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Post-hypoxic myoclonus induces Fos expression in the reticular thalamic nucleus and neurons in the brainstem
Brain Research 1059 (2005) 122 – 128 www.elsevier.com/locate/brainres
Research Report
Post-hypoxic myoclonus induces Fos expression in the reticular thalamic nucleus and neurons in the brainstem Kwok-Keung Tai, Daniel D. Truong* The Parkinson’s and Movement Disorder Research Laboratory, Long Beach Memorial Medical Center, 2625 Pasadena Avenue, Long Beach, CA 90806, USA The Parkinson’s and Movement Disorder Institute, Fountain Valley, CA 92708, USA Accepted 5 August 2005 Available online 28 September 2005
Abstract Post-hypoxic myoclonus is a movement disorder characterized by brief, sudden involuntary muscle jerks. Although the mechanism underlying this disorder remains unclear, earlier pharmacological studies indicated that aberrant activity of specific neuronal circuitry in the central nervous system causes this disorder. In the present study, Fos protein, an immediate-early gene product, was used as a marker of neuronal activity to identify the brain nuclei possibly involved in post-hypoxic myoclonus. We found that Fos protein was immunologically detected in the reticular thalamic nucleus (RT), the medial longitudinal fasciculus (MLF) as well as in the locus coeruleus (LC) and the periventricular gray substance (PVG) in post-hypoxic rats that developed myoclonus in response to auditory stimuli. Fos was not detected in these nuclei from rats that underwent 4 min of cardiac arrest without myoclonus. Electrolytic lesions of the RT or MLF but not the LC/PVG significantly reduced auditory stimulated myoclonus in the post-hypoxic rats. The results suggest that neuronal activity in the RT and the MLF plays a contributing role in post-hypoxic myoclonus. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Post-hypoxic myoclonus; Fos; Immediate-early gene; Reticular thalamic nucleus; Medial longitudinal fasciculus; Locus coeruleus; Periventricular gray substance; Electrolytic lesion
1. Introduction Post-hypoxic myoclonus is neurological disorder characterized by brief, sudden, shock-like involuntary movements caused by active muscle contractions or inhibitions [9]. Cerebral ischemic/hypoxic insults during cardiac arrest are one of the main causes of post-hypoxic myoclonus. Although the mechanism underlying post-hypoxic myoclonus remains obscure, the response of this condition to certain pharmacological agents provides implications on the mechanism underlying this disorder. For example, administration * Corresponding author. The Parkinson’s and Movement Disorder Research Laboratory, Long Beach Memorial Medical Center, 2625 Pasadena Avenue, Long Beach, CA 90806, USA. Fax: +1 562 426 8903. E-mail address: [email protected] (D.D. Truong). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.08.027
of classical anti-epileptic agents such as clonazapam [27] and valproate [4,18,28] in patients with post-hypoxic myoclonus improves their myoclonus symptoms. The principal action of these anti-epileptic agents is to enhance the inhibitory GABAergic neurotransmission of the neural circuitries in the brain by increasing the GABA receptor activity. Furthermore, in animal studies, activation of GABA receptors has anti-myoclonic effects, whereas blockade of the GABA receptors by intraventricular infusion of GABAA receptor antagonists such as bicuculline elicits myoclonus in a dose-related manner [14]. These results support the notion that post-hypoxic myoclonus is the result of hyperactivity of specific neural circuits in the brain whose activities are substantially elevated following cerebral hypoxic injury. Identification of these brain nuclei will, therefore, provide new insights into the mechanism of this disorder.
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Since the early 90s, neuronal activity is known to cause a rapid induction of a class of proteins coded for by immediate-early genes such as c-fos [12]. Fos protein is synthesized during certain forms of neuronal activation as a result of intraneuronal metabolic changes. This unique feature of c-fos expression enables it to be a valuable marker to identify the neuronal cell groups that are activated in response to exogenous stimuli. It has been shown that neuronal activity whether it is triggered electrically [1,3,23] or chemically such as administration of chemical convulsants [11,13] induces a rapid and transient increase of c-fos expression in rat brain. In the present study, Fos protein was used a functional marker for neuronal activation to identify hyperactive brain nuclei in an established animal model of post-hypoxic myoclonus [7,8,24,25]. Using this approach, we have identified discrete nuclei in the thalamus and the brainstem whose hyperactivity following cerebral hypoxic insult may cause post-hypoxic myoclonus.
2. Methods and experimental procedures 2.1. Animal model of cardiac arrest-induced post-hypoxic myoclonus An animal model of post-hypoxic myoclonus originally developed by Truong et al. [24] was used in this study. In brief, Sprague – Dawley rats of 220– 240 g were anesthetized with ketamine (85 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.). Atropine (0.04 mg/kg, i.p.) was administrated to minimize respiratory secretion. The trachea was intubated with an 18gauge catheter, which was then attached to a ventilator (settings: 425 ml/min; 60 strokes/min). The rat was placed on a heating pad, and ECG electrodes were attached. Body temperature was maintained at 37 -C with a heating lamp. The left femoral artery and vein were catheterized to monitor arterial blood pressure and for administration of drugs, respectively. An incision of an inch length was made on the skin along the rat cavity at a location above the heart. The skin was pulled apart to facilitate the insertion of an Lshaped metal loop into the rat cavity. An L-shaped metal loop was inserted through the muscle of the rat body cavity into an area underneath the aorta and the surrounding major blood vessels. Cardiac arrest was initiated and maintained by mechanically obstructing all the major cardiac blood vessels including the aorta by applying pressure on the surface of the rat cavity with the palm of one hand while pulling the inserted L-shaped loop up against the rat cavity with another hand. The arterial blood pressure was maintained at 0 –10 mm Hg. Under such low systemic arterial blood pressure, cerebral perfusion came to a halt. Post-hypoxic rats developed myoclonus only after a critical duration of cerebral hypoxia. A cardiac arrest for a duration of 9 min 30 s is a compromise at which myoclonus reliably develops following recovery, the survival rate is acceptable, and the severity of other neurological conditions was manageable.
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Resuscitation began at 9 min 30 s following cardiac arrest by resuming manual thoracic compression and by intravenous injection of 10 mg/kg epinephrine and 4 mEq/ kg sodium bicarbonate. Following resuscitation, rats were weaned from the ventilator, the catheters were removed, and wounds were sutured. The animals were placed on a heating pad to facilitate recovery from surgical coma. 2.2. Behavior and evaluation of post-hypoxic myoclonus in rat One of the clinical features of post-hypoxic myoclonus is the trigger of myoclonic jerks in response to exogenous stimuli such as sound. Jerking movements in response to auditory stimuli were ranked according to their intensity as previously described [24]. For the Fos staining experiments, 2 days after cardiac arrest, rats were given auditory stimuli. For the purpose of evaluating myoclonic jerks in response to auditory stimuli, the rat was placed in a clear plastic cage for 10 min to let it habituate to the new environment prior to evaluation. The rat was then presented with 45 clicks of a metronome as the auditory stimulus. Each click had a sound intensity of 96 dB with a duration of 40 ms at a frequency of 0.75 Hz. The involuntary muscle jerks in response to each click were scored with the following criteria: 0 = no jerks; 1 = ear twitch; 2 = ear and head jerk; 3 = ear, head and shoulder jerk; 4 = whole body jerk; and 5 = whole body jerk with jumping [7,24]. The myoclonus scores shown in Figs. 1 and 5 are the sum of the 45 scores of the rats in response to the 45 clicks. One hour after auditory stimulus, rats were sacrificed. Brain sections were prepared for detection of Fos protein as described below. Rats that underwent cardiac arrest for 4 min did not develop myoclonus when given auditory stimuli. This group would serve as a negative control for the study.
Fig. 1. Rats subjected to cardiac arrest for 9 min 30 s but not for 4 min developed myoclonus in response to auditory stimuli. Myoclonus scores recorded from rats before and following cardiac arrest for 9 min 30 s ( ) or 4 min (D). The arrow indicates the day on which cardiac arrest surgery was performed. Values are mean T SE. **Indicates significant difference from control at P < 0.01. Five animals were used in each group.
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2.3. Procedures for detection of Fos protein in rat brain sections Fos protein was detected in brain sections using the avidin– biotin peroxidase (ABC) method. Fos in brain sections was detected by primary anti-Fos antibody with a biotin-conjugated secondary antibody followed by ABC solution and then visualized by enhanced DAB immunocytochemistry. In brief, coronal brain sections of ¨30 Am were collected in phosphate buffer (PBS) with 0.1% sodium azide. Then sections were transferred and incubated in methanol with 0.3% H2O2 for 20 min. Sections were rinsed in PBS three times, 10 min each. Sections were incubated in 5% goat normal serum in 0.3% Triton-X100 PBS for 2 h at room temperature with gentle agitation on a shaker. Then sections were incubated in anti-Fos antibody (1:10,000) (Santa Cruz Biotechnology) in 0.3% TritonX100 PBS in blocking serum with gentle agitation on a shaker in the 4 -C refrigerator for a duration of 48 h. Sections were then rinsed in PBS four times, 10 min each. Sections were then incubated in biotinylated goat antirabbit antibody (1:1000) in 0.3% Triton-X100 PBS in blocking serum with agitation on a shaker at room temperature for 2 h. Sections were then rinsed in PBS four times, 10 min each. Sections were incubated in Vectastain ABC solution at room temperature for 1 h with gentle shaking and then were rinsed in PBS, 3 times, 10 min each. Sections were then incubated in freshly prepared DAB solution with H2O2 solution (0.01%) and 5 drops of nickel chloride, then mixed well in dishes on a shaker until an adequate level of staining was reached. It took 7 to 9 min for the reaction to complete. DAB reaction was stopped by rinsing the sections seven times in PBS for a duration of 5 min each. Sections were stored in PBS buffer in the 4 -C refrigerator overnight. Then sections were mounted on gel-coated slides and dried for 2 days. Sections were then dehydrated with the following steps: purified water for 2 min; 50% ethanol for 2 min; 70% ethanol for 2 min; 95% ethanol for 2 min; 100% ethanol for 2 min; 100% ethanol for 2 min; xylene for 2 min. Then slides were covered with cover slip with Permount. Fos protein is
recognized as dark staining in the neuronal cell nucleus under a microscope. Two groups of animals were used in the fos staining experiments. The experimental group was subjected to cardiac arrest for a duration of 9 min 30 s, the other group is their respective control which was subjected to cardiac arrest for a duration of 4 min. Five animals were used in each group. 2.4. Electrolytic lesion of rat brain nuclei Electrolytic lesion to functionally evaluate the contribution of Fos expressed nuclei to audiogenic post-hypoxic myoclonus was performed according to the standard procedures of Cubero et al. [2] with some modifications. In brief, a rat was anesthetized with ketamine hydrochloride (85 mg/kg, i.p.) and xylazine (15 mg/kg i.p.). The rat was mounted in a stereotaxic frame (KOPF Instruments). The incisor bar was adjusted until the head was in the flat-skull position about 3.3 mm below horizontal, thus allowing the animal’s stereotaxic coordinates to be referred to a standard brain atlas [17]. With the head properly positioned, a midline incision was made from the level of the eyes to the neck using a sterile scalpel. Using a millimeter scale on the stereotaxic frame, the coordinates of the intended lesion site were located according the brain map. These coordinates would be marked using a marker on the skull. With a high speed dental drill, a 1-mm hole was made in each hemisphere to the reference sutures (Bregma and Lambda) to allow insertion of a microelectrode for electrolytic lesions. The brain nucleus was destroyed by electrolytic lesion using a stainless steel microelectrode (FHC, Inc., ME) to the site of lesion by applying a constant current of 0.3 mA for 4 s from a DS3 constant current stimulator (Harvard Apparatus, MA). After the lesions, the electrode would be removed and incisions in the scalp closed. Sham controls followed the same procedures as described except that no current was applied to the electrode. Seven days after electrolytic lesions, both lesioned rats and sham-operated control rats underwent cardiac arrest for 9 min 30 s. Myoclonus scores were obtained from the rats on a daily
Fig. 2. Fos protein was detected in the LC and the PVG of the rats that developed myoclonus in response to auditory stimuli 48 h after cardiac arrest for 9 min 30 s (A). Fos was not detected in the LC and the PVG from rats that were subjected to 4 min of cardiac arrest and did not develop myoclonus (B).
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basis for the next 3 days as described above. Rats were then sacrificed, and their brains were fixed for sectioning. Cresyl violet staining was used to verify the site of electrolytic lesion as previously described [21]. The coordinates for the lesion of the locus coeruleus (LC)/ periventricular gray substance (PVG) are Bregma: 10 mm, Lateral: T1 mm, Depth: 7.5 mm; the coordinates for the lesion of the reticular thalamic nucleus (RT) are Bregma: 2.5 mm, Lateral: T2.5 mm, Depth: 7 mm, and the coordinates for the lesion of the medial longitudinal fasciculus (MLF) are Bregma: 11 mm, Lateral: T0.5 mm, Depth: 8.5 mm. A total of six groups were used in the lesion study, one each for the lesion of the LC/PVG, the RT, and the MLF. The other three groups are their respective sham-operated control groups. Each group consists of five animals. All experiments involving animal use were approved by the institutional animal care and use committee. 2.5. Statistical analysis Brain sections for Fos staining were obtained from rats that underwent cardiac arrest for 9 min 30 s and 4 min. The mean myoclonus scores between these two groups of posthypoxic rat at each time point were compared using Student’s t test. A P < 0.05 was considered as significant. In the electrolytic lesion studies, the myoclonus scores between lesioned animals and the corresponding sham-
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operated controls were compared using one-way analysis of variance (ANOVA). If there were significant differences, a post hoc Tukey’s test was used to determine which sample means were different.
3. Results Auditory stimuli induced myoclonus in rats that underwent cardiac arrest for 9 min 30 s. As expected from our previous experience, rats that underwent cardiac arrest for 4 min did not develop myoclonus in response to auditory stimuli (Fig. 1). In post-hypoxic rats with myoclonus, Fos was detected in the LC, the PVG (Fig. 2), the reticular thalamic nucleus (RT) (Fig. 3), and the MLF (Fig. 3). To evaluate whether these nuclei or cell groups contribute to the generation of myoclonus, we examined the effects of electrolytic lesions of these nuclei on the severity of posthypoxic myoclonus. Lesions of the nuclei were verified using cresyl violet staining in coronal brain sections. We were not able to destroy the LC and the PVG individually using electrolytic lesions as the LC and the PVG are so close together. Therefore, the LC and the PVG were destroyed together (Fig. 4A). Discrete regions of the RT (Fig. 4C) and the MLF (Fig. 4E) were destroyed by electrolytic lesions. Lesions of the LC/PVG did not alter cardiac arrest-induced post-hypoxic myoclonus (Fig. 5A). However, lesions of the RT (Fig. 5B) and the MLF (Fig. 5C) significantly reduced
Fig. 3. Post-hypoxic myoclonus induced Fos protein in the RT and the MLF in coronal brain sections. Fos protein was detected in the RT (A) and the MLF (C) of the rats that developed myoclonus in response to auditory stimuli 2 days after cardiac arrest-induced cerebral hypoxic injury. Fos protein was not detected in the RT (B) and the MLF (D) from the rats that did not develop myoclonus in response to auditory stimuli 2 days after cardiac arrest for 4 min.
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Fig. 4. Electrolytic lesions of the LC/PVG, the RT, and the MLF. Cresyl violet staining of the rat coronal brain sections showing electrolytic lesions of the LC/ PGV (A), the RT (C) and the MLF (E) and their respective sham-operated control brain sections (B, D and F). The arrows indicate the sites of lesions. The coordinates used for each of these sites are specified in Methods and experimental procedures section.
cardiac arrest-induced post-hypoxic myoclonus when compared with their respective sham controls (Figs. 5B, C).
4. Discussion In the present study, we detected Fos protein in the RT, LC, PVG, and the MLF of the brainstem in post-hypoxic rats that developed myoclonus in response to auditory stimuli. Fos was not detected in these nuclei of rats that were subjected to a short duration of cardiac arrest of 4 min and did not exhibit myoclonus in response to auditory stimuli. The results indicate that these nuclei may be involved in post-hypoxic myoclonus. Using an electrolytic lesion technique to further verify the involvement of these nuclei in post-hypoxic myoclonus, we found that lesion of the LC/PVG had no significant effects on cardiac arrest-induced post-hypoxic myoclonus. This result suggests that the LC/PVG neuronal activity is not directly related to the generation of myoclonic jerks. The physiological function of the LC is associated with anxiety and stress responses [10], and the PVG is related with modulation of nociception signaling [20]. The Fos expression in these two nuclei could have resulted from the fact that rats were under stress during auditory stimuli-induced myoclonic
jerks. The route of signaling of fear and threat was probably relayed from the sensory cortex through the thalamus to the brain stem. The elevated noradrenergic activity in the LC would increase the alertness and attention of the rats to the challenging environment. We understand that inserting the electrode without passing current in sham-operated controls may cause some mechanical damage to the brain. As a result, we evaluated the effects of lesions on post-hypoxic myoclonus 1 week after the induction of lesion so that the rats can recover from the injury. Histological examinations of the brain sections 10 days after the induction of lesion do not reveal damage along the path through which the lesion electrode was inserted (Figs. 4B, D, F). In addition, we do not observe differences in the animal behavior such as feeding pattern, body weight change, and movement between the sham-operated rats and normal untreated rats. These observations suggest that the sham-operated rats are not different from the normal untreated rats, and thus, they can be used as a proper control for the lesion study. The expression of Fos in the RT is consistent with previous pharmacological studies [14] which showed that the RT is one of the generator centers for post-hypoxic myoclonus. As predicted from these earlier studies, ablation of the RT reduced post-hypoxic myoclonus as demonstrated in the present study. This result further
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Fig. 5. Effects of electrolytic lesions of rat brain nuclei on post-hypoxic myoclonus. Electrolytic lesion of the RT (B), the MLF (C) but not the LC/PGV (A) reduced auditory stimuli provoked myoclonus from rats which underwent cardiac arrest for 9 min 30 s. Values are mean T SE. *, **Indicate significant difference at P < 0.05 and P < 0.01, respectively. Five animals were used in each group.
substantiates the notion of the functional role of the RT in post-hypoxic myoclonus. It also suggests that the RT is a target for future possible intervention with deep brain stimulation. Indeed, electrical stimulation of the RT has recently been showed to suppress limbic motor seizures [16]. Recently, Frucht et al. [5] reported the findings in 4 patients with post-hypoxic myoclonus and noted an increase of glucose metabolism in the ventrolateral thalamic nucleus (VL) and pontine tegmentum in patients relative to controls. We do not detect significant Fos expression in the VL following post-hypoxic myoclonus in our animal model. In addition, lesion of the VL (5/5 rats) does not alter the severity of post-hypoxic myoclonus when compared with sham-operated controls. These results argue against the role of the VL in post-hypoxic myoclonus. The neuronal activity in the MLF as indicated by Fos expression suggested that the MLF was involved in the observed myoclonic jerks. The MLF, located on each side of the brainstem, carries information about the direction that the eyes should move. The MLF connects the several cranial nerve nuclei III, IV, and VI, as well as the gaze centers and information about head movement from cranial nerve VIII [15]. The MLF descends into the cervical spinal cord and innervates some muscles of the neck [19] in coordination of gaze with head and neck movements [22]. It is not clear why the MLF was activated in post-hypoxic rats showing myoclonic jerks. According to the scoring criteria, rats that
showed involuntary muscle jerks in the head and shoulders in response to each stimulus received a score of 3. One possibility that could account for the activation of the MLF is that the head movement during myoclonic jerks triggered vestibular signals from each of the labyrinthine canals. Because the observed post-hypoxic myoclonus-induced Fos protein expression occurred mostly in the brainstem region, regardless of the neuronal pathways through which these nuclei are involved, the results suggest that the post-hypoxic myoclonus animal model has a significant brainstem component. This finding further supports the relevance of this animal model to human post-hypoxic myoclonus which is believed to have brainstem origin [6] when characterized with generalized massive body jerks [26,30]. Future studies directed towards elucidating the neuronal connections and the nature of neurotransmission of the MLF that displayed Fos expression will give us useful information regarding the signaling pathway underlying post-hypoxic myoclonus in this animal model. It has been hypothesized that the inferior olive activity may be involved in the generation of post-hypoxic myoclonus [29]. We were not able to detect neuronal activity in the inferior olive following post-hypoxic myoclonus using Fos as a marker. This observation does not rule out the role of inferior olive in the generation of posthypoxic myoclonus as induction of immediate-early gene expression is related to the stimuli strength. Furthermore, it is possible that post-hypoxic myoclonus could induce a
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variety of immediate-early gene products in different nuclei, Fos is one of them whose expression was elevated in the certain nuclei whose activity is associated with auditory stimulated myoclonic jerks. The use of other immediateearly gene products as functional markers for neuronal activity will likely reveal more brain nuclei whose aberrant activity may underlie post-hypoxic myoclonus. Acknowledgments The study was supported by funding from the Parkinson’s and Movement Disorder Foundation, and the Long Beach Memorial Foundation. References [1] A.J. Cole, S. Abu-Shakra, D.W. Saffen, J.M. Baraban, P.F. Worley, Rapid rise in transcription factor mRNAs in rat brain after electroshock-induced seizures, J. Neurochem. 55 (1990) 1920 – 1927. [2] I. Cubero, T.E. Thiele, I.L. Berstein, Insular cortex lesions and taste aversion learning: effects of conditioning method and timing of lesion, Brain Res. 839 (1999) 323 – 330. [3] M. Dragunow, H.A. Robertson, Kindling stimulation induces c-fos protein(s) in granule cells of the rat dentate gyrus, Nature 329 (1987) 441 – 442. [4] S. Fahn, Posthypoxic action myoclonus: review of the literature and report of two new cases with response to valproate and estrogen, Adv. Neurol. 26 (1979) 49 – 84. [5] S.J. Frucht, M. Trost, Y. Ma, D. Eidelberg, The metabolic topography of posthypoxic myoclonus, Neurology 62 (2004) 1879 – 1881. [6] M. Hallett, Neurophysiology of brainstem myoclonus, Adv. Neurol. 89 (2002) 99 – 102. [7] A.G. Kanthasamy, R.R. Matsumoto, D.D. Truong, Animal models of myoclonus, Clin. Neurosci. 3 (1995 – 1996) 236 – 245. [8] A.G. Kanthasamy, B.Q. Nguyen, D.D. Truong, Animal model of posthypoxic myoclonus: II. Neurochemical, pathologic, and pharmacologic characterization, Mov. Disord. 1 (2000) 31 – 38. [9] J.W. Lance, R.D. Adams, The syndrome of intention or action myoclonus as a sequel to hypoxic encephalopathy, Brain 86 (1963) 111 – 136. [10] S. Ma, D.A. Morilak, Induction of FOS expression by acute immobilization stress is reduced in locus coeruleus and medial amygdala of Wistar – Kyoto rats compared to Sprague – Dawley rats, Neuroscience 124 (2004) 963 – 972. [11] R. Maggio, P. Lanaud, D.R. Grayson, K. Gale, Expression of c-fos mRNA following seizures evoked from an epileptogenic site in the deep prepiriform cortex: regional distribution in brain as shown by in situ hybridization, Exp. Neurol. 119 (1993) 11 – 19.
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Research Report
Post-hypoxic myoclonus induces Fos expression in the reticular thalamic nucleus and neurons in the brainstem Kwok-Keung Tai, Daniel D. Truong* The Parkinson’s and Movement Disorder Research Laboratory, Long Beach Memorial Medical Center, 2625 Pasadena Avenue, Long Beach, CA 90806, USA The Parkinson’s and Movement Disorder Institute, Fountain Valley, CA 92708, USA Accepted 5 August 2005 Available online 28 September 2005
Abstract Post-hypoxic myoclonus is a movement disorder characterized by brief, sudden involuntary muscle jerks. Although the mechanism underlying this disorder remains unclear, earlier pharmacological studies indicated that aberrant activity of specific neuronal circuitry in the central nervous system causes this disorder. In the present study, Fos protein, an immediate-early gene product, was used as a marker of neuronal activity to identify the brain nuclei possibly involved in post-hypoxic myoclonus. We found that Fos protein was immunologically detected in the reticular thalamic nucleus (RT), the medial longitudinal fasciculus (MLF) as well as in the locus coeruleus (LC) and the periventricular gray substance (PVG) in post-hypoxic rats that developed myoclonus in response to auditory stimuli. Fos was not detected in these nuclei from rats that underwent 4 min of cardiac arrest without myoclonus. Electrolytic lesions of the RT or MLF but not the LC/PVG significantly reduced auditory stimulated myoclonus in the post-hypoxic rats. The results suggest that neuronal activity in the RT and the MLF plays a contributing role in post-hypoxic myoclonus. D 2005 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Post-hypoxic myoclonus; Fos; Immediate-early gene; Reticular thalamic nucleus; Medial longitudinal fasciculus; Locus coeruleus; Periventricular gray substance; Electrolytic lesion
1. Introduction Post-hypoxic myoclonus is neurological disorder characterized by brief, sudden, shock-like involuntary movements caused by active muscle contractions or inhibitions [9]. Cerebral ischemic/hypoxic insults during cardiac arrest are one of the main causes of post-hypoxic myoclonus. Although the mechanism underlying post-hypoxic myoclonus remains obscure, the response of this condition to certain pharmacological agents provides implications on the mechanism underlying this disorder. For example, administration * Corresponding author. The Parkinson’s and Movement Disorder Research Laboratory, Long Beach Memorial Medical Center, 2625 Pasadena Avenue, Long Beach, CA 90806, USA. Fax: +1 562 426 8903. E-mail address: [email protected] (D.D. Truong). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.08.027
of classical anti-epileptic agents such as clonazapam [27] and valproate [4,18,28] in patients with post-hypoxic myoclonus improves their myoclonus symptoms. The principal action of these anti-epileptic agents is to enhance the inhibitory GABAergic neurotransmission of the neural circuitries in the brain by increasing the GABA receptor activity. Furthermore, in animal studies, activation of GABA receptors has anti-myoclonic effects, whereas blockade of the GABA receptors by intraventricular infusion of GABAA receptor antagonists such as bicuculline elicits myoclonus in a dose-related manner [14]. These results support the notion that post-hypoxic myoclonus is the result of hyperactivity of specific neural circuits in the brain whose activities are substantially elevated following cerebral hypoxic injury. Identification of these brain nuclei will, therefore, provide new insights into the mechanism of this disorder.
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Since the early 90s, neuronal activity is known to cause a rapid induction of a class of proteins coded for by immediate-early genes such as c-fos [12]. Fos protein is synthesized during certain forms of neuronal activation as a result of intraneuronal metabolic changes. This unique feature of c-fos expression enables it to be a valuable marker to identify the neuronal cell groups that are activated in response to exogenous stimuli. It has been shown that neuronal activity whether it is triggered electrically [1,3,23] or chemically such as administration of chemical convulsants [11,13] induces a rapid and transient increase of c-fos expression in rat brain. In the present study, Fos protein was used a functional marker for neuronal activation to identify hyperactive brain nuclei in an established animal model of post-hypoxic myoclonus [7,8,24,25]. Using this approach, we have identified discrete nuclei in the thalamus and the brainstem whose hyperactivity following cerebral hypoxic insult may cause post-hypoxic myoclonus.
2. Methods and experimental procedures 2.1. Animal model of cardiac arrest-induced post-hypoxic myoclonus An animal model of post-hypoxic myoclonus originally developed by Truong et al. [24] was used in this study. In brief, Sprague – Dawley rats of 220– 240 g were anesthetized with ketamine (85 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.). Atropine (0.04 mg/kg, i.p.) was administrated to minimize respiratory secretion. The trachea was intubated with an 18gauge catheter, which was then attached to a ventilator (settings: 425 ml/min; 60 strokes/min). The rat was placed on a heating pad, and ECG electrodes were attached. Body temperature was maintained at 37 -C with a heating lamp. The left femoral artery and vein were catheterized to monitor arterial blood pressure and for administration of drugs, respectively. An incision of an inch length was made on the skin along the rat cavity at a location above the heart. The skin was pulled apart to facilitate the insertion of an Lshaped metal loop into the rat cavity. An L-shaped metal loop was inserted through the muscle of the rat body cavity into an area underneath the aorta and the surrounding major blood vessels. Cardiac arrest was initiated and maintained by mechanically obstructing all the major cardiac blood vessels including the aorta by applying pressure on the surface of the rat cavity with the palm of one hand while pulling the inserted L-shaped loop up against the rat cavity with another hand. The arterial blood pressure was maintained at 0 –10 mm Hg. Under such low systemic arterial blood pressure, cerebral perfusion came to a halt. Post-hypoxic rats developed myoclonus only after a critical duration of cerebral hypoxia. A cardiac arrest for a duration of 9 min 30 s is a compromise at which myoclonus reliably develops following recovery, the survival rate is acceptable, and the severity of other neurological conditions was manageable.
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Resuscitation began at 9 min 30 s following cardiac arrest by resuming manual thoracic compression and by intravenous injection of 10 mg/kg epinephrine and 4 mEq/ kg sodium bicarbonate. Following resuscitation, rats were weaned from the ventilator, the catheters were removed, and wounds were sutured. The animals were placed on a heating pad to facilitate recovery from surgical coma. 2.2. Behavior and evaluation of post-hypoxic myoclonus in rat One of the clinical features of post-hypoxic myoclonus is the trigger of myoclonic jerks in response to exogenous stimuli such as sound. Jerking movements in response to auditory stimuli were ranked according to their intensity as previously described [24]. For the Fos staining experiments, 2 days after cardiac arrest, rats were given auditory stimuli. For the purpose of evaluating myoclonic jerks in response to auditory stimuli, the rat was placed in a clear plastic cage for 10 min to let it habituate to the new environment prior to evaluation. The rat was then presented with 45 clicks of a metronome as the auditory stimulus. Each click had a sound intensity of 96 dB with a duration of 40 ms at a frequency of 0.75 Hz. The involuntary muscle jerks in response to each click were scored with the following criteria: 0 = no jerks; 1 = ear twitch; 2 = ear and head jerk; 3 = ear, head and shoulder jerk; 4 = whole body jerk; and 5 = whole body jerk with jumping [7,24]. The myoclonus scores shown in Figs. 1 and 5 are the sum of the 45 scores of the rats in response to the 45 clicks. One hour after auditory stimulus, rats were sacrificed. Brain sections were prepared for detection of Fos protein as described below. Rats that underwent cardiac arrest for 4 min did not develop myoclonus when given auditory stimuli. This group would serve as a negative control for the study.
Fig. 1. Rats subjected to cardiac arrest for 9 min 30 s but not for 4 min developed myoclonus in response to auditory stimuli. Myoclonus scores recorded from rats before and following cardiac arrest for 9 min 30 s ( ) or 4 min (D). The arrow indicates the day on which cardiac arrest surgery was performed. Values are mean T SE. **Indicates significant difference from control at P < 0.01. Five animals were used in each group.
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2.3. Procedures for detection of Fos protein in rat brain sections Fos protein was detected in brain sections using the avidin– biotin peroxidase (ABC) method. Fos in brain sections was detected by primary anti-Fos antibody with a biotin-conjugated secondary antibody followed by ABC solution and then visualized by enhanced DAB immunocytochemistry. In brief, coronal brain sections of ¨30 Am were collected in phosphate buffer (PBS) with 0.1% sodium azide. Then sections were transferred and incubated in methanol with 0.3% H2O2 for 20 min. Sections were rinsed in PBS three times, 10 min each. Sections were incubated in 5% goat normal serum in 0.3% Triton-X100 PBS for 2 h at room temperature with gentle agitation on a shaker. Then sections were incubated in anti-Fos antibody (1:10,000) (Santa Cruz Biotechnology) in 0.3% TritonX100 PBS in blocking serum with gentle agitation on a shaker in the 4 -C refrigerator for a duration of 48 h. Sections were then rinsed in PBS four times, 10 min each. Sections were then incubated in biotinylated goat antirabbit antibody (1:1000) in 0.3% Triton-X100 PBS in blocking serum with agitation on a shaker at room temperature for 2 h. Sections were then rinsed in PBS four times, 10 min each. Sections were incubated in Vectastain ABC solution at room temperature for 1 h with gentle shaking and then were rinsed in PBS, 3 times, 10 min each. Sections were then incubated in freshly prepared DAB solution with H2O2 solution (0.01%) and 5 drops of nickel chloride, then mixed well in dishes on a shaker until an adequate level of staining was reached. It took 7 to 9 min for the reaction to complete. DAB reaction was stopped by rinsing the sections seven times in PBS for a duration of 5 min each. Sections were stored in PBS buffer in the 4 -C refrigerator overnight. Then sections were mounted on gel-coated slides and dried for 2 days. Sections were then dehydrated with the following steps: purified water for 2 min; 50% ethanol for 2 min; 70% ethanol for 2 min; 95% ethanol for 2 min; 100% ethanol for 2 min; 100% ethanol for 2 min; xylene for 2 min. Then slides were covered with cover slip with Permount. Fos protein is
recognized as dark staining in the neuronal cell nucleus under a microscope. Two groups of animals were used in the fos staining experiments. The experimental group was subjected to cardiac arrest for a duration of 9 min 30 s, the other group is their respective control which was subjected to cardiac arrest for a duration of 4 min. Five animals were used in each group. 2.4. Electrolytic lesion of rat brain nuclei Electrolytic lesion to functionally evaluate the contribution of Fos expressed nuclei to audiogenic post-hypoxic myoclonus was performed according to the standard procedures of Cubero et al. [2] with some modifications. In brief, a rat was anesthetized with ketamine hydrochloride (85 mg/kg, i.p.) and xylazine (15 mg/kg i.p.). The rat was mounted in a stereotaxic frame (KOPF Instruments). The incisor bar was adjusted until the head was in the flat-skull position about 3.3 mm below horizontal, thus allowing the animal’s stereotaxic coordinates to be referred to a standard brain atlas [17]. With the head properly positioned, a midline incision was made from the level of the eyes to the neck using a sterile scalpel. Using a millimeter scale on the stereotaxic frame, the coordinates of the intended lesion site were located according the brain map. These coordinates would be marked using a marker on the skull. With a high speed dental drill, a 1-mm hole was made in each hemisphere to the reference sutures (Bregma and Lambda) to allow insertion of a microelectrode for electrolytic lesions. The brain nucleus was destroyed by electrolytic lesion using a stainless steel microelectrode (FHC, Inc., ME) to the site of lesion by applying a constant current of 0.3 mA for 4 s from a DS3 constant current stimulator (Harvard Apparatus, MA). After the lesions, the electrode would be removed and incisions in the scalp closed. Sham controls followed the same procedures as described except that no current was applied to the electrode. Seven days after electrolytic lesions, both lesioned rats and sham-operated control rats underwent cardiac arrest for 9 min 30 s. Myoclonus scores were obtained from the rats on a daily
Fig. 2. Fos protein was detected in the LC and the PVG of the rats that developed myoclonus in response to auditory stimuli 48 h after cardiac arrest for 9 min 30 s (A). Fos was not detected in the LC and the PVG from rats that were subjected to 4 min of cardiac arrest and did not develop myoclonus (B).
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basis for the next 3 days as described above. Rats were then sacrificed, and their brains were fixed for sectioning. Cresyl violet staining was used to verify the site of electrolytic lesion as previously described [21]. The coordinates for the lesion of the locus coeruleus (LC)/ periventricular gray substance (PVG) are Bregma: 10 mm, Lateral: T1 mm, Depth: 7.5 mm; the coordinates for the lesion of the reticular thalamic nucleus (RT) are Bregma: 2.5 mm, Lateral: T2.5 mm, Depth: 7 mm, and the coordinates for the lesion of the medial longitudinal fasciculus (MLF) are Bregma: 11 mm, Lateral: T0.5 mm, Depth: 8.5 mm. A total of six groups were used in the lesion study, one each for the lesion of the LC/PVG, the RT, and the MLF. The other three groups are their respective sham-operated control groups. Each group consists of five animals. All experiments involving animal use were approved by the institutional animal care and use committee. 2.5. Statistical analysis Brain sections for Fos staining were obtained from rats that underwent cardiac arrest for 9 min 30 s and 4 min. The mean myoclonus scores between these two groups of posthypoxic rat at each time point were compared using Student’s t test. A P < 0.05 was considered as significant. In the electrolytic lesion studies, the myoclonus scores between lesioned animals and the corresponding sham-
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operated controls were compared using one-way analysis of variance (ANOVA). If there were significant differences, a post hoc Tukey’s test was used to determine which sample means were different.
3. Results Auditory stimuli induced myoclonus in rats that underwent cardiac arrest for 9 min 30 s. As expected from our previous experience, rats that underwent cardiac arrest for 4 min did not develop myoclonus in response to auditory stimuli (Fig. 1). In post-hypoxic rats with myoclonus, Fos was detected in the LC, the PVG (Fig. 2), the reticular thalamic nucleus (RT) (Fig. 3), and the MLF (Fig. 3). To evaluate whether these nuclei or cell groups contribute to the generation of myoclonus, we examined the effects of electrolytic lesions of these nuclei on the severity of posthypoxic myoclonus. Lesions of the nuclei were verified using cresyl violet staining in coronal brain sections. We were not able to destroy the LC and the PVG individually using electrolytic lesions as the LC and the PVG are so close together. Therefore, the LC and the PVG were destroyed together (Fig. 4A). Discrete regions of the RT (Fig. 4C) and the MLF (Fig. 4E) were destroyed by electrolytic lesions. Lesions of the LC/PVG did not alter cardiac arrest-induced post-hypoxic myoclonus (Fig. 5A). However, lesions of the RT (Fig. 5B) and the MLF (Fig. 5C) significantly reduced
Fig. 3. Post-hypoxic myoclonus induced Fos protein in the RT and the MLF in coronal brain sections. Fos protein was detected in the RT (A) and the MLF (C) of the rats that developed myoclonus in response to auditory stimuli 2 days after cardiac arrest-induced cerebral hypoxic injury. Fos protein was not detected in the RT (B) and the MLF (D) from the rats that did not develop myoclonus in response to auditory stimuli 2 days after cardiac arrest for 4 min.
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Fig. 4. Electrolytic lesions of the LC/PVG, the RT, and the MLF. Cresyl violet staining of the rat coronal brain sections showing electrolytic lesions of the LC/ PGV (A), the RT (C) and the MLF (E) and their respective sham-operated control brain sections (B, D and F). The arrows indicate the sites of lesions. The coordinates used for each of these sites are specified in Methods and experimental procedures section.
cardiac arrest-induced post-hypoxic myoclonus when compared with their respective sham controls (Figs. 5B, C).
4. Discussion In the present study, we detected Fos protein in the RT, LC, PVG, and the MLF of the brainstem in post-hypoxic rats that developed myoclonus in response to auditory stimuli. Fos was not detected in these nuclei of rats that were subjected to a short duration of cardiac arrest of 4 min and did not exhibit myoclonus in response to auditory stimuli. The results indicate that these nuclei may be involved in post-hypoxic myoclonus. Using an electrolytic lesion technique to further verify the involvement of these nuclei in post-hypoxic myoclonus, we found that lesion of the LC/PVG had no significant effects on cardiac arrest-induced post-hypoxic myoclonus. This result suggests that the LC/PVG neuronal activity is not directly related to the generation of myoclonic jerks. The physiological function of the LC is associated with anxiety and stress responses [10], and the PVG is related with modulation of nociception signaling [20]. The Fos expression in these two nuclei could have resulted from the fact that rats were under stress during auditory stimuli-induced myoclonic
jerks. The route of signaling of fear and threat was probably relayed from the sensory cortex through the thalamus to the brain stem. The elevated noradrenergic activity in the LC would increase the alertness and attention of the rats to the challenging environment. We understand that inserting the electrode without passing current in sham-operated controls may cause some mechanical damage to the brain. As a result, we evaluated the effects of lesions on post-hypoxic myoclonus 1 week after the induction of lesion so that the rats can recover from the injury. Histological examinations of the brain sections 10 days after the induction of lesion do not reveal damage along the path through which the lesion electrode was inserted (Figs. 4B, D, F). In addition, we do not observe differences in the animal behavior such as feeding pattern, body weight change, and movement between the sham-operated rats and normal untreated rats. These observations suggest that the sham-operated rats are not different from the normal untreated rats, and thus, they can be used as a proper control for the lesion study. The expression of Fos in the RT is consistent with previous pharmacological studies [14] which showed that the RT is one of the generator centers for post-hypoxic myoclonus. As predicted from these earlier studies, ablation of the RT reduced post-hypoxic myoclonus as demonstrated in the present study. This result further
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Fig. 5. Effects of electrolytic lesions of rat brain nuclei on post-hypoxic myoclonus. Electrolytic lesion of the RT (B), the MLF (C) but not the LC/PGV (A) reduced auditory stimuli provoked myoclonus from rats which underwent cardiac arrest for 9 min 30 s. Values are mean T SE. *, **Indicate significant difference at P < 0.05 and P < 0.01, respectively. Five animals were used in each group.
substantiates the notion of the functional role of the RT in post-hypoxic myoclonus. It also suggests that the RT is a target for future possible intervention with deep brain stimulation. Indeed, electrical stimulation of the RT has recently been showed to suppress limbic motor seizures [16]. Recently, Frucht et al. [5] reported the findings in 4 patients with post-hypoxic myoclonus and noted an increase of glucose metabolism in the ventrolateral thalamic nucleus (VL) and pontine tegmentum in patients relative to controls. We do not detect significant Fos expression in the VL following post-hypoxic myoclonus in our animal model. In addition, lesion of the VL (5/5 rats) does not alter the severity of post-hypoxic myoclonus when compared with sham-operated controls. These results argue against the role of the VL in post-hypoxic myoclonus. The neuronal activity in the MLF as indicated by Fos expression suggested that the MLF was involved in the observed myoclonic jerks. The MLF, located on each side of the brainstem, carries information about the direction that the eyes should move. The MLF connects the several cranial nerve nuclei III, IV, and VI, as well as the gaze centers and information about head movement from cranial nerve VIII [15]. The MLF descends into the cervical spinal cord and innervates some muscles of the neck [19] in coordination of gaze with head and neck movements [22]. It is not clear why the MLF was activated in post-hypoxic rats showing myoclonic jerks. According to the scoring criteria, rats that
showed involuntary muscle jerks in the head and shoulders in response to each stimulus received a score of 3. One possibility that could account for the activation of the MLF is that the head movement during myoclonic jerks triggered vestibular signals from each of the labyrinthine canals. Because the observed post-hypoxic myoclonus-induced Fos protein expression occurred mostly in the brainstem region, regardless of the neuronal pathways through which these nuclei are involved, the results suggest that the post-hypoxic myoclonus animal model has a significant brainstem component. This finding further supports the relevance of this animal model to human post-hypoxic myoclonus which is believed to have brainstem origin [6] when characterized with generalized massive body jerks [26,30]. Future studies directed towards elucidating the neuronal connections and the nature of neurotransmission of the MLF that displayed Fos expression will give us useful information regarding the signaling pathway underlying post-hypoxic myoclonus in this animal model. It has been hypothesized that the inferior olive activity may be involved in the generation of post-hypoxic myoclonus [29]. We were not able to detect neuronal activity in the inferior olive following post-hypoxic myoclonus using Fos as a marker. This observation does not rule out the role of inferior olive in the generation of posthypoxic myoclonus as induction of immediate-early gene expression is related to the stimuli strength. Furthermore, it is possible that post-hypoxic myoclonus could induce a
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