Tissue SetUp

Quit Marijuana The Complete Guide

New Treatment for Cannabis Dependence

Get Instant Access

The next two sections describe the electrical stimulation procedures used in this laboratory (Subheadings 3.2.1. and 3.2.2.). Whether cannabinoids are to be assayed using electrically stimulated tissue (Subheading 3.3.) or not (Subheading 3.4.), vasa deferentia are first subjected to an equilibration procedure that always involves electrical stimulation, as described in Subheading 3.2.3.

3.2.1. Electrical Stimulation Conditions

Contractions are usually evoked by applying 0.5-s trains of pulses of 110% maximal voltage (train frequency 0.1 Hz; pulse frequency 5 Hz; pulse duration 0.5 ms) (see Note 4 and Table 2 for more details). The amplitude of each monophasic contraction so induced appears to be determined more by released ATP than noradrenaline (Subheading 1.), as we have found that twitch amplitude is attenuated to a significantly greater extent by PPADS (a P2 receptor antagonist) than by prazosin (an ai-adrenoceptor antagonist) (12). Different stimulation conditions have been described by another laboratory (see Note 5).

Table 2

Simplified Electrical Stimulation Conditions on Grass S48 Stimulator (Grass Medical Instruments, Quincy, MA)

Parameter

Setting

Train rate Train duration Stimulation rate Delay

Pulse duration Pulse size (Volts)

0.1 train/s

500 ms

5 pulse/s 0.01 ms

3.2.2. Electrically Induced Contractions

Tissue contractions are usually monitored by computer (Apple Macintosh or PC) connected to a data recording and analysis system (MacLab or PowerLab). These are linked via preamplifiers to Pioden UF1 isometric transducers (Harvard Apparatus), to which the tissue is attached. Electrical stimuli are generated by a Grass S48 stimulator, are amplified (channel attenuator; MedLab Instruments), and then divided to yield separate outputs to four organ baths (StimuSplitter; MedLab Instruments). As this laboratory sets up eight organ baths, two four-organ bath set-ups are employed. The stimuli are applied through a stainless steel electrode attached to the lower end of each bath and a platinum electrode attached to the upper end. Although this laboratory prefers to monitor tissue contractions by computer (see also Note 6), it is also possible to use flatbed recorders.

3.2.3. Equilibration Procedure

Each tissue is electrically stimulated over a period of 10 min, starting with a submaximal voltage and systematically increasing this output until a supramaximal voltage is achieved (110% maximal). Electrical stimulation is then

Table 3

Dose Cycles and Typical Concentrations for Some Common Cannabinoids

Table 3

Dose Cycles and Typical Concentrations for Some Common Cannabinoids

Compound

Dose cycle (minutes)a

Typical concentrations (nM)

A9-THC

30

1 ^ 1000

tf-(+)-WIN55,212

15

0.1 ^ 1000

CP55940

15

0.1 ^ 1000

Anandamide

15

1 ^ 10,000

SR141716A

_b

32, 100

a The dose cycle (interval between doses) is dependent on the length of time taken for the cannabinoid to produce the maximum effect.

b SR141716A is usually incubated for 30 min before the construction of a cannabinoid concentration-response curve is commenced (see Subheading 3.3.2).

a The dose cycle (interval between doses) is dependent on the length of time taken for the cannabinoid to produce the maximum effect.

b SR141716A is usually incubated for 30 min before the construction of a cannabinoid concentration-response curve is commenced (see Subheading 3.3.2).

stopped and the tissue rested for 10 min before subjecting it to further electrical stimulation for 2 min. This cycle of 10 min of rest followed by 2 min of stimulation is repeated until the tissue contractions exhibit a constant amplitude.

Was this article helpful?

0 0

Post a comment