Bee Venom Alleviates Motor Deficits and Modulates the Transfer of Cortical Information through the Basal Ganglia in Rat Models of Parkinson's Disease

Abstract

Recent evidence points to a neuroprotective action of bee venom on nigral dopamine neurons in animal models of Parkinson's disease (PD). Here we examined whether bee venom also displays a symptomatic action by acting on the pathological functioning of the basal ganglia in rat PD models. Bee venom effects were assessed by combining motor behavior analyses and in vivo electrophysiological recordings in the substantia nigra pars reticulata (SNr, basal ganglia output structure) in pharmacological (neuroleptic treatment) and lesional (unilateral intranigral 6-hydroxydopamine injection) PD models. In the hemi-parkinsonian 6-hydroxydopamine lesion model, subchronic bee venom treatment significantly alleviates contralateral forelimb akinesia and apomorphine-induced rotations. Moreover, a single injection of bee venom reverses haloperidol-induced catalepsy, a pharmacological model reminiscent of parkinsonian akinetic deficit. This effect is mimicked by apamin, a blocker of small conductance Ca2+-activated K+ (SK) channels, and blocked by CyPPA, a positive modulator of these channels, suggesting the involvement of SK channels in the bee venom antiparkinsonian action. In vivo electrophysiological recordings in the substantia nigra pars reticulata (basal ganglia output structure) showed no significant effect of BV on the mean neuronal discharge frequency or pathological bursting activity. In contrast, analyses of the neuronal responses evoked by motor cortex stimulation show that bee venom reverses the 6-OHDA- and neuroleptic-induced biases in the influence exerted by the direct inhibitory and indirect excitatory striatonigral circuits. These data provide the first evidence for a beneficial action of bee venom on the pathological functioning of the cortico-basal ganglia circuits underlying motor PD symptoms with potential relevance to the symptomatic treatment of this disease.

Trial registration: ClinicalTrials.gov NCT01341431.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Time schedule for bee venom injections and behavioral testing in 6-OHDA lesioned rats.

Time points at which 6-OHDA lesion and bee venom (1 μg/kg for BV1 group or 3 μg/kg for BV3 group) or saline i.p. injections were carried out. Note that bee venom or saline was given every 3 days, starting 15–21 days after the 6-OHDA injection. Cyl. and Apo. indicate time points at which rats were taken for the cylinder test (Cyl.) or apomorphine-induced rotations (Apo.), respectively. After the apomorphine-induced rotations test, all rats were killed for brain processing and analysis.

Fig 2. Raw recording traces from a SNr cell illustrating the short spike duration (A), its spontaneous discharge (B) and its response to one single orofacial motor cortex stimulation (C).

(A) Magnified view of recording traces from a SNr neuron illustrating that its spikes are narrow (< 2 ms). (B) The spontaneous activity is displayed following injection of haloperidol; the discharge of the cell is represented as the raw recording trace (top), the result of the Poisson Surprise spike analysis (S ≥ 2) indicating bursts (middle) and the corresponding sequence of spikes (bottom). (C) Response evoked by a single cortical stimulation illustrated as a raw trace (top) and the corresponding sequence of spikes (bottom). Arrow indicates the artifact stimulation.

Fig 3. Bee venom alleviates parkinsonian-like deficits.

(A) Effects of bee venom and apamin on haloperidol-induced catalepsy in rats. Rats received a single injection of haloperidol (1 mg/kg i.p.). After 30 min, animals received either an i.p. administration of BV (1, and 3 μg/kg; n = 7/group) or apamin (0.1 mg/kg; n = 8/group) in combination or not with CyPPA (1 μg, i.c.v.). Haloperidol-injected animals receiving vehicle served as reference. Catalepsy was measured 30 min later for 120 min. The histograms represent the mean latencies to step down the rod ± S.E.M. during the 120-min test. * p < 0.05 vs. haloperidol + vehicle, Kruskal-Wallis one-way ANOVA followed by multiple comparisons versus control group (Dunn's Method). # p < 0.01, Mann-Whitney Rank Sum Test. (B) Effects of acute and repeated bee venom injections on 6-OHDA lesion-induced forelimb akinesia evaluated using the cylinder test. The data presented in the histogram are the means ± SEM of the numbers of double contacts made with the forepaws on the wall of the cylinder, expressed as the percentage of the total number of contacts, determined from n animals. Statistical comparison was performed using two-way repeated measures ANOVA with treatment (saline, BV 1 μg/kg and BV 3 μg/kg) as the between-subject factor and repetitions of injections as within subject factor followed by multiple comparisons versus pre-treatment (Holm-Sidak method) with an overall significance level of 0.05. * Statistically different from pre-treatment score. The numbers of animals per group were as follows: controls = 7, 6-OHDA + Saline = 7, 6-OHDA + BV 1 μg/kg = 5 and 6-OHDA + BV 3 μg/kg = 7. (C) Effects of bee venom on apomorphine-induced circling behavior. Unilateral 6-OHDA-lesioned rats were treated with vehicle or BV (9 injections, i.p, n = 5/group), and 30 min after the last injection they received apomorphine (0.1 mg/kg s.c.). The total numbers of ipsilateral and contralateral rotations were measured for 70 min immediately after apomorphine injection. The graphs represent the means ± S.E.M. of net rotations (number of contralateral minus ipsilateral rotations) for every 5-min interval. The histograms (inset) represent the means ± S.E.M. of the total net rotations for the 70 min period. * p < 0.01 vs. vehicle group for time, two-way repeated measures ANOVA; # p < 0.05 vs. vehicle group, t-test.

Fig 4. Experimental design (A) and effect of systemic injection of haloperidol alone (B) or followed by bee venom injection (C) on cortically-evoked responses on SNr neurons.

(A) Schematic representation illustrating the three main basal ganglia pathways activated by cortical stimulation and connecting the cerebral cortex to the SNr. It includes the hyperdirect cortico-subthalamo-nigral pathway (#1) and the direct (#2) and the indirect (#3) striato-nigral pathways. Str, striatum; GPe, external globus pallidus; STN, subthalamic nucleus. (B, top) In this SNr neuron, orofacial sensori-motor cortex stimulation in control conditions evoked a complex response composed of an inhibition followed by a late excitation. (B, bottom) 60 minutes after haloperidol injection (1 mg/kg), the late excitation of the cortically-evoked response was markedly increased. (C, top) Classical triphasic excitatory-inhibitory-excitatory sequence evoked by stimulation of the orofacial sensori-motor cortex in control conditions in another SNr cell. In this cell, the increase of the late excitatory component was prevented by injecting BV 30 minutes after haloperidol as shown by comparing the cortical responses elicited 60 minutes after haloperidol (C, bottom) to the control response of the same SNr cell (C, top). Red numbers in B-C indicate which pathway in A is responsible for each component (excitation-inhibition-excitation) of the cortically-evoked responses. The same number of cortical stimulations (red bar, n = 50) was applied in B-C.

Fig 5. The effect of systemic injection of dopaminergic antagonists of D1- and D2-like receptors on the pattern of responses evoked by cortical stimulation in a SNr neuron is reversed by systemic injection of bee venom.

(A) Classical triphasic excitatory-inhibitory-excitatory sequence evoked by stimulation of the orofacial sensori-motor cortex in control condition. (B) 25 minutes after injection of raclopride (1 mg/kg) plus SCH-23390 (0.5 mg/kg), the inhibitory component of the response of the same SNr neuron presented a marked reduction, whereas the late excitatory component was increased. (C) By 30 minutes following the systemic injection of BV, the inhibitory component of the cortically-evoked response was restored and the late excitatory component was decreased. Red numbers in A-C are as in Fig 4. The same number of cortical stimulations (red bar, n = 50) was applied in A-C.

Fig 6. Effect of systemic injection of bee venom on the pattern of responses evoked by cortical stimulation in a SNr neuron from a 6-OHDA lesioned rat.

(A) Triphasic excitatory-inhibitory-excitatory sequence evoked in a SNr neuron by stimulation of the orofacial sensori-motor cortex in the 6-OHDA condition. (B) 30 minutes after systemic injection of bee venom (3 μg/kg), the inhibitory component of the response of the same SNr neuron was markedly increased, whereas the early and late excitatory components were not modified. Red numbers in A-C are as in Fig 4. The same number of cortical stimulations (red bar, n = 70) was applied in A and B.