The modulation of striatonigral and nigrotectal pathways by CB1 signalling in the substantia nigra pars reticulata regulates panic elicited in mice by urutu-cruzeiro lancehead pit vipers

Abstract

Cannabinoid receptor type 1 (CB₁R) is widely distributed in the substantia nigra pars reticulata (SNpr). However, the role of CB₁R at the SNpr level in threatening situations is poorly understood. We investigated the role of CB₁R in the SNpr on the expression of fear responses in mice confronted with urutu-cruzeiro pit vipers. First, a bidirectional neurotracer was injected into the SNpr; then, immunostaining of the vesicular GABA transporter was conducted at the levels of the striatum (CPu) and deep layers of the superior colliculus (dlSC). In addition, CB₁R immunostaining and GABA labelling were performed in the SNpr. Using a prey-versus-snake paradigm, mice were pretreated with the CB₁R antagonist AM251 (100 pmol) and treated with the endocannabinoid anandamide (AEA, 5 pmol) in the SNpr, followed by bicuculline (40 ng) in the dlSC, and were then confronted with a snake. Bidirectional neural tract tracers associated with immunofluorescence showed the GABAergic striatonigral disinhibitory and nigrotectal inhibitory pathways. Furthermore, we showed that CB₁R labelling was restricted to axonal fibres surrounding SNpr GABAergic cells. We also demonstrated a decrease in the defensive behaviours of mice treated with AEA in the SNpr, but this effect was blocked by pre-treatment with AM251 in this structure. Taken together, our results show that the panicolytic consequences of the AEA enhancement in the SNpr are signalled by CB₁R, suggesting that CB₁R localised in axon terminals of CPu GABAergic neurons in the SNpr modulates the activity of the nigrotectal GABAergic pathway during the expression of defensive behaviours in threatening situations.

Introduction

The substantia nigra consists of heterogeneous groups of cells, whose cytoarchitecture is divided into three different regions: the pars lateralis (SNpl), pars compacta (SNpc) and pars reticulata (SNpr). Dopaminergic neurons in the SNpc mainly project to the neostriatum (nigrostriatal pathway), while the SNpr sends GABAergic projections to the ventromedial thalamic nucleus (nigrothalamic pathway) and the dorsal midbrain (nigrotectal pathway), reaching the periaqueductal grey matter (PAG), the deep layers of the superior colliculus (dlSC) and the inferior colliculus [[1], [2], [3], [4], [5]].

There is a substantial amount of evidence for the involvement of the nigrotectal pathway in the organisation of defensive behaviour processing [[6], [7], [8], [9]]. For instance, inactivation of fibres of passage at the level of the SNpr increase defensive behaviours elicited by injections of the GABA_A receptor selective antagonist bicuculline in the dlSC [10]. Using optogenetic methods, we recently showed that the activation of SNpr afferents at the level of the SC reduces threat recognition [9]. The modulation of the neural processes of aversion in the dorsal midbrain is largely associated with GABAergic mechanisms from the SNpr [6,[10], [11], [12]]. Moreover, morphological approaches have shown high expression of cannabinoid receptor type 1 (CB₁R) in the striatum (CPu), PAG, dlSC and substantia nigra [[13], [14], [15], [16]]. CB₁Rs are present in both glutamatergic and GABAergic projections [17,18]; however, the majority of CB₁Rs detected in the CPu are localised on GABAergic axons [16].

These findings lead to an interesting hypothesis that endocannabinoids can serve as important neuromodulators to control GABAergic striatonigral disinhibitory and nigrotectal inhibitory pathways activity during the organisation of innate fear-related behaviours. We have already shown that endocannabinoid (eCB) signalling in the SNpr modulates GABAergic striatonigral-nigrotectal pathways during the generation and expression of innate fear in threatening situations [7]. Injections of the endocannabinoid anandamide (AEA) in the SNpr causes a decrease in the expression of defensive behaviour elicited by either the GABA_A receptor antagonist bicuculline or confrontation with the urutu-cruzeiro venomous lancehead pit viper [7]. Although it has been proposed that AEA at the SNpr level may be important for the modulation of innate fear-related behaviours [7], a role for CB₁Rs has yet to be established. Using neuropharmacological, behavioural and morphological approaches, we tested the hypothesis that AEA modulates the striatonigral disinhibitory and nigrotectal inhibitory pathways by decreasing the activity of GABAergic inputs to the SNpr through CB₁R recruitment during threatening situations. The antipredatory behaviour neural circuitry [19] and the neural substrates related to midbrain tectum GABAergic disinhibition with bicuculline is not similar [20], but they can be overlapped during the limbic system activation during prey versus snakes confrontation [21,22]. In addition, there is evidence that structures related to the organisation of oriented escape behaviours are more rostral in the neuro-axis, such the medial hypothalamic nuclei [23,24], while the non-oriented/explosive escape behaviour is organised by the periaqueductal grey matter and dlSC [25]. In the present work, we combined the threatening of mice with a potential predator and the GABAergic disinhibition of the dlSC with bicuculline in an enriched polygonal arena for snakes with burrow and elevated platforms for escape to enhance panic attack-like defensive reactions in prey and to allow discrimination between oriented and non-oriented escape behaviours.