can just be discerned. Heavy blue bars denote the winter months.

2 .8 .6 A n a ly sis

Raw data was digitized at a frequency of 4kHz and 12 second epochs were stored electronically (including 2 seconds pre-stimulus). The latency and duration of the response were determined directly from recordings of the raw data. Data for analysis of latency was obtained from reflexes evoked by electrical stimulation only. Integrating software (Chart, Maclab ADI) calculated the peak response and area under the rectified (RMS) signal. The area under the RMS signal was plotted against stimulus strength and the resulting curve integrated to calculate an area under the curve. This value termed “Reflex Responsiveness” was used as a summary statistic for much of the

pharmacodynamic analysis and reflects the overall sensitivity and responsiveness of the withdrawal reflex.

The use of this summary statistic that combines individual data obtained over a range of stimulus strengths (i.e. a range of von Frey hairs) has the effect of increasing the discriminative power of the recorded data. The use of this measure has several advantages including simplicity and some degree of intuitive relevance to the reflex being measured (Mathews et al., 1990). The latter can be obtained from a visual representation of a volume on a three dimensional plot of time (ms), RMS signal amplitude (mV) and stimulus strength (gm) see Diagram 10. More importantly, the distribution of this measure is not obviously non-Gaussian and therefore lends itself more easily to parametric statistical analysis.

Critical to the use of this measure (reflex responsiveness) is the range of stimulus strengths used for the calculation. The lower limit must obviously be below the threshold for eliciting the withdrawal reflex. The selection of an upper limit to the stimulus strength is based on the change from a simple (single limb) withdrawal to a more complicated crossed and ascending reflex response. This cut-off lies just below the strength at which a stimulus ceases to be merely “potentially noxious” and becomes overtly tissue damaging. This range (5-50gm weight) was found to be consistent across the age groups. Of interest is the relationship between ranges used in different

experimental preparations. The range of stimulus strengths used in these experiments is almost identical to that used by Pertovaara et al (Pertovaara et al., 1998) in experiments on pentobarbitone anaesthetised adult rats undergoing dorsal horn cell recording. The range chosen by Pertovaara was specifically chosen to be supra-threshold.

REFLEX RESPONSIVENESS