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Effects of cervical vagus nerve stimulation on amygdala-evoked responses of the medial prefrontal cortex neurons in rat
Neuroscience Research 65 (2009) 122–125
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Rapid communication
Effects of cervical vagus nerve stimulation on amygdala-evoked responses of the medial prefrontal cortex neurons in rat Olga Lyubashina *, Sergey Panteleev Laboratory of Cortico-Visceral Physiology, Pavlov Institute of Physiology, Russian Academy of Sciences, nab. Makarova 6, 199034 Sankt-Petersburg, Russia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2008 Received in revised form 29 May 2009 Accepted 1 June 2009 Available online 11 June 2009
In experiments on urethane-anaesthetized rats, the effects of repetitive vagus nerve stimulation (VNS) on responses of medial prefrontal cortex (mPFC) neurons to electrical stimulation of the basal nucleus of the amygdala were examined before and after intracerebroventricular administration of the neuronal nitric oxide synthase inhibitor 7-nitroindasole (7-NI). It was shown that the amygdala-evoked responses of cortical neurons were inhibited by repetitive VNS (pulses 50–150 mA, 0.5 ms, frequency 10 Hz). 7-NI administration did not change the amygdala-evoked neuronal reactions but reversed the effect of VNS on them. The present results suggest that the inhibitory action of VNS on amygdala–mPFC neurotransmission may involve a cortical NO-dependent mechanism. ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Amygdala Medial prefrontal cortex Neuronal activity Vagus nerve stimulation Nitric oxide
The amygdala has widespread cortical and subcortical connections (Pitkanen, 2000) and plays an integrative role in the emotional and motivational aspects of behaviour (LeDoux, 2000; Davis and Whalen, 2001). At the same time, the amygdala is often considered as a primary focus of seizure activity in temporal lobe epilepsy and its outputs are involved in the propagation of paroxysmal discharges to other brain areas (Hirsch et al., 1997; Sitcoske O‘Shea et al., 2000; Benini et al., 2003). An important pathway through which the discharges can spread to the cortical level and cause concomitant behavioural and autonomic manifestations of the seizures is the amygdala input to the medial prefrontal cortex (mPFC). The projection arises from the parvicellular part of the basal nucleus of the amygdala (BNA) and terminates mostly within deep layers of the prelimbic and infralimbic cortical areas (McDonald, 1991; Bacon et al., 1996; Ishikawa and Nakamura, 2003). Currently, for preventing and treating epileptic seizures, vagus nerve stimulation (VNS) is applied (Binnie, 2000; Groves and Brown, 2005). The precise nervous and neurochemical mechanisms involved in the anticonvulsant action of VNS are still unclear. A plausible assumption is that VNS can prevent the propagation of paroxysmal discharges from the amygdala to the cortex by altering neurotransmission in the BNA–mPFC pathway. However, experimental data supporting this hypothesis are lacking. That is why it
* Corresponding author. Tel.: +7 812 70 72 686; fax: +7 812 70 72 485. E-mail address: [email protected] (O. Lyubashina).
would be interesting to examine the effects of VNS on the amygdala-induced responses of mPFC neurons. Meanwhile, both clinical and animal studies indicate that nitric oxide (NO) is involved in the pathophysiology of epilepsy, and NOergic substances can modify the susceptibility to seizures (Del-Bel et al., 1997; Wojtal et al., 2003). Indeed, various brain areas involved in epileptogenesis, including the amygdala and cerebral cortex, contain NO-producing neurons (McDonald et al., 1993; Valtschanoff et al., 1993; Bertini et al., 1996). Moreover, expression of the neuronal nitric oxide synthase (nNOS) in cortical neurons and the nNOS activity in the amygdala significantly increase after experimental epilepsy (Talavera et al., 1997; Huh et al., 2000). At the same time, the nitric oxide interaction with glutamate has been shown to play a crucial role in the production of seizures and control of cortical excitability (Ferraro et al., 1999). On the basis of that, we hypothesized that the action of VNS on the amygdala– cortical neurotransmission may be mediated by NO-dependent mechanisms. In this study, we investigated the effects of VNS on the BNAevoked responses of neurons within the prelimbic and infralimbic areas of the mPFC. To determine whether vagal stimulation may interact with NO-mediated events, we examined the VNS action on the responses of cortical neurons before and after intracerebroventricular administration of the nNOS inhibitor 7-nitroindasole (7-NI). Experiments were performed on 28 adult male Wistar rats (body weight 250–360 g) anaesthetized with urethane (1.5 g/kg, i.p.). In accordance with the European Community Council
0168-0102/$ – see front matter ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2009.06.002
O. Lyubashina, S. Panteleev / Neuroscience Research 65 (2009) 122–125
Directive (86/609/EEC), adequate measures were taken to minimize pain or discomfort and to limit the numbers of animals used. The rat under surgery level of anaesthesia was placed on a thermostatically controlled heating pad. The femoral artery and vein were cannulated for the monitoring of arterial blood pressure and for the administration of the anaesthetic when it was necessary. Afterwards, the animal was fixed in a stereotaxic unit. The cervical portion of the left vagal nerve trunk was isolated and cut, its central end was put on silver bipolar stimulating electrodes. Two holes were drilled in the skull at the positions appropriate for the lowering of stimulating and recording electrodes with coordinates estimated from the stereotaxic atlas of the rat brain (Paxinos and Watson, 1998). A varnished insulated tungsten stimulating electrode with a tip diameter of 50–60 mm and a resistance of 80–100 kV was lowered into the parvicellular part of the left BNA (3.3 mm caudal to the bregma, 5.0 mm lateral from the midline, and 7.1 mm down from the dorsal surface of the cerebral hemisphere). Stimulation of the amygdala was achieved by single rectangular pulses of 30–60 mA with a duration of 0.3 ms. The current intensity of the vagus nerve stimulation was approximately 0.8 of its threshold to induce 10–15% changes in arterial blood pressure and was not more than 200 mA. Typically, we used rectangle current pulses of 50–150 mA with duration of 0.5 ms and frequency of 10 Hz. Varnished insulated microelectrodes (Science Products GMBH, Germany) with a tip diameter of 1 mm and resistance of 5 MV at 1 kHz were used for extracellular recording of neuronal activity. The electrodes were lowered into the deep layers of the left mPFC parallel to its medial surface at a distance between 0.5 and 1.0 mm from the midline. Recording was limited to a region defined by a caudo-rostral direction from 2.0 to 4.0 mm rostral to the bregma and dorsoventrally from 2.0 to 5.0 mm relative to the dorsal surface of the cerebral hemisphere. Stimulation, data acquisition, and processing were under on-line computer control. Results were presented as peri-stimulus histograms accumulated with 50 sweeps with bin 1 ms. Background and evoked activity of neurons were estimated within 50 ms before and 50 ms after BNA stimulation, respectively. The response intensity was estimated from the histogram as mean number of spikes per second reduced to one trial. The response latency was determined as the time interval between the onset of the stimulus and appearance of the maximum peak of the histogram. In each of the 28 animal preparations, the responses of the prelimbic and infralimbic cortical neurons to the amygdala singlepulse stimulation were investigated before and under repetitive stimulation of the left vagus nerve. In 23 animals, the same experimental protocol was continued after 7-NI (Sigma, USA) was administered stereotaxically with a Hamilton syringe into the anterior horn of the left lateral ventricle at a dose of 5 mg (0.3 ml of the suspension in 5% dimethyl sulfoxide). Recordings were performed within 45 min after the administration. The remaining 5 animals received intracerebroventricular 5% dimethyl sulfoxide and were used as control to determine the effects of 7-NI. At the end of each experiment, the animal was sacrificed with a lethal dose of urethane. After electrolytic destruction of tissue via electrodes, the brain was removed and stored in 10% paraformaldehyde for a week. The locations of stimulating and recording electrodes were examined on 100-mm-thick thionin-stained sections using the atlas of the rat brain (Paxinos and Watson, 1998). Data obtained under VNS before and after 7-NI administration were compared using ANOVA with Tukey’s test and expressed as mean value SEM and F-statistic. The mean rate of background activity of cortical neurons was 5.2 0.4 spikes/s (n = 119; Fig. 1). Single-pulse stimulation of the BNA-induced a short discharge in these cells that occurred with a mean latency of 16.1 0.5 ms. We did not observe any differences
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Fig. 1. Diagrams demonstrating changes in background and amydala-evoked activity of the prefrontal cortical neurons after intracerebroventricular 7nitroindasole administration (7-NI), under vagus nerve stimulation (VNS) and under vagus nerve stimulation after 7-nitroindasole administration (VNS + 7-NI). *, **—the mean rates of background and amygdala-evoked firing are significantly different from the corresponding initial levels (P < 0.005).
between neuronal reactions recorded in the prelimbic and infralimbic areas of the mPFC. The mean rate of the amygdala-evoked firing was 28.0 1.6 spikes/s and significantly differed from the background level (F = 269.92; P < 0.005; n = 119). Repetitive stimulation of the vagus nerve did not change the ongoing firing of the mPFC cells (4.2 0.4 spikes/s; n = 64; Fig. 1). However, neuronal responses to the BNA stimulation were reduced (Fig. 2). Under VNS, the mean rate of amygdala-evoked firing of mPFC neurons significantly decreased in comparison with the value prior to nerve stimulation and was
Fig. 2. Average peri-stimulus time histograms demonstrating responses of the prefrontal cortical neurons to the amygdala stimulation (initial) and their changes under vagus nerve stimulation before (VNS) and after intracerebroventricular 7nitroindasole administration (VNS + 7-NI). Bin—1 ms, sweeps—50. Ordinate— number of spikes in each bin per sweep per neuron. Abscissa—time in ms, 0— moment of the amygdala stimulation.
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O. Lyubashina, S. Panteleev / Neuroscience Research 65 (2009) 122–125
17.2 1.3 spikes/s (F = 19.75; P < 0.005; n = 64; Fig. 1). The mean latency of the responses did not significantly change (17.3 0.9 ms; n = 64). The intracerebroventricular 7-NI administration did not cause any noticeable changes in background activity of mPFC neurons (3.6 0.7 spikes/s; n = 59; Fig. 1). The mean rate of amygdala-evoked firing after 7-NI was 24.6 1.2 spikes/s and did not significantly differ from the value before the administration (F = 1.93; P > 0.05; n = 59). The mean latency of the responses after 7-NI administration did not significantly change (16.9 1.0 ms; n = 59). However, the VNS effects on the activity of the recorded cortical neurons were altered. Under nNOS inhibition, the repetitive vagus nerve stimulation resulted in a significant increase in background firing of mPFC neurons in comparison with its initial level before 7-NI administration (10.3 2.6 spikes/s; F = 7.07; P < 0.005; n = 42; Fig. 1). Simultaneously, the VNS effects on responses of mPFC neurons to the BNA stimulation were reversed. Following 7-NI administration, the amygdala-induced activity of cortical neurons was increased by the vagus nerve stimulation (Fig. 2). The mean rate of the evoked firing was 39.5 3.4 spikes/s and significantly differed from the initial value prior to 7-NI administration (F = 12.49; P < 0.005; n = 42; Fig. 1). The mean latency of the responses did not significantly change (18.0 1.1 ms; n = 42). Intracerebroventricular administration of 5% dimethyl sulfoxide, the vehicle for 7-NI, had no noticeable effect on the background and amygdala-evoked activity of the recorded mPFC neurons, nor did it alter the effect of VNS on them. Thus, this study showed for the first time that the electrical stimulation of the left cervical vagus nerve resulted in the inhibition of prelimbic and infralimbic neuronal responses to stimulation of the basal nucleus of the amygdala. The most probable pathway mediating the effect of VNS on the amygdalaevoked responses of cortical neurons is the ascending projection of the nucleus of the solitary tract (NTS) to the parabrachial nucleus, which directly or via the thalamus innervates the medial prefrontal cortex (Schachter and Saper, 1998). The NTS projection to the amygdala is a less attractive candidate because of its termination mostly within the central nucleus (Reyes and Van Bockstaele, 2006), which gives meager projections to other nuclei of the amygdala and does not innervate the cerebral cortex (Pitkanen, 2000). We consider that a possible mechanism by which VNS can change the amygdala-evoked responses of cortical neurons may be realized at the cortical level by decreasing the cortical excitability. This is supported by studies demonstrating that VNS elicits slow hyperpolarization in rat cortical neurons (Zagon and Kemeny, 2000) and increases cortical inhibition in epileptic patients (Di Lazzaro et al., 2004). Thus, in this study, we demonstrated the inhibitory effects of the vagus nerve stimulation on the amygdalaevoked activity of cortical neurons. Probably this effect of VNS can promote the prevention or interruption of the spread of paroxysmal activity from the amygdala–hippocampal region to the mPFC in epileptic patients. Results of this study suggest that the inhibitory effect of VNS on the amygdala-induced responses of mPFC neurons is probably related to endogenous nitric oxide owing to its dependence on 7NI, a preferred inhibitor of nNOS. It has been shown that a number of GABA-ergic cortical neurons contain NOS (Valtschanoff et al., 1993) and a release of GABA in the cortex is under NO-dependent control (Ohkuma et al., 1996). Moreover, it has been demonstrated that VNS increases the level of free GABA in the cerebrospinal fluid as well as GABAA receptor density in the cerebral cortex (BenMenachem et al., 1994; Marrosu et al., 2003). We consider that the inhibitory effect of the vagus nerve stimulation on the mPFC excitability can be linked to the increase in GABA release from cortical neurons owing to the activation of local NO-ergic
mechanisms. If this is true, a depression of these mechanisms will lead to a decrease in cortical GABA concentration, and as a result, to an increase in cortical excitability. Indeed, the reduction in NO in the brain has been shown to cause an increase in the discharge activity of the cortex (Ferraro et al., 1999). Consistent with this, in this study, after 7-NI administration, we observed an increase of the amygdala-induced responses of mPFC neurons under vagus nerve stimulation. Moreover, we cannot rule out that the intraventricular administration of 7-NI results in a suppression of NO-dependent inhibitory mechanisms not only in the cortex but in other brain structures mediating the effect of VNS on cortical neurons. In this study, we did not reveal significant changes in responses of the mPFC neurons to the BNA stimulation before and after intracerebroventricular 7-NI administration. This indicates that BNA–mPFC neurotransmission is probably NO-independent. In addition, data on Fos/NADPH diaphorase histochemistry recently obtained in our laboratory showed that VNS did not trigger NOergic neurons in the nucleus of the solitary tract (Osharina et al., 2006) as well as in the parabrachial nucleus and hypothalamus (unpublished data). Taken together, this allows us to assume that the main role in mediating the inhibitory effect of the vagus nerve stimulation on the BNA-induced responses of the mPFC neurons belongs to cortical NO-dependent mechanisms. In conclusion, our results suggest that a possible mechanism by which subthreshold vagus nerve stimulation can inhibit amygdala–mPFC neurotransmission and prevent a spread of paroxysmal activity to the cortical level may be the activation of cortical NOdependent mechanisms. However, this assumption needs to be confirmed in experimental models of epilepsy. References Bacon, S.J., Headlam, A.J., Gabbott, P.L., Smith, A.D., 1996. Amygdala input to medial prefrontal cortex (mPFC) in the rat: a light and electron microscope study. Brain Res. 720, 211–219. Ben-Menachem, E., Manon-Espaillat, R., Ristanovic, R., Wilder, B.J., Stefan, H., Mirza, W., Tarver, W.B., Wernicke, J.F., 1994. Vagus nerve stimulation for treatment of partial seizures. I. A controlled study of effect on seizures. Epilepsia 35, 616–626. Benini, R., D’Antuono, M., Pralong, E., Avoli, M., 2003. Involvement of amygdala networks in epileptiform synchronization in vitro. Neuroscience 120, 75–84. Bertini, G., Peng, Z.C., Bentivoglio, M., 1996. The chemical heterogeneity of cortical interneurons: nitric oxide synthase vs. calbindin and parvalbumin immunoreactivity in the rat. Brain Res. Bull. 39, 261–266. Binnie, C.D., 2000. Vagus nerve stimulation for epilepsy: a review. Seizure 9, 161–169. Davis, M., Whalen, P.J., 2001. The amygdala: vigilance and emotion. Mol. Psychiatry 6, 13–34. Del-Bel, E.A., Oliveira, P.R., Oliveira, J.A., Mishra, P.K., Jobe, P.C., Garcia-Cairasco, N., 1997. Anticonvulsant and proconvulsant roles of nitric oxide in experimental epilepsy models. Braz. J. Med. Biol. Res. 30, 971–979. Di Lazzaro, V., Oliviero, A., Pilato, F., Saturno, E., Dileone, M., Meglio, M., Colicchio, G., Barba, C., Papacci, F., Tonali, P.A., 2004. Effects of vagus nerve stimulation on cortical excitability in epileptic patients. Neurology 62, 2310–2312. Ferraro, G., Montalbano, M.E., La Grutta, V., 1999. Nitric oxide and glutamate interaction in the control of cortical and hippocampal excitability. Epilepsia 40, 830–836. Groves, D.A., Brown, V.J., 2005. Vagal nerve stimulation: a review of its applications and potential mechanisms that mediate its clinical effects. Neurosci. Biobehav. Rev. 29, 493–500. Hirsch, E., Danober, L., Simler, S., Pereira de Vasconcelos, A., Maton, B., Nehlig, A., Marescaux, C., Vergnes, M., 1997. The amygdala is critical for seizure propagation from brainstem to forebrain. Neuroscience 77, 975–984. Huh, Y., Heo, K., Park, C., Ahn, H., 2000. Transient induction of neuronal nitric oxide synthase in neurons of rat cerebral cortex after status epilepticus. Neurosci. Lett. 281, 49–52. Ishikawa, A., Nakamura, S., 2003. Convergence and interaction of hippocampal and amygdalar projections within the prefrontal cortex in the rat. J. Neurosci. 23, 9987–9995. LeDoux, J.E., 2000. Emotion circuits in the brain. Annu. Rev. Neurosci. 23, 155–184. Marrosu, F., Serra, A., Maleci, A., Puligheddu, M., Biggio, G., Piga, M., 2003. Correlation between GABA(A) receptor density and vagus nerve stimulation in individuals with drug-resistant partial epilepsy. Epilepsy Res. 55, 59–70. McDonald, A.J., 1991. Organization of amygdaloid projections to the prefrontal cortex and associated striatum in the rat. Neuroscience 44, 1–14.
O. Lyubashina, S. Panteleev / Neuroscience Research 65 (2009) 122–125 McDonald, A.J., Payne, D.R., Mascagni, F., 1993. Identification of putative nitric oxide producing neurons in the rat amygdala using NADPH-diaphorase histochemistry. Neuroscience 52, 97–106. Osharina, V., Bagaev, V., Wallois, F., Larnicol, N., 2006. Autonomic response and Fos expression in the NTS following intermittent vagal stimulation: importance of pulse frequency. Auton. Neurosci. 126–127, 72–80. Ohkuma, S., Katsura, M., Guo, J.L., Narihara, H., Hasegawa, T., Kuriyama, K., 1996. Role of peroxynitrite in [3H] gamma-aminobutyric acid release evoked by nitric oxide and its mechanism. Eur. J. Pharmacol. 301, 179–188. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York, p. 71. Pitkanen, A., 2000. Connectivity of the rat amygdaloid complex. In: Aggleton, J.P. (Ed.), The Amygdala: A Functional Analysis. Oxford University Press, New York, pp. 31–115. Reyes, B.A., Van Bockstaele, E.J., 2006. Divergent projections of catecholaminergic neurons in the nucleus of the solitary tract to limbic forebrain and medullary autonomic brain regions. Brain Res. 1117, 69–79. Schachter, S.C., Saper, C.B., 1998. Vagus nerve stimulation. Epilepsia 39, 677–686.
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Sitcoske O‘Shea, M., Rosen, J.B., Post, R.M., Weiss, S.R.B., 2000. Specific amygdaloid nuclei are involved in suppression or propagation of epileptiform activity during transition stage between oral automatisms and generalized clonic seizures. Brain Res. 873, 1–17. Talavera, E., Martinez-Lorenzana, G., Corkidi, G., Leon-Olea, M., Condes-Lara, M., 1997. NADPH-diaphorase-stained neurons after experimental epilepsy in rats. Nitric Oxide 1, 484–493. Valtschanoff, J.G., Weinberg, R.J., Kharasia, V.N., Schmidt, H.H., Nakane, M., Rustioni, A., 1993. Neurons in rat cerebral cortex that synthesize nitric oxide: NADPHdiaphorase histochemistry, NOS immunocytochemistry and colocalization with GABA. Neurosci. Lett. 157, 157–161. Wojtal, K., Gniatkowska-Nowakowska, A., Czuczwar, S.J., 2003. Is nitric oxide involved in the anticonvulsant action of antiepileptic drugs? Pol. J. Pharmacol. 55, 535–542. Zagon, A., Kemeny, A.A., 2000. Slow hyperpolarization in cortical neurons: a possible mechanism behind vagus nerve stimulation therapy for refractory epilepsy? Epilepsia 41, 1382–1389.
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
Rapid communication
Effects of cervical vagus nerve stimulation on amygdala-evoked responses of the medial prefrontal cortex neurons in rat Olga Lyubashina *, Sergey Panteleev Laboratory of Cortico-Visceral Physiology, Pavlov Institute of Physiology, Russian Academy of Sciences, nab. Makarova 6, 199034 Sankt-Petersburg, Russia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2008 Received in revised form 29 May 2009 Accepted 1 June 2009 Available online 11 June 2009
In experiments on urethane-anaesthetized rats, the effects of repetitive vagus nerve stimulation (VNS) on responses of medial prefrontal cortex (mPFC) neurons to electrical stimulation of the basal nucleus of the amygdala were examined before and after intracerebroventricular administration of the neuronal nitric oxide synthase inhibitor 7-nitroindasole (7-NI). It was shown that the amygdala-evoked responses of cortical neurons were inhibited by repetitive VNS (pulses 50–150 mA, 0.5 ms, frequency 10 Hz). 7-NI administration did not change the amygdala-evoked neuronal reactions but reversed the effect of VNS on them. The present results suggest that the inhibitory action of VNS on amygdala–mPFC neurotransmission may involve a cortical NO-dependent mechanism. ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Amygdala Medial prefrontal cortex Neuronal activity Vagus nerve stimulation Nitric oxide
The amygdala has widespread cortical and subcortical connections (Pitkanen, 2000) and plays an integrative role in the emotional and motivational aspects of behaviour (LeDoux, 2000; Davis and Whalen, 2001). At the same time, the amygdala is often considered as a primary focus of seizure activity in temporal lobe epilepsy and its outputs are involved in the propagation of paroxysmal discharges to other brain areas (Hirsch et al., 1997; Sitcoske O‘Shea et al., 2000; Benini et al., 2003). An important pathway through which the discharges can spread to the cortical level and cause concomitant behavioural and autonomic manifestations of the seizures is the amygdala input to the medial prefrontal cortex (mPFC). The projection arises from the parvicellular part of the basal nucleus of the amygdala (BNA) and terminates mostly within deep layers of the prelimbic and infralimbic cortical areas (McDonald, 1991; Bacon et al., 1996; Ishikawa and Nakamura, 2003). Currently, for preventing and treating epileptic seizures, vagus nerve stimulation (VNS) is applied (Binnie, 2000; Groves and Brown, 2005). The precise nervous and neurochemical mechanisms involved in the anticonvulsant action of VNS are still unclear. A plausible assumption is that VNS can prevent the propagation of paroxysmal discharges from the amygdala to the cortex by altering neurotransmission in the BNA–mPFC pathway. However, experimental data supporting this hypothesis are lacking. That is why it
* Corresponding author. Tel.: +7 812 70 72 686; fax: +7 812 70 72 485. E-mail address: [email protected] (O. Lyubashina).
would be interesting to examine the effects of VNS on the amygdala-induced responses of mPFC neurons. Meanwhile, both clinical and animal studies indicate that nitric oxide (NO) is involved in the pathophysiology of epilepsy, and NOergic substances can modify the susceptibility to seizures (Del-Bel et al., 1997; Wojtal et al., 2003). Indeed, various brain areas involved in epileptogenesis, including the amygdala and cerebral cortex, contain NO-producing neurons (McDonald et al., 1993; Valtschanoff et al., 1993; Bertini et al., 1996). Moreover, expression of the neuronal nitric oxide synthase (nNOS) in cortical neurons and the nNOS activity in the amygdala significantly increase after experimental epilepsy (Talavera et al., 1997; Huh et al., 2000). At the same time, the nitric oxide interaction with glutamate has been shown to play a crucial role in the production of seizures and control of cortical excitability (Ferraro et al., 1999). On the basis of that, we hypothesized that the action of VNS on the amygdala– cortical neurotransmission may be mediated by NO-dependent mechanisms. In this study, we investigated the effects of VNS on the BNAevoked responses of neurons within the prelimbic and infralimbic areas of the mPFC. To determine whether vagal stimulation may interact with NO-mediated events, we examined the VNS action on the responses of cortical neurons before and after intracerebroventricular administration of the nNOS inhibitor 7-nitroindasole (7-NI). Experiments were performed on 28 adult male Wistar rats (body weight 250–360 g) anaesthetized with urethane (1.5 g/kg, i.p.). In accordance with the European Community Council
0168-0102/$ – see front matter ß 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2009.06.002
O. Lyubashina, S. Panteleev / Neuroscience Research 65 (2009) 122–125
Directive (86/609/EEC), adequate measures were taken to minimize pain or discomfort and to limit the numbers of animals used. The rat under surgery level of anaesthesia was placed on a thermostatically controlled heating pad. The femoral artery and vein were cannulated for the monitoring of arterial blood pressure and for the administration of the anaesthetic when it was necessary. Afterwards, the animal was fixed in a stereotaxic unit. The cervical portion of the left vagal nerve trunk was isolated and cut, its central end was put on silver bipolar stimulating electrodes. Two holes were drilled in the skull at the positions appropriate for the lowering of stimulating and recording electrodes with coordinates estimated from the stereotaxic atlas of the rat brain (Paxinos and Watson, 1998). A varnished insulated tungsten stimulating electrode with a tip diameter of 50–60 mm and a resistance of 80–100 kV was lowered into the parvicellular part of the left BNA (3.3 mm caudal to the bregma, 5.0 mm lateral from the midline, and 7.1 mm down from the dorsal surface of the cerebral hemisphere). Stimulation of the amygdala was achieved by single rectangular pulses of 30–60 mA with a duration of 0.3 ms. The current intensity of the vagus nerve stimulation was approximately 0.8 of its threshold to induce 10–15% changes in arterial blood pressure and was not more than 200 mA. Typically, we used rectangle current pulses of 50–150 mA with duration of 0.5 ms and frequency of 10 Hz. Varnished insulated microelectrodes (Science Products GMBH, Germany) with a tip diameter of 1 mm and resistance of 5 MV at 1 kHz were used for extracellular recording of neuronal activity. The electrodes were lowered into the deep layers of the left mPFC parallel to its medial surface at a distance between 0.5 and 1.0 mm from the midline. Recording was limited to a region defined by a caudo-rostral direction from 2.0 to 4.0 mm rostral to the bregma and dorsoventrally from 2.0 to 5.0 mm relative to the dorsal surface of the cerebral hemisphere. Stimulation, data acquisition, and processing were under on-line computer control. Results were presented as peri-stimulus histograms accumulated with 50 sweeps with bin 1 ms. Background and evoked activity of neurons were estimated within 50 ms before and 50 ms after BNA stimulation, respectively. The response intensity was estimated from the histogram as mean number of spikes per second reduced to one trial. The response latency was determined as the time interval between the onset of the stimulus and appearance of the maximum peak of the histogram. In each of the 28 animal preparations, the responses of the prelimbic and infralimbic cortical neurons to the amygdala singlepulse stimulation were investigated before and under repetitive stimulation of the left vagus nerve. In 23 animals, the same experimental protocol was continued after 7-NI (Sigma, USA) was administered stereotaxically with a Hamilton syringe into the anterior horn of the left lateral ventricle at a dose of 5 mg (0.3 ml of the suspension in 5% dimethyl sulfoxide). Recordings were performed within 45 min after the administration. The remaining 5 animals received intracerebroventricular 5% dimethyl sulfoxide and were used as control to determine the effects of 7-NI. At the end of each experiment, the animal was sacrificed with a lethal dose of urethane. After electrolytic destruction of tissue via electrodes, the brain was removed and stored in 10% paraformaldehyde for a week. The locations of stimulating and recording electrodes were examined on 100-mm-thick thionin-stained sections using the atlas of the rat brain (Paxinos and Watson, 1998). Data obtained under VNS before and after 7-NI administration were compared using ANOVA with Tukey’s test and expressed as mean value SEM and F-statistic. The mean rate of background activity of cortical neurons was 5.2 0.4 spikes/s (n = 119; Fig. 1). Single-pulse stimulation of the BNA-induced a short discharge in these cells that occurred with a mean latency of 16.1 0.5 ms. We did not observe any differences
123
Fig. 1. Diagrams demonstrating changes in background and amydala-evoked activity of the prefrontal cortical neurons after intracerebroventricular 7nitroindasole administration (7-NI), under vagus nerve stimulation (VNS) and under vagus nerve stimulation after 7-nitroindasole administration (VNS + 7-NI). *, **—the mean rates of background and amygdala-evoked firing are significantly different from the corresponding initial levels (P < 0.005).
between neuronal reactions recorded in the prelimbic and infralimbic areas of the mPFC. The mean rate of the amygdala-evoked firing was 28.0 1.6 spikes/s and significantly differed from the background level (F = 269.92; P < 0.005; n = 119). Repetitive stimulation of the vagus nerve did not change the ongoing firing of the mPFC cells (4.2 0.4 spikes/s; n = 64; Fig. 1). However, neuronal responses to the BNA stimulation were reduced (Fig. 2). Under VNS, the mean rate of amygdala-evoked firing of mPFC neurons significantly decreased in comparison with the value prior to nerve stimulation and was
Fig. 2. Average peri-stimulus time histograms demonstrating responses of the prefrontal cortical neurons to the amygdala stimulation (initial) and their changes under vagus nerve stimulation before (VNS) and after intracerebroventricular 7nitroindasole administration (VNS + 7-NI). Bin—1 ms, sweeps—50. Ordinate— number of spikes in each bin per sweep per neuron. Abscissa—time in ms, 0— moment of the amygdala stimulation.
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17.2 1.3 spikes/s (F = 19.75; P < 0.005; n = 64; Fig. 1). The mean latency of the responses did not significantly change (17.3 0.9 ms; n = 64). The intracerebroventricular 7-NI administration did not cause any noticeable changes in background activity of mPFC neurons (3.6 0.7 spikes/s; n = 59; Fig. 1). The mean rate of amygdala-evoked firing after 7-NI was 24.6 1.2 spikes/s and did not significantly differ from the value before the administration (F = 1.93; P > 0.05; n = 59). The mean latency of the responses after 7-NI administration did not significantly change (16.9 1.0 ms; n = 59). However, the VNS effects on the activity of the recorded cortical neurons were altered. Under nNOS inhibition, the repetitive vagus nerve stimulation resulted in a significant increase in background firing of mPFC neurons in comparison with its initial level before 7-NI administration (10.3 2.6 spikes/s; F = 7.07; P < 0.005; n = 42; Fig. 1). Simultaneously, the VNS effects on responses of mPFC neurons to the BNA stimulation were reversed. Following 7-NI administration, the amygdala-induced activity of cortical neurons was increased by the vagus nerve stimulation (Fig. 2). The mean rate of the evoked firing was 39.5 3.4 spikes/s and significantly differed from the initial value prior to 7-NI administration (F = 12.49; P < 0.005; n = 42; Fig. 1). The mean latency of the responses did not significantly change (18.0 1.1 ms; n = 42). Intracerebroventricular administration of 5% dimethyl sulfoxide, the vehicle for 7-NI, had no noticeable effect on the background and amygdala-evoked activity of the recorded mPFC neurons, nor did it alter the effect of VNS on them. Thus, this study showed for the first time that the electrical stimulation of the left cervical vagus nerve resulted in the inhibition of prelimbic and infralimbic neuronal responses to stimulation of the basal nucleus of the amygdala. The most probable pathway mediating the effect of VNS on the amygdalaevoked responses of cortical neurons is the ascending projection of the nucleus of the solitary tract (NTS) to the parabrachial nucleus, which directly or via the thalamus innervates the medial prefrontal cortex (Schachter and Saper, 1998). The NTS projection to the amygdala is a less attractive candidate because of its termination mostly within the central nucleus (Reyes and Van Bockstaele, 2006), which gives meager projections to other nuclei of the amygdala and does not innervate the cerebral cortex (Pitkanen, 2000). We consider that a possible mechanism by which VNS can change the amygdala-evoked responses of cortical neurons may be realized at the cortical level by decreasing the cortical excitability. This is supported by studies demonstrating that VNS elicits slow hyperpolarization in rat cortical neurons (Zagon and Kemeny, 2000) and increases cortical inhibition in epileptic patients (Di Lazzaro et al., 2004). Thus, in this study, we demonstrated the inhibitory effects of the vagus nerve stimulation on the amygdalaevoked activity of cortical neurons. Probably this effect of VNS can promote the prevention or interruption of the spread of paroxysmal activity from the amygdala–hippocampal region to the mPFC in epileptic patients. Results of this study suggest that the inhibitory effect of VNS on the amygdala-induced responses of mPFC neurons is probably related to endogenous nitric oxide owing to its dependence on 7NI, a preferred inhibitor of nNOS. It has been shown that a number of GABA-ergic cortical neurons contain NOS (Valtschanoff et al., 1993) and a release of GABA in the cortex is under NO-dependent control (Ohkuma et al., 1996). Moreover, it has been demonstrated that VNS increases the level of free GABA in the cerebrospinal fluid as well as GABAA receptor density in the cerebral cortex (BenMenachem et al., 1994; Marrosu et al., 2003). We consider that the inhibitory effect of the vagus nerve stimulation on the mPFC excitability can be linked to the increase in GABA release from cortical neurons owing to the activation of local NO-ergic
mechanisms. If this is true, a depression of these mechanisms will lead to a decrease in cortical GABA concentration, and as a result, to an increase in cortical excitability. Indeed, the reduction in NO in the brain has been shown to cause an increase in the discharge activity of the cortex (Ferraro et al., 1999). Consistent with this, in this study, after 7-NI administration, we observed an increase of the amygdala-induced responses of mPFC neurons under vagus nerve stimulation. Moreover, we cannot rule out that the intraventricular administration of 7-NI results in a suppression of NO-dependent inhibitory mechanisms not only in the cortex but in other brain structures mediating the effect of VNS on cortical neurons. In this study, we did not reveal significant changes in responses of the mPFC neurons to the BNA stimulation before and after intracerebroventricular 7-NI administration. This indicates that BNA–mPFC neurotransmission is probably NO-independent. In addition, data on Fos/NADPH diaphorase histochemistry recently obtained in our laboratory showed that VNS did not trigger NOergic neurons in the nucleus of the solitary tract (Osharina et al., 2006) as well as in the parabrachial nucleus and hypothalamus (unpublished data). Taken together, this allows us to assume that the main role in mediating the inhibitory effect of the vagus nerve stimulation on the BNA-induced responses of the mPFC neurons belongs to cortical NO-dependent mechanisms. In conclusion, our results suggest that a possible mechanism by which subthreshold vagus nerve stimulation can inhibit amygdala–mPFC neurotransmission and prevent a spread of paroxysmal activity to the cortical level may be the activation of cortical NOdependent mechanisms. However, this assumption needs to be confirmed in experimental models of epilepsy. References Bacon, S.J., Headlam, A.J., Gabbott, P.L., Smith, A.D., 1996. Amygdala input to medial prefrontal cortex (mPFC) in the rat: a light and electron microscope study. Brain Res. 720, 211–219. Ben-Menachem, E., Manon-Espaillat, R., Ristanovic, R., Wilder, B.J., Stefan, H., Mirza, W., Tarver, W.B., Wernicke, J.F., 1994. Vagus nerve stimulation for treatment of partial seizures. I. A controlled study of effect on seizures. Epilepsia 35, 616–626. Benini, R., D’Antuono, M., Pralong, E., Avoli, M., 2003. Involvement of amygdala networks in epileptiform synchronization in vitro. Neuroscience 120, 75–84. Bertini, G., Peng, Z.C., Bentivoglio, M., 1996. The chemical heterogeneity of cortical interneurons: nitric oxide synthase vs. calbindin and parvalbumin immunoreactivity in the rat. Brain Res. Bull. 39, 261–266. Binnie, C.D., 2000. Vagus nerve stimulation for epilepsy: a review. Seizure 9, 161–169. Davis, M., Whalen, P.J., 2001. The amygdala: vigilance and emotion. Mol. Psychiatry 6, 13–34. Del-Bel, E.A., Oliveira, P.R., Oliveira, J.A., Mishra, P.K., Jobe, P.C., Garcia-Cairasco, N., 1997. Anticonvulsant and proconvulsant roles of nitric oxide in experimental epilepsy models. Braz. J. Med. Biol. Res. 30, 971–979. Di Lazzaro, V., Oliviero, A., Pilato, F., Saturno, E., Dileone, M., Meglio, M., Colicchio, G., Barba, C., Papacci, F., Tonali, P.A., 2004. Effects of vagus nerve stimulation on cortical excitability in epileptic patients. Neurology 62, 2310–2312. Ferraro, G., Montalbano, M.E., La Grutta, V., 1999. Nitric oxide and glutamate interaction in the control of cortical and hippocampal excitability. Epilepsia 40, 830–836. Groves, D.A., Brown, V.J., 2005. Vagal nerve stimulation: a review of its applications and potential mechanisms that mediate its clinical effects. Neurosci. Biobehav. Rev. 29, 493–500. Hirsch, E., Danober, L., Simler, S., Pereira de Vasconcelos, A., Maton, B., Nehlig, A., Marescaux, C., Vergnes, M., 1997. The amygdala is critical for seizure propagation from brainstem to forebrain. Neuroscience 77, 975–984. Huh, Y., Heo, K., Park, C., Ahn, H., 2000. Transient induction of neuronal nitric oxide synthase in neurons of rat cerebral cortex after status epilepticus. Neurosci. Lett. 281, 49–52. Ishikawa, A., Nakamura, S., 2003. Convergence and interaction of hippocampal and amygdalar projections within the prefrontal cortex in the rat. J. Neurosci. 23, 9987–9995. LeDoux, J.E., 2000. Emotion circuits in the brain. Annu. Rev. Neurosci. 23, 155–184. Marrosu, F., Serra, A., Maleci, A., Puligheddu, M., Biggio, G., Piga, M., 2003. Correlation between GABA(A) receptor density and vagus nerve stimulation in individuals with drug-resistant partial epilepsy. Epilepsy Res. 55, 59–70. McDonald, A.J., 1991. Organization of amygdaloid projections to the prefrontal cortex and associated striatum in the rat. Neuroscience 44, 1–14.
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