The pharmacological blockade of medial forebrain bundle induces an acute pathological synchronization of the cortico-subthalamic nucleus-globus pallidus pathway

Abstract

Pathological oscillations characterize the firing discharge of different basal ganglia (BG) stations in rat models of Parkinson's disease. Most recent literature focused on the prominence of the beta frequency band in awake rats. Yet, in 6-hydroxydopamine-lesioned animals, the firing discharge of the globus pallidus (GP) and the substantia nigra reticulata are in phase with urethane-induced slow wave cortical activity. The neuronal basis of this pathological synergy at low frequency is widely debated. In order to understand the role of substantia nigra pars compacta (SNc) signalling in the development of pathological synchronization, we performed a pharmacological inactivation of the medial forebrain bundle (MFB) through tetrodotoxin (TTX), which led to a dramatic, but reversible, reduction of the dopamine content in the striatum. This procedure caused a significant contralateral akinesia, detectable as soon as anaesthesia vanished, and lasting about 3-4 h. We sought to determine the electrophysiological counterpart of this transient Parkinsonian-like hypokinetic syndrome. Hence, we obtained the electrocorticogram (ECoG) and single unit recordings from GP and subthalamic nucleus (STN) in normal rats before and after the TTX injection in MFB. Intriguingly, the TTX-mediated inactivation of MFB induced a fast developing coherence between cortex and GP and a significant increase of the cortex/STN synchronization. The intra-GP iontophoretic delivery of haloperidol or the GABA(A) receptor antagonist bicuculline induced a short term cortex/GP synchronization. Strikingly, STN inactivation by local muscimol reversed both haloperidol- and TTX-mediated coherence between cortex and GP. Our data show that an abnormal cortical/BG synchronization, at low frequency, can be reproduced also without SNc neuronal loss and striatal cytoarchitectonic alterations. In addition, our results, which represent an acute and reversible Parkinsonism based upon impaired cable properties, seem compatible with the interpretation of acute changes of the functional interplay between cortex and the STN-GP pathway as a key factor mechanism underlying the fast deep brain stimulation-induced acute Off-On transitions.

Figures

Figure 1. Schematic representation of the utilized electrophysiological procedures and the impact of the pharmacological blockade of medial forebrain bundle (MFB) on motor performance assessed by the step test (Olsson et al. 1995) and by the elevated body swing test (EBST) (Borlongan & Sanberg, 1995)

A, scheme showing electrophysiological setting on a parasagittal section of rat brain. Extracellular action potentials from globus pallidus (GP) or substantia nigra pars reticulata (SNr) or subthalamic nucleus (STN) were simultaneously acquired with ECoG before and after TTX injection into the MFB. B, diagram depicting the micro-iontophoretic experiments by which a GP neuron is recorded during local (or intra-STN) drug delivery. C, micrograph of three antero-posterior sections showing the needle track targeting MFB. The inset depicts a magnified view of the needle target in the MFB area as shown in the joined atlas section (coronal section −2.80 mm from the bregma). Note that the MFB target was reached through a trajectory angled 1 deg in the anterior–posterior plane. Protocols: D, dramatic increase of the initiation step time in the right body forelimb (controlled by the left injected hemisphere) 1 h after MFB block. Deficit is still significant after 3 h. E, increase of the right forelimb stepping time evaluated 1 h after the TTX injection into the left MFB. F, clear-cut decrease of the right forelimb step length following the pharmacological blockade of the left MFB. G, following pharmacological blockade of the left MFB, rats tend to move toward right.

Figure 2. Microdialytic measurement of dopamine (DA) and homovanillic acid (HVA) striatal levels before and after TTX infusion and assessment of dopaminergic nigrostriatal damage after TTX-mediated MFB blockade

A, effect of infusion of 2 μl of 10 μm TTX (•) or saline (○) into the MFB on the DA levels in striatal dialysate. Numbers on the abscissa represent successive 20 min dialysate samples after 4 h of stabilization. TTX or saline was applied at the end of the 5th sample. B, effect of infusion of 2 μl of 10 μm TTX (•) or saline (○) into the MFB on the HVA levels in striatal dialysate. Numbers on the abscissa represent successive 20 min dialysate samples after 4 h of stabilization. TTX or saline was applied at the end of the 5th sample. *P < 0.05 versus basal values. C, example of a histological section at the level of the left substantia nigra pars compacta (SNc) (TTX-injected side) compared to the right SNc (control side) performed 5 weeks after the pharmacological blockade of the MFB: no substantial differences are evidenced. D, quantitative analysis of neuronal loss within SNc carried out at 1, 2 and 5 weeks following the TTX injection of MFB compared to control side. E, histological section of the left dorsal striatum (injected side) substantially similar to the right control side at 5 weeks from the TTX-mediated blockade of the MFB. F, quantitative analysis of dopaminergic fibre content within the striatum performed 1, 2 and 5 weeks after the TTX injection of MFB, compared to control side.

Figure 3. Example ECoG traces during urethane-induced slow wave cortical activity (SWA) before and after TTX-induced MFB blockade

A, note the active component superimposed on the 1 Hz SWA. B and C, after TTX injection the active component of SWA expressed a higher frequency as highlighted by the grey boxes. D, in the course of MFB blockade no significant differences of the dominant frequency and its power were observed.

Figure 4. The impact of TTX-induced MFB blockade on GP activity and ECoG/GP coherence

A and B, an example change of synchronization revealed by the simultaneous acquisition of ECoG and GP single unit before (A) and after (B) TTX infusion. The lower insets represent the GP spike-triggered waveform average (AvWv) and the autocorrelogram histograms (AutoCrl). Before the pharmacological MFB manipulation (A) the AvWv's do not present any phase between cortex and GP. Accordingly, the AutoCrl is lacking a clear oscillatory activity. In the post-TTX period (B), the GP firing pattern changed (AutoCrl) demonstrating an undoubted coherence with the cortex as shown in AvWv. C, the corresponding contour plot of the neuron in A and B describing the mean power changes time course following the TTX injection into the MFB. D is the mean of ECoG/GP coherence power before (○, n= 45) and following TTX (•, n= 59). Dashed line in D is P= 0.05; in the same figure * is P < 0.003 within analysis; § is P < 0.003 between analysis.

Figure 5. Pipette insertion as well as vehicle infusion into MFB did not cause any synchronization between cortex and GP

A, in basal condition the GP firing is regular and not correlated with the cortex as demonstrated by AvWv and coherence histograms. B, further, the simple pipette insertion as well as the vehicle infusion into the MFB field did not cause any GP firing pattern change. C, however, riluzole (injected into MFB) mimicked the TTX-mediated behaviour of GP neurons producing good synchronization between the two structures with a clear phase in AvWv and in the coherence histogram.

Figure 6. The impact of TTX blockade on STN activity and ECoG/STN coherence

A and B, following TTX, STN units already synchronized with the cortex in normal conditions (A) displayed a more profound coherence (B). In the pre- (A) and post-TTX (B) period the AvWv's are suggestive of a synchronized activity between cortex and STN and the AutoCrls display an oscillatory behaviour of the discharge. C, the corresponding contour plot of the STN activity described above (A and B) in which the mean power is represented during the time course following the TTX injection into the MFB. D is the mean of ECoG/STN coherence power before (○, n= 26) and following TTX (•, n= 46). Dashed line in D is P= 0.05; in the same figure * is P < 0.003 within analysis; § is P < 0.003 between analysis.

Figure 8. Bicuculline micro-iontophoretically delivered within GP induced an ECoG/GP peak of coherence

A and B, the GABAA antagonist, bicuculline caused the shift of the GP firing pattern from regular (A) to clustered (B). Changes of discharge are highlighted by the AvWv and AutoCrl showing a clear oscillation (A and B). C, the corresponding contour plot of the GP activity described in A and B showing the mean power in the course of bicuculline micro-iontophoretically injected at 50 nA within GP. D is the mean of ECoG/GP coherence power before (○, n= 18) and post-bicuculline (•, n= 19). Dashed line in D is P= 0.05; in the same figure * is P < 0.003 within analysis; § is P < 0.003 between analysis.

Figure 7. Pharmacological inactivation of STN abolished the TTX-induced ECoG/GP synchronization

A–C, the impact of pharmacological blockade of MFB and the subsequent inactivation of activity of STN is illustrated by means of traces and the AvWv and AutoCrl series (see Results). The GP firing does not show any synchronization with the ECoG in basal condition (A) while it is in phase after the TTX protocol (B). However, as a consequence of the STN inactivation by micro-iontophoresis of muscimol, the GP reacquired the pre-TTX firing pattern (C). D is the corresponding contour plot of A–C, showing the mean power in the course of the TTX injection into the MFB and the subsequent inactivation of the STN by muscimol. E, mean of ECoG/GP coherence power before (○, n= 4), following TTX (•, n= 4) and post-muscimol (▴, n= 4). Dashed line in E is P= 0.05; in the same figure * is P < 0.003 within analysis; § is P < 0.003 between analysis.

Figure 9. Changes of ECoG/GP coherence induced by D2 antagonist haloperidol only when locally applied

A, the systemic treatment with haloperidol and SCH23390 caused a significant firing rate reduction not associated with changes in firing pattern as evidenced in the corresponding insets (on the right). B–D, when haloperidol was micro-iontophoretically applied, basal GP pattern (B) was shifted towards ‘clustered’ discharge (C); the D1 antagonist SCH23390 failed to cause clear pattern changing (D). E, the corresponding contour plot of the GP activity shown in B and C, picturing the mean power in the course of haloperidol micro-iontophoretically injected at 50 nA within GP. F, mean of ECoG/GP coherence power in control (○, n= 18), post-haloperidol (•, n= 16), post-SCH23390 (▴, n= 10) and post-systemic treatment of both drugs (▪, n= 10). Dashed line in F is P= 0.05; in the same figure * is P < 0.003 within analysis; § is P < 0.003 between analysis.