Microdialysis methods for in vivo neuropeptide measurement in the stalk-median eminence in the Rhesus monkey

Abstract

Direct measurement of neuropeptides in the hypothalamus is essential for neuroendocrine studies. However, the small quantities of peptides released at their neuroterminals and relatively large molecular sizes make these measurements difficult. We have evaluated microdialysis probes with two membrane materials (polycarbonate and polyarylethersulfone, both: molecular cut off 20,000 Da) in vitro, and adapted the method for in vivo hypothalamic sample collection in non-human primates. The results of in vitro experiments showed that the polyarylethersulfone membrane yielded a several fold higher recovery rate than the polycarbonate membrane. In in vivo experiments, a guide cannula with stylet was inserted into the medial basal hypothalamus through the permanently implanted cranial pedestal under light sedation. The stylet was replaced by a microdialysis probe and artificial CSF was infused. The results indicated that the neuropeptide luteinizing hormone-releasing hormone was readily measurable in dialysates collected at 10 min-intervals, and responded to neuroactive substances applied through the probe. The animals were fully conscious except for the initial hour of sampling. After the experiment the animal was returned to the home cage, and later similarly examined during several additional experiments. Therefore, the microdialysis method described here is a highly useful tool for neuroendocrine studies in non-human primates.

Figure 1

Schematic illustration of the microdialysis system in non-human primates. A custom-made microdialysis probe (insert) for the rhesus monkey was inserted into the medial basal hypothalamus through the guide cannula. The inlet cannula of the microdialysis probe was connected to the infusion pump with a gas tight syringe and the outlet cannula was connected to the fraction collector. Insert: the dialysis membrane made of either polycarbonate or polyarylethersulfone (molecular cut off of the membranes are 20,000 daltons) is glued on the bottom of concentric inlet and outlet cannulae, through which perfusion fluid enters and comes out, respectively. The permeable membrane allows for chemical equilibrium between the fluid inside the probe and the extracellular tissue. Note that prior to the probe insertion, a guide cannula with a stylet was inserted into the medial basal hypothalamus through a permanently implanted cranial pedestal on the monkey skull using a hydraulic microdrive unit and x-ray ventriculographs under light sedation. After insertion the monkey was placed in a chair, to which the monkey was well adapted, the stylet was removed, and the probe was inserted into the aiming point through the guide cannula.

Figure 2

An assessment of the recovery rate in vitro. Artificial CSF was infused through microdialysis probes with either the polycarbonate (PC, open circle) or polyarylethersulfone (PAES, closed circle) membrane, which were placed in a reservoir containing LHRH at 0.01, 0.1 and 1 nM concentrations, while dialysates were continuously collected at 10 min intervals. LHRH concentrations in dialysates were measured by RIA. Note that the recovery rate with the PAES membrane was much higher than that with the PC membrane at both 0.1 and 1 nM reservoir concentrations. Neither membrane yielded detectable levels of LHRH at 0.01 nM reservoir concentration. It is also of interest to note that the PAES membrane allowed quicker clearance than the PC membrane.

Figure 3

Examples of in vivo LHRH release in the stalk-median eminence measured by the microdialysis method with a PC membrane probe in an adult female ovariectomized rhesus monkey at 9 years of age (A) and with a PAES membrane probe in a pubertal female rhesus monkey at 2.7 years of age (B). Perfusate samples were collected at 10 min intervals for a 9–10 h period, and LHRH concentration in perfusates were measured by RIA. Arrowheads indicate LHRH pulses depicted by the PULSAR algorithm.

Figure 4

Effects of an LHRH agonist (Des-Gly¹⁰, [D-Ala⁶]-LHRH Ethylamide) at 0.1 μM and 1 μM in an adult female ovariectomized rhesus monkey at 10 years of age. LHRH agonist was infused for a period of 20 min (indicated by shading) while perfusates were continuously collected. Approximately 1 % of the LHRH agonist was diffused through the microdialysis membrane and thus the LHRH agonist at these concentrations was not cross-reactive with our LHRH assay. Note that the infusion of both doses of the LHRH agonist at 0.1 and 1 μM (which was estimated as 1 and 10 nM LHRH agonist concentration, respectively) caused a significant LHRH increase.