Unilateral low-frequency stimulation of central piriform cortex inhibits amygdaloid-kindled seizures in Sprague-Dawley rats

The central piriform cortex (cPC) is considered to be critically involved in the generation and propagation of kindled seizures. Our previous study found that low-frequency stimulation (LFS) of the cPC inhibits the development process of amygdala kindling. In this study, we determined whether unilateral LFS of the cPC had an inhibitory effect on amygdaloid-kindled seizures in Sprague-Dawley rats. When fully-kindled seizures were achieved by daily amygdala electrical stimulation (2 s train of 1 ms pulses at 60 Hz and 150-300 microA), LFS (15 min train of 0.1 ms pulses at 1 Hz and 50-150 microA) was applied to the ipsilateral or contralateral cPC 1 s after cessation of kindling stimulation for 10 days. LFS of the ipsilateral cPC significantly decreased the incidence of generalized seizures and seizure stage, and shortened cumulative afterdischarge duration and cumulative generalized seizure duration. LFS of the contralateral cPC also significantly decreased the expression of seizure stage, but had no appreciable effect on the generalized seizure incidence, cumulative afterdischarge duration and cumulative generalized seizure duration. On the other hand, LFS of the ipsilateral cPC significantly increased the afterdischarge threshold and further increased the differences of current intensity between afterdischarge threshold and generalized seizure threshold. Our data suggest that LFS of the cPC may be an effective method of inhibiting kindled seizures by preventing both afterdischarge generation and propagation. It provide further evidence that brain regions like the cPC, other than the seizure focus, can serve as targets for deep brain stimulation treatment of epilepsy.     

Decreased susceptibility to pentylenetetrazol-induced seizures after low-frequency transcranial magnetic stimulation in rats

We studied the effects of low-frequency repetitive transcranial magnetic stimulation (rTMS) on seizure susceptibility in rats. rTMS of 1000 pulses at 0.5 Hz led to a prolonged latency for seizure development after an intraperitoneal injection of pentylenetetrazol. The rTMS effectively prevented the development of status epilepticus of pentylenetetrazol-induced convulsions. These findings indicate that low-frequency rTMS affects the neural excitability, in the direction of anticonvulsive, and therefore, suggest the possibility of therapeutic use of rTMS in epilepsy.     

Low-frequency stimulation of the hippocampal CA3 subfield is anti-epileptogenic and anti-ictogenic in rat amygdaloid kindling model of epilepsy

Neuromodulation with low-frequency stimulation (LFS), of brain structures other than epileptic foci, is effective in inhibiting seizures in animals and patients, whereas selection of targets for LFS requires further investigation. The hippocampal CA₃ subfield is a key site in the circuit of seizure generation and propagation. The present study aimed to illustrate the effects of LFS of the CA₃ region on seizure acquisition and generalization in the rat amygdaloid kindling model of epilepsy. We found that LFS (monophasic square-wave pulses, 1 Hz, 100 μA and 0.1 ms per pulse) of the CA₃ region significantly depressed the duration of epileptiform activity and seizure acquisition by retarding progression from focal to generalized seizures (GS). Moreover, GS duration was significantly shortened and its latency was significantly increased in the LFS group demonstrating an inhibition of the severity of GS and the spread of epileptiform activity. Furthermore, LFS prevented the decline of afterdischarge threshold (ADT) and elevated GS threshold indicating an inhibition of susceptibility to GS. These results suggest that LFS of the hippocampal CA₃ subfield is anti-epileptogenic and anti-ictogenic. Neuromodulation of CA₃ activity using LFS may be an alternative potential approach for temporal lobe epilepsy treatment.     

Anticholinesterase-induced epileptiform activity in immature rat piriform cortex slices, in vitro

Purpose: Acute in vitro brain slice models are commonly used to study epileptiform seizure generation and to test anti-epileptic drug action. Seizure-like activity can be readily induced by manipulating external ionic concentrations or by adding convulsant agents to the bathing medium. We previously showed that epileptiform bursting was induced in slices of immature (P14–28) rat piriform cortex (PC) by applying oxotremorine-M, a potent muscarinic receptor agonist. Here, we examined whether raising levels of endogenous acetylcholine (ACh) by exposure to anticholinesterases, could also induce epileptiform events in immature (P12–14) or early postnatal (P7–9) rat PC brain slices. Methods: The effects of anticholinesterases were investigated in rat PC neurons using both extracellular MEA (P7–9 slices) and intracellular (P12–14 slices) recording methods. Results: In P7–9 slices, eserine (20 μM) or neostigmine (20 μM) induced low amplitude, low frequency bursting activity in all three PC cell layers (I–III), particularly layer III, where neuronal muscarinic responsiveness is known to predominate. In P12–14 neurons, neostigmine produced a slow depolarization together with an increase in input resistance and evoked cell firing. Depolarizing postsynaptic potentials evoked by intrinsic fibre stimulation were selectively depressed although spontaneous bursting was not observed. Neostigmine effects were blocked by atropine (1 μM), confirming their muscarinic nature. We conclude that elevation of endogenous ACh by anticholinesterases can induce bursting in early postnatal PC brain slices, further highlighting the epileptogenic capacity of this brain region. However, this tendency declines with further development, possibly as local inhibitory circuit mechanisms become more dominant.     

Relationship of some open-field behaviors to amygdaloid kindled convulsions in Wistar rats

Repeated low-intensity electrical stimulation (kindling) of the amygdala eventually produces convulsive behavior in animals. The present study examined the relationship between behaviors displayed in a novel open-field situation with behavioral characteristics of the kindled clonic convulsion (CC). Wistar rats were given two open-field tests and were subsequently kindled to clonic convulsions. A multiple regression analysis indicated that rats which urinated more often in the open-field tests tended to show longer latencies to CC onset. Thus, open-field urination was a significant predictor of latency to CC onset. It is suggested that an emotionality construct may be related to rate of kindling.     

Selective bilateral destruction of substantia nigra has no effect on kindled seizures induced from stimulation of amygdala or piriform cortex in rats

Enhancement of GABAergic transmission in the substantia nigra has been shown to attenuate motor manifestations of diverse seizure models, including kindling. Similar anticonvulsant effects were reported after bilateral lesions of the substantia nigra, supporting the view that the nigra efferents constitute a critical gating mechanism in the propagation of seizure activity. However, in the lesion studies reported so far the nigra was not destroyed selectively so that regions destroyed in addition to the nigra could have been involved in the anticonvulsant effects observed. We destroyed the nigra selectively in fully kindled rats by bilateral microinjection of small amounts of the neurotoxin ibotenic acid. Two groups of rats were studied; one was kindled from stimulation of the basolateral amygdala, the other from stimulation of the piriform cortex. In both groups, there was no indication of a reduction in seizure susceptibility, seizure severity or seizure duration after bilateral destruction of the nigra. The data thus indicate that, at least in kindled rats, the substantia nigra might be less important for seizure generation and/or propagation than previously thought.     

Expression of highly polysialylated NCAM in the neocortex and piriform cortex of the developing and the adult rat

Summary The expression of a highly polysialylated form of the neural cell adhesion molecule (NCAM-H) has been investigated in the neocortex and piriform cortex of the developing and the adult rat by using a monoclonal antibody 12E3, which has been found to recognize the polysialic acid portion of NCAM-H. Immunoblot analysis of the cortical homogenates showed that NCAM-H was temporarily expressed during the late embryonic and early postnatal stages. Further, immunohistochemical observations revealed that NCAM-H appeared at embryonic day 13 (E13) in the plexiform primordium in horizontally-oriented cells, probably Cajal-Retzius cells, which are the first neurons to differentiate. During the late embryonic stage, the marginal zone, subplate, and intermediate zone strongly stained, whereas the ventricular zone stained weakly. After birth, the NCAM-H expression was progressively attenuated from a week onwards, and almost vanished in the adult neocortex. In the primordium of the piriform cortex, NCAM-H immunoreactivity also became positive at E13. The time sequences of the NCAM-H expression in these neurons were similar to those of the neurons in the neocortical area. In the piriform cortex, however, the expression remained in a number of neurons in the layer II, which receives a large number of olfactory fibers from the olfactory bulb, where prolonged neurogenesis and construction of neural circuits take place in adulthood. These results suggest that NCAM-H not only plays an important role in the developing rat cortex, but also may be involved in some functions related to reorganization in the adult piriform cortex.     

Physiology of modulation of motor cortex excitability by low-frequency suprathreshold repetitive transcranial magnetic stimulation

Many studies show consistently that repetitive transcranial magnetic stimulation (rTMS) with a frequency of 1 Hz and an intensity above the resting motor threshold (RMT) performed for several minutes over the primary motor cortex (M1) leads to a depression of cortical excitability. Furthermore, most studies concur on a facilitation of the non-stimulated contralateral M1. Little is known, however, about the physiological mechanisms underlying these effects. In 11 healthy volunteers, we stimulated the left M1 for 15 min with 1 Hz-rTMS of 115% RMT. Before, immediately after, and 30 min after the rTMS train, we examined short-interval intracortical inhibition (SICI; interstimulus interval (ISI) of 2 and 4 ms), intracortical facilitation (ICF; ISI 10 ms), and short-interval intracortical facilitation (SICF; ISI 1.5 ms) with established paired-pulse protocols. Mean unconditioned motor evoked potential (MEP) amplitudes and RMT were also measured. Two sessions were run at least 1 week apart, in one excitability of the stimulated M1 was tested, in the other one excitability of the non-stimulated M1. rTMS led to the expected reduction of MEP amplitude of the stimulated M1, which was significant only immediately after the rTMS train. rTMS increased MEP amplitude of the non-stimulated M1, which lasted for at least 30 min. RMT, SICI, ICF and SICF did not show any significant change in either M1, except for a long lasting increase of SICF in the non-stimulated M1. In conclusion, the MEP increase in the non-stimulated M1 lasted longer than the MEP decrease in the stimulated M1. Only the long-lasting MEP increase was associated with a specific change in intracortical excitability (increase in SICF). Modulation of motor cortical inhibition did not play a role in explaining the rTMS induced changes in MEP amplitude.     

Plasticity of the medial prefrontal cortex: Facilitated acquisition of intracranial self-stimulation by pretraining stimulation

Prior electrical stimulation of the medial prefrontal cortex MFC facilitated the subsequent acquisition of intracranial self-stimulation (ICSS) from the same MFC electrode site. Stimulations that were spaced over a period of six days were more effective in producing this facilitation than the same number of stimulations delivered over a two day period. These data suggest that the rewarding effects of MFC stimulation may involve some process akin to the kindling phenomenon and as such may provide insights in the neuronal modifications thought to underlie learning and memory.     

Influences of bilateral hippocampal lesions upon kindled amygdaloid convulsive seizure in rats

In this study, rat hippocampus was lesioned bilaterally after completion of amygdaloid kindling, to examine how hippocampus affects the kindling permanency. The ventral hippocampal lesions of the kindled rats inhibited reappearance of any kindled seizures. The other rats with the same lesions showed the regression of generalized convulsion. These results suggest that hippocampus, especially ventral parts of hippocampus, would have rather facilitatory or maintaining influence on the kindled neural circuits, relating to the catecholaminergic system in rat forebrain.     

Modification of the human motor cortex by associative stimulation

Manipulation of afferent input is capable of inducing reorganisation of the motor cortex. For example, following 1 h of paired electrical stimulation to the motor point of two hand muscles (associative stimulation) the excitability of the corticospinal projection to the stimulated muscles is increased. Here we investigated the mechanisms responsible for such change using transcranial magnetic stimulation (TMS). Cortical excitability changes were investigated by measuring motor evoked potentials (MEPs), intracortical inhibition (ICI), intracortical facilitation (ICF), and short-interval intracortical facilitation (SICF). Following 1 h of associative stimulation MEP amplitudes in the stimulated muscles significantly increased. Additionally, there was a significant increase in ICF and of SICF at interstimulus intervals in the range of 2.3–3.3 ms. There was no significant change in ICI. These findings confirm previous observations that a 1-h period of associative stimulation can increase the excitability of the cortical projection to stimulated muscles. Additionally, these results suggest that the observed modifications of excitability are due to changes in intracortical excitatory circuits.     

Motor cortex inhibition induced by acoustic stimulation

The influence of the brainstem motor system on cerebral motor areas may play an important role in motor control in health and disease. A new approach to investigate this interaction in man is combining acoustic stimulation activating the startle system with transcranial magnetic stimulation (TMS) over the motor cortex. However, it is unclear whether the inhibition of TMS responses following acoustic stimulation occurs at the level of the motor cortex through reticulo-cortical projections or subcortically, perhaps through reticulo-spinal projections. We compared the influence of acoustic stimulation on motor effects elicited by TMS over motor cortical areas to those evoked with subcortical electrical stimulation (SES) through depth electrodes in five patients treated with deep brain stimulation for Parkinsons disease. SES bypasses the motor cortex, demonstrating any interaction with acoustic stimuli at the subcortical level. EMG was recorded from the contralateral biceps brachii muscle. Acoustic stimulation was delivered binaurally through headphones and used as a conditioning stimulus at an interstimulus interval of 50 ms. When TMS was used as the test stimulus, the area and amplitude of the conditioned motor response was significantly inhibited (area: 57.5±12.9%, amplitude: 47.9±7.4%, as percentage of unconditioned response) whereas facilitation occurred with SES (area: 110.1±4.3%, amplitude: 116.9±6.9%). We conclude that a startle-evoked activation of reticulo-cortical projections transiently inhibits the motor cortex.     

经颅磁刺激对部位相关癫癎患者运动皮质功能的评估

目的:采用经颅磁刺激技术(TMS)探讨症状性运动部位相关癫癎患者发作间期运动皮质的兴奋性.方法:对诊断明确的34例癫癎患者(分治疗组和未治疗组)及20例年龄、性别匹配的正常对照组进行单脉冲经颅磁刺激,刺激部位头颅相应的运动手区和颈7棘突外侧,并于对侧小指外展肌记录运动诱发电位(MEP),分析其阈强度(TI)、周围潜伏期(PL)及皮质潜伏期(CL)、中枢传导时间(CCT)和静息期(SP).结果:所有癫癎患者PL、CL及CCT均在正常范围内,但TI和SP明显低于正常对照组(P< 0.01).在癫癎患者中,未治疗组TI及SP明显低于治疗组(P< 0.01),致癎灶侧TI及SP低于非致癎灶侧(P< 0.05),但非致癎灶侧SP亦缩短.结论:单脉冲低频TMS能有效地反映中枢运动皮质的功能状态,用于症状性运动部位相关癫癎患者发作间期运动皮质兴奋性研究具有重要的实用价值.     

Suppression of the motor cortex by magnetic stimulation of the cerebellum

Conditioning magnetic stimulation of the cerebellum suppresses the motor cortex 5-8 ms later, probably through activation of cerebellar Purkinje cells, which inhibit the dentatothalamocortical pathway. To further characterize this pathway, we examined several factors that may modulate its excitability. We tested the effects of different test motor evoked potential (MEP) amplitudes, voluntary activation of the target muscle, and arm extension that required activation of proximal arm muscles while maintaining relaxation of hand muscles. Surface electromyography was recorded from the right first dorsal interosseous (FDI) muscle. A double-cone coil centered 3 cm lateral to the inion was used for right cerebellar stimulation. The stimulus intensity was set at 5% below the threshold for activation of the corticospinal tract. A figure-of-eight coil was used for left motor cortex stimulation. Interstimulus intervals (ISIs) between 3 and 15 ms were studied. Small test MEPs of about 0.5 mV were markedly inhibited at ISIs of 5-8 ms, but there was much less inhibition for test MEPs of about 2 mV. There was no significant MEP suppression during voluntary activation of the FDI muscle or during right arm extension. Left arm extension did not affect inhibition. Our findings indicate that cerebellar stimulation has a much stronger effect on motor cortex neurons activated near threshold intensities than those activated at higher intensities. Activation of contralateral but not ipsilateral proximal arm muscles during arm extension reduced the excitability of the cerebellothalamocortical projections to the hand area of the motor cortex.     

Serine/threonine protein phosphatases have no role in the inhibitory effects of low-frequency stimulation in perforant path kindling acquisition in rats

The use of low-frequency stimulation (LFS) as a therapy for epilepsy is currently being studied in experimental animals and patients with epilepsy. In the present study, the role of serine/threonine protein phosphatases in the inhibitory effects of LFS on perforant path kindling acquisition was investigated in rats. Animals were kindled by stimulation of perforant path in a stimulation using rapid kindling procedure (six stimulations per day). LFS (1 Hz) was applied immediately after termination of each kindling stimulation. FK506 (1 μM; i.c.v.), a serine/threonine protein phosphatase PP2B inhibitor and okadaic acid (1 μM; i.c.v.), a serine/threonine protein phosphatases PP1/2A inhibitor, were daily microinjected into the left ventricle 10 min before starting the stimulation protocol. Application of LFS retarded the kindling acquisition and delayed the expression of different kindled seizure stages significantly. In addition, LFS reduced the increment of daily afterdischarge duration during kindling development. Neither FK506 nor okadaic acid microinjection interfere with the antiepileptogenic effect of LFS on kindling parameters. Obtained results showed that activation of PP1/2A and PP2B, which play a critical role in LFS induced down-regulation of synaptic strength, had no role in mediating the inhibitory effects of LFS on perforant path kindling acquisition.     

Unilateral low-frequency stimulation of central piriform cortex delays seizure development induced by amygdaloid kindling in rats

Low-frequency stimulation of the kindling site interferes with the course of kindling epileptogenesis. The present study examined the effect of unilateral low-frequency stimulation of the central piriform cortex on seizure development induced by amygdaloid kindling in rats. The ipsilateral or contralateral central piriform cortex received low-frequency stimulation (15 min train of 0.1 ms pulses at 1 Hz and 50-150 muA) immediately after termination of once daily kindling stimulation (2 s train of 1 ms pulses at 60 Hz and 150-300 microA) in the right amygdala for 30 days. Low-frequency stimulation of either the ipsilateral or contralateral central piriform cortex significantly suppressed the progression of seizure stages and reduced afterdischarge duration throughout the course of amygdaloid kindling. The marked suppression induced by low-frequency stimulation of the central piriform cortex on either side was predominantly due to the significant retardation of progression from stage 0 to stage 1 and stage 3 to stage 4 seizures. In addition, the suppressive effect of low-frequency stimulation did not disappear when the stimulation was stopped; it could persist for at least 10 days. These findings indicate that brain areas other than the kindling focus, such as the central piriform cortex on both sides, can also be used as reasonable targets for low-frequency stimulation to retard seizure development induced by amygdaloid kindling. Secondly, like the ipsilateral central piriform cortex, the contralateral central piriform cortex may also participate in the progression and secondary generalization of focal seizures. The study suggests that unilateral low-frequency stimulation of the central piriform cortex may have a significant antiepileptogenic effect, and may be helpful for exploring effective and long-lasting therapies for human temporal lobe epilepsy.     

Anodal transcranial direct current stimulation of prefrontal cortex enhances working memory

Previous studies have claimed that weak transcranial direct current stimulation (tDCS) induces persisting excitability changes in the human motor cortex that can be more pronounced than cortical modulation induced by transcranial magnetic stimulation, but there are no studies that have evaluated the effects of tDCS on working memory. Our aim was to determine whether anodal transcranial direct current stimulation, which enhances brain cortical excitability and activity, would modify performance in a sequential-letter working memory task when administered to the dorsolateral prefrontal cortex (DLPFC). Fifteen subjects underwent a three-back working memory task based on letters. This task was performed during sham and anodal stimulation applied over the left DLPFC. Moreover seven of these subjects performed the same task, but with inverse polarity (cathodal stimulation of the left DLPFC) and anodal stimulation of the primary motor cortex (M1). Our results indicate that only anodal stimulation of the left prefrontal cortex, but not cathodal stimulation of left DLPFC or anodal stimulation of M1, increases the accuracy of the task performance when compared to sham stimulation of the same area. This accuracy enhancement during active stimulation cannot be accounted for by slowed responses, as response times were not changed by stimulation. Our results indicate that left prefrontal anodal stimulation leads to an enhancement of working memory performance. Furthermore, this effect depends on the stimulation polarity and is specific to the site of stimulation. This result may be helpful to develop future interventions aiming at clinical benefits.Felipe Fregni and Paulo S. Boggio contributed equally to this work.     

Electrical stimulation of the motor cortex: Theoretical considerations

The aim of this paper is to present the results of a theoretical analysis of the intracranial fields produced by electrical stimulation of the unexposed motor cortex with surface electrodes in humans. Simulations of a first approximation model of the head indicate that the intensity and the spatial configuration of the intracranial fields can be controlled, to a great extent, by proper choice of the location and of the number of the stimulating electrodes. Fields are shown to be reasonably insensitive to changes of some crucial parameters, like the number of the stimulating electrodes and the ratio between the conductivity of the skull and that of the other tissues.     

Changes in muscle responses to stimulation of the motor cortex induced by peripheral nerve stimulation in human subjects

The aim of this study was to determine whether prolonged, repetitive mixed nerve stimulation (duty cycle 1 s, 500 ms on-500 ms off, 10 Hz) of the ulnar nerve leads to a change in excitability of primary motor cortex in normal human subjects. Motor-evoked potentials (MEPs) generated in three intrinsic hand muscles [abductor digiti minimi (ADM), first dorsal interosseous (FDI) and abductor pollicis brevis (APB)] by focal transcranial magnetic stimulation were recorded during complete relaxation before and after a period of prolonged repetitive ulnar nerve stimulation at the wrist. Transcranial magnetic stimuli were applied at seven scalp sites separated by 1 cm: the optimal scalp site for eliciting MEPs in the target muscle (FDI), three sites medial to the optimal site and three sites lateral to the optimal stimulation site. The area of the MEPs evoked in the ulnar-(FDI, ADM) but not the median-innervated (APB) muscles was increased after prolonged ulnar nerve stimulation. Centre of gravity measures demonstrated that there was no significant difference in the distribution of cortical excitability after the peripheral stimulation. F-wave responses in the intrinsic hand muscles were not altered after prolonged ulnar nerve stimulation, suggesting that the changes in MEP areas were not the result of stimulus-induced increases in the excitability of spinal motoneurones. Control experiments employing transcranial electric stimulation provided no evidence for a spinal origin for the excitability changes. These results demonstrate that in normal human subjects the excitability of the cortical projection to hand muscles can be altered in a manner determined by the peripheral stimulus applied.     

The effect on corticospinal volleys of reversing the direction of current induced in the motor cortex by transcranial magnetic stimulation

Descending corticospinal volleys were recorded from a bipolar electrode inserted into the cervical epidural space of four conscious human subjects after monophasic transcranial magnetic stimulation over the motor cortex with a figure-of-eight coil. We examined the effect of reversing the direction of the induced current in the brain from the usual posterior-anterior (PA) direction to an anterior-posterior (AP) direction. The volleys were compared with D waves evoked by anodal electrical stimulation (two subjects) or medio-lateral magnetic stimulation (two subjects). As reported previously, PA stimulation preferentially recruited I1 waves, with later I waves appearing at higher stimulus intensities. AP stimulation tended to recruit later I waves (I3 waves) in one of the subjects, but, in the other three, I1 or D waves were seen. Unexpectedly, the descending volleys evoked by AP stimulation often had slightly different peak latencies and/or longer duration than those seen after PA stimulation. In addition the relationship between the size of the descending volleys and the subsequent EMG response was often different for AP and PA stimulation. These findings suggest that AP stimulation does not simply activate a subset of the sites activated by PA stimulation. Some sites or neurones that are relatively inaccessible to PA stimulation may be the low-threshold targets of AP stimulation.