• Amygdaloid kindling;
  • Nucleus of the solitary tract;
  • Vagus nerve;
  • Cat


  1. Top of page
  2. Abstract

Summary:  Purpose: This work analyzed the effect of electrical stimulation of the nucleus of the solitary tract (NTS) on the development of electrical amygdaloid kindling (AK) in freely moving cats.

Methods: Nine male adult cats with implanted electrodes in both amygdalae (basolateral nucleus), both lateral geniculate bodies, left NTS, and both prefrontal cortices were used. Electromyogram and electrooculogram also were recorded. The AK was performed every 24 h (1-s train, 1-ms pulses, 60 Hz, 300–600 μA). The NTS was stimulated previously for 1 min (0.5-ms pulses, 30 Hz, 150–300 μA), just before the AK at 10:00 a.m., and then every 60 min, 4 times, from 11:00 a.m. to 2:00 p.m. On different days, all NTS stimulation was suspended, and AK was continued until stage VI kindling was reached.

Results: Behavioral changes produced by the stimulation of the NTS were blinking, immobility periods with upward sight, licking, and swallowing. Animals with simultaneous stimulation of NTS and AK did not reach stage VI, remaining in behavioral stages I–III. Stage VI was reached after NTS stimulation was intentionally suspended. The amplitude, duration, and the propagation of the amygdaloid afterdischarge did not exhibit progressive evolution during NTS stimulation. A regression analysis was performed between the number of days with only AK stimulation and days with simultaneous NTS stimulation, which showed a positive correlation (values of r = 0.84).

Conclusions: Our results suggest that NTS stimulation interferes with the development of convulsive evolution and secondary generalization. This delay effect may be due to the activation of the locus ceruleus and some areas of the midbrain reticular formation, among other structures, which has been demonstrated to inhibit experimental convulsive seizures.

Kindling is an experimental model of partial seizures with secondary generalization, characterized by the increase of electrographic afterdischarge duration and complexity produced by the repeated low-intensity electrical stimulation of several brain areas, mainly the limbic system (1). Excitability of the stimulated sites remains altered once the generalized seizures are manifested. Kindling in forebrain structures is well known, whereas the stimulation of brainstem nuclei does not produce the kindling effect (1,2), or even has an antikindling effect (3).

Conversely, the electrical stimulation or local cooling of the pontine and midbrain reticular formations modifies the cortical convulsive discharge (4–6). The nucleus of the solitary tract (NTS) receives afferent projections from the vagus nerve and in turn projects rostrally. Thus it possibly has anticonvulsant effects as well, such as those shown for vagus nerve stimulation (VNS). Vagal stimulation is capable of inhibiting experimental seizures induced by systemic application of pentylenetetrazole (Metrazol; PTZ) (7), penicillin (8), strychnine (9–11), electroshock (12,13), and topical application of aluminum gel (14). We also reported a delay in the amygdaloid kindling in cat caused by repeated VNS (15,16). VNS is currently used in the treatment of partial and generalized seizures in patients resistant to pharmacologic therapy (17–19).

Not much is known about the role of vagus nerve ascending projections in the control of generalized convulsive seizures. About 95% of the vagus nerve afferent fibers project toward the NTS, located in the caudal portion of the medulla oblongata. The objective of this study was to analyze the effect of NTS electrical stimulation on the development of amygdaloid kindling in freely moving cats.


  1. Top of page
  2. Abstract

Nine freely moving cats weighing 3,800–4,100 g were used. All experiments were performed in accordance with the technical specifications for the production, care, and use of laboratory animals of the Ministry of Agriculture, Livestock and Rural Development (NORM-062-ZOO-2000) and approved by the Ethics Committee of the Instituto Nacional de Psiquiatría. Surgery was carried out under pentobarbital sodium anesthesia (33 mg/kg, i.v.). Stainless steel bipolar electrodes were stereotaxically implanted, oriented (20) to both lateral geniculate bodies, to both temporal lobe amygdalae, and to the left NTS. Epidural electrodes were directed to both prefrontal cortices for the electroencephalogram (EEG) recording. Nail-shaped electrodes were implanted in the supraorbital cavity for electrooculogram (EOG) recording. The electromyogram (EMG) was recorded with flexible electrodes inserted in the nape lateral muscles. Electrodes were welded to a DB25 connector and fixed to the skull with acrylic cement. After surgery, the animals were placed in the sound-damped chambers used for recording, during a 21-day recovery that served as habituation to the experimental environment. Food was placed ad libitum. Two 78E and 78D model Grass polygraphs were used to record brain electric activity.

The electrical stimulation threshold of the amygdala (AK) and NTS was determined with an S88 stimulator (Grass). The current was delivered by using monophasic square-wave signals. For the amygdala (basolateral and central nuclei), stimulation was repeated every 5 min, increasing intensity until a wink and ipsilateral facial contractions were manifested, followed by a brief afterdischarge. The threshold intensity of AK varied between 300 and 600 μA. For the NTS, the threshold was established by the appearance of swallowing, licking, vomiting reflex, and immobility. The current varied between 150 and 300 μA. Once the threshold was obtained, the AK was performed every 24 h (10:00 a.m.) with a stimulation of the left amygdala (1-s train, 1-ms pulses, 60-Hz frequency). The NTS was stimulated previously for 1 min (0.5-ms pulses, 30-Hz frequency) just before the AK and then every 60 min, 4 times (with the same parameters from 11:00 a.m. to 2:00 p.m.). Electrical stimulation of the NTS was intentionally suspended at different days for each cat, whereas the AK was daily applied until kindling behavioral stage VI was reached.

The progressive behavioral stages of kindling were analyzed by using the method of Wada and Sato (21): stage I, ipsilateral facial contraction; stage II, bilateral facial contraction; stage III, head vertical movements; stage IV, marching in circles; stage V, generalized myoclonias of the four limbs; and stage VI, generalized tonic–clonic convulsive seizures. These behavioral changes were compared with AK in five control animals previously obtained at our laboratory (16).

Animal behavior was analyzed with a video camera. The analyzed variables in the experiment were duration and propagation of amygdaloid afterdischarge. Histologic verification of the electrode position was done at the end of the experiment by using the rapid procedure technique (22).

Statistical analysis

The mean amygdaloid-stimulation days required to reach the first generalized convulsive seizure in animals stimulated in a different bulbar structure adjacent to the NTS were subjected to a variance analysis (Kruskal–Wallis). A regression analysis between the number of days with simultaneous stimulation of the NTS during the AK, and the following days with stimulation of the AK alone after suspending NTS stimulation was applied.


  1. Top of page
  2. Abstract

NTS electrical stimulation induces the following behavioral changes: licking, swallowing, vomiting reflex, immobility with upward sight, and occasional yawning. The histologic examination showed that electrodes directed to the NTS were located at the NTS medial portion (NTSm) in five cats, in the nucleus intercalatus (NI) in two, and in another two at the lateral tegmental field (LTF). Electrodes directed to the amygdala were located in seven cats at the basolateral nucleus, in one at the basal nucleus, and in one in the amygdala central nucleus (Fig. 1).


Figure 1. A: Brainstem sagittal plane at 1.2 and 2 mm of midline, with location of different stimulation sites in cats used in this study. Medial nucleus tractus solitarius (NTS; circle); lateral tegmental field (LTF; square); nucleus intercalatus (NI; rhomb); FC, cuneate fasciculus; AP, area postrema; DMVN, dorsal motor vagus nucleus; ST, solitary tract; C, cuneate nucleus; P, pyramidal tract. B: Brain coronal plane, amygdala basolateral, basal, and central nuclei (diamond).

Download figure to PowerPoint

The electrical stimulation of the NI and LTF did not demonstrate any anticonvulsant effect. The animals displayed a behavioral evolution similar to that in control animals. These animals also showed a progressive increase in the afterdischarge duration (Figs. 2 and 3). The amount of mean stimulation to reach stage VI in five control animals previously obtained in our laboratory was 23.41 ± 3.7 days (16); in the animals with simultaneous electrical stimulation of the NI and AK, 27.5 ± 2.5 days; and in the animals with simultaneous stimulation of the LTF and AK, 23.5 ± 5.5 days.


Figure 2. Behavioral stages of kindling of amygdala (AK). The four animals with electrodes located in structures (nucleus intercalatus and lateral tegmental field) adjacent to nucleus tractus solitarius (NTS) had a behavioral evolution similar to control animals to reach kindling stage VI. The animals simultaneously stimulated in the NTS and AK showed a delay in behavioral evolution. The arrow and its number indicate the suspension day of NTS stimulation, which was followed by the AK only until animals reached stage VI.

Download figure to PowerPoint


Figure 3. Evolution of amygdaloid afterdischarge duration in seconds. The left column shows the control animal, and four with electrodes located in structures adjacent to the nucleus tractus solitarius (NTS; lateral tegmental field and nucleus intercalatus). The right column shows the effect of NTS plus amygdaloid kindling (AK) stimulation. The arrow and its number indicate the suspension day of NTS stimulation. The asterisk indicates the first kindling stage VI in all animals. The afterdischarge duration does not evolve progressively during NTS stimulation.

Download figure to PowerPoint

Development of AK was remarkably delayed in five animals with simultaneous stimulation of the NTSm and AK. The evolution of behavioral stages was retarded, with an increase in the number of stimuli required for stages I–III. The delay in kindling also was reflected in the amygdaloid afterdischarge duration; these animals showed no progressive increase. Animals reached stage VI after intentional suspension of NTSm stimulation (Figs. 2 and 3). An outstanding fact is that propagation of afterdischarge toward the contralateral amygdala is retarded in animals with NTSm stimulation (Fig. 4). The mean of the amygdaloid-stimulation days required to reach stage VI to complete kindling after intentional suspension of the NTSm stimulation was 41.4 ± 7.01. This period was prolonged and related to the previous days of the NTSm stimulation (Fig. 5).


Figure 4. Representative polygraphic recordings of the evolution of amygdaloid kindling (AK) in a control cat (CAT 1) and one cat with simultaneous stimulation of the nucleus tractus solitarius (NTS) and AK (CAT 3). The electrical recording demonstrates electrographic changes produced by kindling equivalent days (K1, K11, and K25). Note how the convulsive activity has not propagated toward the contralateral amygdala in the animal with simultaneous stimulation of the NTS and AK. In this case, a decrease in frequency and amplitude of the convulsive afterdischarge was observed, evident in the K25. L- and R- Pf Cx, left and right prefrontal cortices; L- and R- AM, left and right amygdala.

Download figure to PowerPoint


Figure 5. Days of simultaneous stimulation of the nucleus tractus solitarius (NTS) and amygdaloid kindling (AK), and days of only AK stimulation to reach kindling stage VI. Note that the amount of AK stimulation required to reach this stage is related to the number of days of NTS stimulation (41.4 ± 7.01 days).

Download figure to PowerPoint

The NTSm simultaneous stimulation days were correlated with the number of amygdaloid-stimulation days required to reach stage VI of kindling. A significant positive correlation of 0.84 (p < 0.023) was detected (Fig. 6).


Figure 6. Regression analysis between the number of days with amygdaloid kindling (AK) stimulation required to reach stage VI of the kindling of the amygdala, and days of simultaneous stimulation of the nucleus tractus solitarius (NTS). A positive correlation was obtained (r values, 0.84).

Download figure to PowerPoint


  1. Top of page
  2. Abstract

Our results demonstrate that the electrical stimulation of the NTSm exerts an inhibitory effect on the development of generalized experimentally induced convulsive seizures by electrical AK in the cat. This observation shows that convulsive seizures of limbic origin are subject to NTSm regulation. This anticonvulsive effect occurred with behavioral changes with no adverse effect on the spontaneous motor activity of cats.

The NTS receives the largest amount of vagus nerve afferent projections and acts as a sensory information relay (23). The ascending projection toward the forebrain is through the parabrachial nucleus, the locus ceruleus (LC), cerebellum, dorsal raphe nucleus, and the reticular formation, among other structures (24,25). Behavioral changes in our animals, such as swallowing and licking, occur because of activation of the NTS rostral portion where somatosensory and gustatory information is processed (26), which in turn projects toward the ambiguous nucleus, which controls the swallowing muscles (27). The vomiting reflex results from the area postrema activation through the NTS (28).

The NTS anatomic relation with the brainstem and forebrain structures involved in regulation of the experimental seizure threshold may have consequences at neurotransmitter or neuromodulator levels. For example, C-fos studies in rats during VNS have demonstrated an increase in C-fos expression in amygdala, hypothalamus, LC, and NTS (29). Recently an increase in γ-aminobutyric acid (GABA) transmission or a decrease in glutamate transmission in the NTSm was reported to reduce the severity of experimentally induced limbic seizures by topic or systemic application of PTZ and bicuculline (30). Conversely, it was reported that LC electrical stimulation prevents secondary generalization of convulsive activity induced by the AK in the rat (3). It also was reported that an LC lesion suppresses the anticonvulsive effects achieved by VNS in the rat (12). The fact that LC electrical stimulation has anticonvulsive effects is consistent with our results. Unilateral NTSm electrical stimulation may activate the LC, which would suffice to prevent generalization of convulsive activity induced by the AK in the cat.

Our results also suggest that this delay of convulsive generalization may be caused by the activation of the midbrain reticular formation, which results in a “waking electroencephalographic reaction” via the ascending reticular activating system (31). Fernández-Guardiola et al. (5) showed that electrical stimulation of the midbrain reticular formation inhibits the spinal monosynaptic reflex and inhibits the convulsive activity unchained by the systemic administration of PTZ. Furthermore, different strategies have been tested to eliminate convulsive activity by activation of multiple sensory modalities. These include reduction of absence seizures due to acoustic stimuli (32), and reduction of interictal spikes of absence seizures due to thermal stimulation (8). High-frequency electrical stimulation of the vagus nerve produces EEG desynchronization (33) and the anticonvulsive effect in experimental models of epilepsy (11,13), and in clinical application of VNS (34,35) would directly activate the ascending reticular activating system via the NTS.

Our results indicate that the NTSm electrical stimulation retards the generalization of convulsive activity caused by the AK in freely moving cats. These results on kindling were similar to those of VNS (15,16); we propose that the NTS mediates the anticonvulsive effects of VNS. This delay effect may be due to the activation of locus ceruleus, and some areas of the midbrain reticular formation, among other structures, which has been demonstrated to inhibit experimental convulsive activity. The long protected effect on kindling by NTS stimulation also was found after VNS in our previous work. To elucidate the cause of this phenomenon, more investigation is necessary.

Acknowledgment: This project was partially supported by CONACyT 31771-N, DGAPA IN 231999, PUIS-UNAM 2035-049-15-II-90. We thank Alejandro Rubio and Alfredo Martínez for their excellent technical assistance; Raúl Cardoso and Jose Luis Calderon for the preparation of the illustration; and Isabel Pérez Monfort for manuscript translation.


  1. Top of page
  2. Abstract
  • 1
    Goddard GV, McIntyre DC, Leech CK. A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 1969;25: 295330.
  • 2
    Fernández-Guardiola A, Jurado JJ, Calvo JM. Repetitive low-intensity electrical stimulation of cat's nonlimbic brain structures: dorsal raphe nucleus kindling. In: WadaJA, ed. Kindling 2. New York: Raven Press, 1981: 12336.
  • 3
    Weiss GK, Lewis J, Jímenez-Rivera C, et al. Antikindling effects of locus coeruleus stimulation: mediation by ascending noradrenergic projections. Exp Neurol 1990;108: 13640.
  • 4
    Fernández-Guardiola A, Alcaraz V, Guzmán CF. Modificación de la descarga convulsiva cortical por estimulación mesencefálica. Bol Estud Med Biol 1956;16: 1520.
  • 5
    Fernández-Guardiola A, Alcaraz VM, Guzmán FC. Inhibition of convulsive activity by the reticular formation. Acta Neurol Latinoamer 1961;7: 306.
  • 6
    Testa G, Gloor P. Generalized penicillin epilepsy in the cat: effect of midbrain cooling. Electroencephalogr Clin Neurophysiol 1974;36: 51724.
  • 7
    Takaya M, Terry WJ, Naritoku DK. Vagus nerve stimulation induces a sustained anticonvulsant effect. Epilepsia 1996;37: 11116.
  • 8
    McLahlan RS. Suppression of interictal spikes and seizures by stimulation of the vagus nerve. Epilepsia 1993;34: 91823.
  • 9
    Zanchetti A, Wang SC, Moruzzi, G. The effect of vagal afferent stimulation on the EEG pattern of the cat. Electroencephalogr Clin Neurophysiol 1952;4: 35761.
  • 10
    Stoica I, Tudor I. Effects of vagus afferents on strychninic focus of coronal gyrus. Rev Roum Neurol 1967;4: 28795.
  • 11
    Zabara J. Inhibition of experimental seizures in canines by repetitive vagal stimulation. Epilepsia 1992;33: 100512.
  • 12
    Krahl SE, Clark KB, Smith DC, et al. Locus coeruleus lesions suppress the seizure-attenuating effects of vagus nerve stimulation. Epilepsia 1998;39: 70914.
  • 13
    Woodbury DM, Woodbury JW. Effects of vagal stimulation on experimentally induced seizures in rats. Epilepsia 1990;31: 719.
  • 14
    Lockard JS, Congdon WC, Ducharm E. Feasibility and safety of vagal stimulation in monkey model. Epilepsia 1990;31: 206.
  • 15
    Fernández-Guardiola A, Martínez-Cervantes A, Valdés-Cruz A, et al. Left vagus nerve stimulation: effects on circadian sleep organization and kindling development in the cat. In: CorcoranME, MosheS, eds. Kindling 5. New York: Plenum Press, 1998: 4329.
  • 16
    Fernández-Guardiola A, Martínez A, et al. Vagus nerve prolonged stimulation in cats: effects on epileptogenesis (amygdala electrical stimulation): behavioral and electrographic changes. Epilepsia 1999;40: 8229.
  • 17
    Hammond EJ, Uthman BM, Reid SA, et al. Vagus nerve stimulation in humans: neurophysiological studies and electrophysiological monitoring. Epilepsia 1990;31: 519.
  • 18
    Ben-Menachem E, Hamberger A, Hedner T, et al. Effects of vagus nerve stimulation on amino acids and other metabolites in the CSF of patients with partial seizures. Epilepsy Res 1995;20: 2217.
  • 19
    Murphy JV. Left vagal nerve stimulation in children with medically refractory epilepsy: the Pediatric VNS Study Group. J Pediatr 1999;134: 5636.
  • 20
    Snider RS, Niemer WT. A stereotaxic atlas of the cat brain. Chicago: University of Chicago Press, 1961.
  • 21
    Wada JA, Sato M. Generalized convulsive seizures induced by daily electrical stimulation of the amygdala in cats: correlative electrographic and behavioral features. Neurology 1974;24: 56574.
  • 22
    Guzmán-Flores C, Alcaraz M, Fernández-Guardiola A. Rapid procedure to localize electrodes in experimental neurophysiology. Bol Estud Med Biol 1958;16: 2931.
  • 23
    Saper CB. The central autonomic system. In: PaxinosG, ed. The rat nervous system. 2nd ed. San Diego: Academic Press, 1995: 10731.
  • 24
    Rutecki P. Anatomical, physiological, and theoretical basis for the antiepileptic effect of vagus nerve stimulation. Epilepsia 1990;31: 16.
  • 25
    George MS, Sackeim HA, Rush AJ, et al. Vagus nerve stimulation: a new tool for brain research and therapy. Biol Psychiatry 2000;47: 28795.
  • 26
    Grabauskas G, Bradley RM. Tetanic stimulation induces short-term potentiation of inhibitory synaptic activity in the rostral nucleus of the solitary tract. J Neurophysiol 1998;79: 595604.
  • 27
    Carpenter MB, Pines J. The rubro-bulbar anatomical relationships: course and terminations in the rhesus monkey. Anat Rec 1957;128: 17185.
  • 28
    Vigier D, Portalier P. Efferent projections of the area postrema demonstrated by autoradiography. Arch Ital Biol 1979;117: 30824.
  • 29
    Naritoku DK, Terry WJ, Helfert RH. Regional induction of fos immunoreactivity in the brain by anticonvulsant stimulation of the vagus nerve. Epilepsy Res 1995;22: 5362.
  • 30
    Walker BR, Easton A, Gale K. Regulation of limbic seizures by GABA and glutamate transmission in nucleus tractus solitarius. Epilepsia 1999;40: 10517.
  • 31
    Moruzzi G, Magoun HW. Brain stem reticular formation and activation of the EEG. Electroencephogr Clin Neurol 1949;1: 44573.
  • 32
    Rajna P, Lona C. Sensory stimulation for inhibition of epileptic seizures. Epilepsia 1989;30: 16874.
  • 33
    Chase MH, Nakamura Y, Clemente CD. Afferent vagal stimulation: neurographic correlates of induced EEG synchronization and desynchronization. Brain Res 1967;5: 23649.
  • 34
    Olejniczak PW, England JD. The effects of vagus nerve stimulation upon EEG as recorded from occipital subdural electrodes in a human case. Soc Neurosci 2000;P:390.15.
  • 35
    Olejniczak PW, Fish BJ, Carey M, et al. The effect of vagus nerve stimulation on epileptiform activity recorded from hippocampal depth electrodes. Epilepsia 2001;43: 4239.