Elsevier

Brain Research

Volume 733, Issue 2, 16 September 1996, Pages 184-192
Brain Research

Research report
Local pressure application of cannabinoid agonists increases spontaneous activity of rat substantia nigra pars reticulata neurons without affecting response to iontophoretically-applied GABA

https://doi.org/10.1016/0006-8993(96)00533-1Get rights and content

Abstract

This study tested the hypothesis that cannabinoid agonists, applied locally into the pars reticulata of substantia nigra (SNpr), could modulate striatonigral transmission, without affecting the response of SNpr neurons to iontophoretically-applied GABA. Multibarreled glass capillary electrode assemblies were used for extracellular recording of the spontaneous electrical activity of single SNpr cells in anesthetized rats. Local pressure ejection of the cannabinoid agonists Win 55212-2 (WIN2) and CP 55940 increased SNpr spontaneous firing rate by 13–46%, similar to the effects of systemic injections. Neither WIN2 nor CP 55940 had an effect on the slowing of SNpr neuron activity in response to iontophoretic GABA. Local pressure application of Win 55212–3 (the much less active enantiomer of WIN2) produced an insignificant decrease in SNpr firing rate. Similarly, locally applied vehicle (45% 2-hydroxypropyl-β-cyclodextrin) produced insignificant decreases in SNpr firing. A second application of cannabinoid agonist produced a much smaller effect, suggesting desensitization. Increasing the interval between CP 55940 applications to 45 min showed recovery of sensitivity to the agonist. Local application of the cannabinoid antagonist, SR 141716A, significantly decreased spontaneous cell firing by 34%. CP 55940, when given immediately following or concurrently with the antagonist application failed to produce the expected increase in discharge rate over baseline. A second application of CP 55940 45 min later produced a 26% increase in firing rate. Bicuculline methiodide (BMI) was applied locally causing a significant increase in SNpr cell firing. CP 55940, when locally administered concurrently with bicuculline methiodide, had no further effect on the firing rate of the cell. Based on the reported presynaptic localization of cannabinoid receptors in SNpr, these findings suggest that cannabinoids act within the SNpr to modulate striatonigral neurotransmission presynaptically. The effect of SR 141716A suggests that an endogenous cannabinoid may mediate striato-nigral transmission.

Introduction

Marijuana has been used since antiquity for medicinal and recreational purposes. Its major active component, Δ9-tetrahydrocannabinol, and related compounds have been found to possess several potentially useful actions. Central nervous system (CNS) actions of cannabinoids include analgesia, drowsiness, antiemesis, and a decrement in psychomotor performance [24]. A significant advance in the understanding of cannabinoids has been brought about by the demonstration of brain cannabinoid receptors [5]and the cloning and characterization of the receptor, which was shown to be a G protein-linked receptor that inhibits adenylyl cyclase [23].

The distribution of cannabinoid binding sites in the CNS has been studied using the synthetic agonists [3H]CP 55940 16, 21and [3H]Win 55212–2 (WIN2) [17]]. Results showed that the distribution is not uniform, and that there are receptors associated with areas such as cerebral cortex and hippocampus, which may be involved in the cannabinoids' psychoactive effects. Dense labeling was also found in the basal ganglia, including areas innervated by the output neurons of the striatum, with the highest concentration being in the pars reticulata of the substantia nigra (SNpr) 15, 20. Lesion and binding studies involving striatonigral and nigrostriatal neurons have shown the receptor to be located on the presynaptic terminals of the striatonigral neurons [14]. The reported loss of cannabinoid receptors in the substantia nigra and the lateral globus pallidus in the brains of patients suffering from Huntington's disease is consistent with their presynaptic localization 11, 31. The function of these receptors has yet to be determined. Their localization in the basal ganglia suggests that cannabinoids may act to regulate striatal output, possibly playing a role in movement and posture. Considering the localization of the receptor on the terminals of GABAergic striatal output neurons, cannabinoid actions in SNpr are most likely to occur presynaptically, possibly involving a change in γ-aminobutyric acid (GABA) release or reuptake.

The effects of a synthetic cannabinoid, WIN2, on striatonigral transmission were studied by Miller and Walker [26], who found that systemic injections of WIN2 increased the rate of spontaneous action potential generation (‘firing rate’) of SNpr neurons, and also attenuated the slowing of SNpr discharge produced by electrical stimulation of the striatum. These findings, coupled with the reported presynaptic localization of cannabinoid receptors, strongly suggested that WIN2 modulated GABA release from striatal nerve terminals. Since the cannabinoid was injected systematically, and cannabinoid receptors exist in many brain areas, including the striatum [21]which is sensitive to cannabinoids [35], the site of action of WIN2 could only be inferred. Moreover, the results could not exclude the possibility that the SNpr neurons, either by direct or indirect WIN2 actions, were made less sensitive to GABA. Though local application of a cannabinoid (i.e., by micro-iontophoresis or pressure application) might provide more information, this is a difficult problem because of the extremely limited aqueous solubility of the cannabinoid agonists currently available.

In the present study, a technique for local application of cannabinoids by pressure application from a multibarreled glass electrode assembly is described. This technique was used to investigate the effects of two cannabinoid agonists, WIN2 and CP 55940, as well as the cannabinoid antagonist SR 141716A [32]on the spontaneous activity of SNpr neurons. The effects of Win 55212–3, the inactive isomer of WIN2 on SNpr neuron spontaneous activity was also examined. The interaction between the GABAA antagonist bicuculline methiodide and CP 55940 was also studied. To evaluate the possibility that cannabinoids might affect the responsiveness of SNpr neurons to GABA, the postsynaptic action of exogenously-applied GABA was also studied, using iontophoretic application of GABA during cannabinoid agonist application.

Section snippets

Rat preparation and electrical recording

Male Sprague-Dawley rats (250–300 g) were anesthetized with chloral hydrate (400 mg/kg) i.p. and placed in a stereotaxic instrument. The rats were kept at a constant level of anesthesia by i.v. infusion of 2.2 mg/kg/min chloral hydrate into the lateral tail vein [38]. The rat was placed on a constant temperature pad (Deltaphase, Braintree Scientific, Braintree, MA) to help maintain body temperature during the course of the experiment. A small diameter burr hole was drilled over the substantia

Effect of vehicle, WIN2 and CP55940 on spontaneous firing rate and GABA inhibition of SNpr cells

The effects of local pressure application of WIN2 and CP 55940 on the spontaneous firing rate of SNpr cells are shown in Fig. 1Fig. 2. Fig. 1 shows oscilloscope tracings and a representative ratemeter recording of the activity of a single SNpr cell and the effects of locally-applied WIN2. Locally-applied WIN2 and CP 55940 increased the spontaneous firing rate of SNpr cells (Fig. 2), but had no significant effect on the inhibition of the cells by iontophoretically-applied GABA (Fig. 3). The

Discussion

Locally-applied WIN2 and CP 55940 increased the spontaneous firing of SNpr neurons, but had no effect on iontophoretically-applied GABA. The lack of interaction between cannabinoids and GABA makes it unlikely that cannabinoids were acting on SNpr cells directly to alter the coupling between GABA binding and inhibition of neuronal activity, or by some indirect mechanism to change the susceptibility of the neuron to the inhibitory effects of GABA. The effects of the two cannabinoid agonists were

Acknowledgements

The authors wish to thank Pfizer, Inc. for the generous gift of CP 55940, and NIDA for the generous gift of SR 141716A. This work was supported by NIH Grant DA02194.

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