Twenty-two male rats ranging in weight from 250 to 350g were housed individually in transparent acrylic cages measuring 44cm×34cm×20cm, each of which contained 5cm sterile sawdust bedding (Harlan, Mexico). Rats were kept with ad libitum feeding and a 12×12hour inverted light–dark cycle (lights were turned on at 20.00). All experiments were carried out in strict accordance with official Mexican guidelines for the care and use of laboratory animals (NOM-062-ZOO-1999) and Guide for the Care and Use of Laboratory Animals (NIH, Washington D.C., USA).

Rats were assigned at random to 3 different groups. The first group (n=8) received IP haloperidol during 14 days (1.5mg/kg) and the second group (n=8) received IP saline vehicle (1.5ml/kg) during the same period. The haloperidol dose and treatment regimen were calculated based on the descriptions by Salamone et al.16 and Trevitt et al.17 The third group (n=6) consisted of animals subjected to a bilateral electrolytic lesion in the VLS.

After 14 days, all rats treated with haloperidol (RBI Research Biochemicals International, Natick, MA, 01760, USA) exhibited TJM. The lesion group presented the same response at 24hours; a 10-minute video recording of the response was made for each study group prior to EMG testing. Subsequently, only 2 rats from each group were selected at random to undergo electromyographic measurement of TJM and contrast responses to pharmacological and electrolytic changes. Rats with VLS lesion were treated with flunixin meglumine as an analgesic (2.5mg/kg) and enrofloxacin as an antibiotic (5mg/kg) in addition to subcutaneous fluid therapy with an electrolyte solution (Hartmann's solution 0.9%).

Rats were anaesthetised with mixture of ketamine (60mg/kg, Bayer) and xylazine (8mg/kg, Pisa). While animals were under deep anaesthesia, researchers drilled burr holes using the following stereotaxic coordinates18: anterior–posterior (AP)=8.74mm anterior to lambda, lateral (L)=±4.4mm, and ventral (V)=3.40mm from the interaural line. Using a stereotaxic instrument (Stoelting Co, USA), researchers then placed a single shaft stainless steel microelectrode 250μm in diameter (Stoelting Co, USA). The electrode delivered 2.5mA direct anodal current during 30seconds using a Grass model S48 stimulator (Astro-Med Inc., USA) connected in series to a Grass model CCU1 constant current unit (Astro-Med Inc., USA).

Rats in all 3 groups were fitted with bipolar silver wire electrodes (Grass EW10AG) in the right temporal muscle 24hours before electromyography. These electrodes measured 254μm in diameter and were approximately 3cm long with a 3mm exposed segment at the end. Electrode leads were routed subcutaneously towards the rostral part of the skull and cemented with dental acrylic (MDC Dental).

Researchers recorded waking EMG activity. Animals with VLS lesion were assessed 48hours after lesion induction. Assessment of animals treated with haloperidol or saline vehicle took place on day 14 of treatment.

Implanted electrodes were connected to a Grass 15A54, 15LT amplifier (Astro-Med Inc., USA) and simultaneously to a Grass AM9 audio monitor (Astro-Med Inc., USA). The amplified analogue signal was relayed through an interface to a PVA-16 PolyVIEW Adaptor Unit to be digitised and then to a PC to calculate and analyse each animal's responses using the Grass PolyVIEW Data Acquisition and Analysis System 16 v 1.0.

After the EMG recording, all animals were injected with high-dose sodium pentobarbital anaesthesia (0.6ml/kg; Sedalpharma, Mexico). Transcardial perfusion was then performed to extract the brain, which was then cryoprotected in graded sucrose concentrations (10%, 20%, and 30%) diluted in a 0.1M phosphate buffer. Researchers used a cryostat at −24°C (LEICA CM1850) to make coronal slices 60μm thick and verify lesion location in those animals with lesions.

Researchers prepared graphs of the distribution of amplitude frequencies and EMG activity frequencies in the temporal muscle at baseline and during TJM in rats and recorded modal values. We also created general linear models with a design of main and nested (hierarchical) factors as indicated for analyses with pseudoreplications19:

Here, y is the response variable (frequency and amplitude), G is the group, R is the rat, and Ps is pseudoreplication of EMG trace. In this analysis, the same model was adjusted by the amplitude of baseline activity in all groups. Response variables were fitted to Poisson-distributed data with a log-link function. All analyses were performed using JMP 6 statistical software (SAS Inc, Cary NC, USA, 2005).

Baseline temporal muscle activity in the control, haloperidol, and electrolytic VLS groups showed different response patterns as evidenced in the 1-second EMG trace (Fig. 1A). Amplitude in control group animals ranged from 0.2 to 0.3mV, while amplitude in the haloperidol group ranged from 0.3 to 0.5mV. Rats with VLS lesions demonstrated the greatest amplitude in baseline activity, from 0.4 to 1.3mV (Fig. 1B).

Figure 1.

(A) One-second EMG trace showing baseline activity in the temporal muscle for control group (C), haloperidol group (H), and electrolytic VLS lesion (L). Horizontal calibration: 200ms. Vertical calibration: 1mV. (B) Amplitude of baseline activity (mV) during the EMG recording in the temporal muscle in the control group (C), the haloperidol group (H), and the electrolytic VLS lesion group (L).

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The modal value of the amplitude in temporal muscle baseline activity differed for each of the 3 groups. The mode was 0.2mV for control group rats, 0.4mV for haloperidol group rats, and 0.5mV for rats in the lesion group (Fig. 1).

We found differences in mean amplitude of baseline EMG activity upon comparing the 3 groups (χ2=6.6, P<.03). Nested factors in the model did not exert an effect (P>.05) given that the greatest amplitude was observed in the group with VLS lesion and not among animals treated with haloperidol. Both of these study groups contrasted with the control group, which showed less activity at 0.3mV (Fig. 2).

Figure 2.

Mean value±SD of the amplitude (mV) of EMG activity in the temporal muscle at baseline in the control group, haloperidol group, and electrolytic VLS lesion group.

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The VLS lesion induced TJMs in all animals as of 24hours after the intervention; systemic exposure to 1.0mg/kg haloperidol also elicited TJMs in all animals by day 14 of treatment.

The frequency distribution for TJM varied between the groups; rats with a ventrolateral striatum lesion displayed higher EMG amplitude and frequency than rats treated with haloperidol (Fig. 3). The modal value of TJM in animals exposed to haloperidol (H) fell in the frequency category of 3Hz and tremor was only recorded within the interval of 3 to 5Hz (Fig. 3A and C). In contrast, rats subjected to VLS lesion displayed 7 categories of TJM frequency between 3 and 9Hz; the mode corresponded to the 4Hz category (Fig. 3A).

Figure 3.

(A) Frequency (Hz) of EMG activity in the temporal muscle during tremulous jaw movement in the haloperidol group (H) and the electrolytic VLS lesion group (L). (B) Amplitude (mV) of EMG activity in the temporal muscle during tremulous jaw movement in the haloperidol group (H) and the electrolytic VLS lesion group (L). (C) One-second sample of EMG trace showing tremulous jaw movements in the haloperidol group (H), and electrolytic VLS lesion group (L). Horizontal calibration: 200ms. Vertical calibration: 1mV.

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EMG activity recordings taken during TJM in rats treated with haloperidol showed amplitudes between 2 and 4mV, while rats with VLS lesion displayed amplitudes between 3 and 9mV (Fig. 3B and C). Furthermore, modal values for TJM occurrence also differed; among animals treated with haloperidol, amplitude was 2mV, while amplitude was recorded in the categories of 3 and 4mV for rats with VLS lesion (Fig. 3B).

Mean values for tremor amplitude (mV) varied between the haloperidol group and the VLS lesion group (χ2=10.3, P<.001). Nested factors in the model did not contribute to the contrast effect (P>.05), which shows that the highest mean amplitude was in subjects with VLS lesion and not in animals treated with haloperidol (Fig. 4A).

Figure 4.

Mean value±SD of the amplitude (mV) and frequency (Hz) of EMG activity in the temporal muscle during tremulous jaw movement in rats subjected to haloperidol exposure or to electrolytic lesion in the VLS.

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Frequency recordings (Hz) also showed the same type of response when the two rat groups were contrasted (χ2=31, P<.001). In this model, the effect of individuals nested within the group provided contrast (χ2=7, P<.03) and pseudoreplications did not vary when nested by rat (χ2=1.7, P<.9). A higher mean frequency value was therefore also recorded in rats with VLS lesion than in rats treated with haloperidol (Fig. 4B).