Three dimensional microelectrodes enable high signal and spatial resolution for neural seizure recordings in brain slices and freely behaving animals.

Neural recordings made to date through various approaches-both in-vitro or in-vivo-lack high spatial resolution and a high signal-to-noise ratio (SNR) required for detailed understanding of brain function, synaptic plasticity, and dysfunction. These shortcomings in turn deter the ability to further design diagnostic, therapeutic strategies and the fabrication of neuro-modulatory devices with various feedback loop systems. We report here on the simulation and fabrication of fully configurable neural micro-electrodes that can be used for both in vitro and in vivo applications, with three-dimensional semi-insulated structures patterned onto custom, fine-pitch, high density arrays. These microelectrodes were interfaced with isolated brain slices as well as implanted in brains of freely behaving rats to demonstrate their ability to maintain a high SNR. Moreover, the electrodes enabled the detection of epileptiform events and high frequency oscillations in an epilepsy model thus offering a diagnostic potential for neurological disorders such as epilepsy. These microelectrodes provide unique opportunities to study brain activity under normal and various pathological conditions, both in-vivo and in in-vitro, thus furthering the ability to develop drug screening and neuromodulation systems that could accurately record and map the activity of large neural networks over an extended time period.

CT perfusion measurement of postictal hypoperfusion: localization of the seizure onset zone and patterns of spread.

PURPOSE: Seizures are often followed by a period of transient neurological dysfunction and postictal alterations in cerebral blood flow may underlie these symptoms. Recent animal studies have shown reduced local cerebral blood flow at the seizure onset zone (SOZ) lasting approximately 1 h following seizures. Using arterial spin labelling (ASL) MRI, we observed postictal hypoperfusion at the SOZ in 75% of patients. The clinical implementation of ASL as a tool to identify the SOZ is hampered by the limited availability of MRI on short notice. Computed tomography perfusion (CTP) also measures blood flow and may circumvent the logistical limitations of MRI. Thus, we aimed to measure the extent of postictal hypoperfusion using CTP. METHODS: Fourteen adult patients with refractory focal epilepsy admitted for presurgical evaluation were prospectively recruited and underwent CTP scanning within 80 min of a habitual seizure. Patients also underwent a baseline scan after they were seizure-free for > 24 h. The acquired scans were qualitatively assessed by two reviewers by visual inspection and quantitatively assessed through a subtraction pipeline to identify areas of significant postictal hypoperfusion. RESULTS: Postictal blood flow reductions of > 15 ml/100 g-1/min-1 were seen in 12/13 patients using the quantitative method of analysis. In 10/12 patients, the location of the hypoperfusion was partially or fully concordant with the presumed SOZ. In all patients, additional areas of scattered hypoperfusion were seen in areas corresponding to seizure spread. CONCLUSION: CTP can reliably measure postictal hypoperfusion which is maximal at the presumed SOZ.

Delta-9-tetrahydrocannabinol (THC) affects forelimb motor map expression but has little effect on skilled and unskilled behavior.

It has previously been shown in rats that acute administration of delta-9-tetrahydrocannabinol (THC) exerts a dose-dependent effect on simple locomotor activity, with low doses of THC causing hyper-locomotion and high doses causing hypo-locomotion. However the effect of acute THC administration on cortical movement representations (motor maps) and skilled learned movements is completely unknown. It is important to determine the effects of THC on motor maps and skilled learned behaviors because behaviors like driving place people at a heightened risk. Three doses of THC were used in the current study: 0.2mg/kg, 1.0mg/kg and 2.5mg/kg representing the approximate range of the low to high levels of available THC one would consume from recreational use of cannabis. Acute peripheral administration of THC to drug naïve rats resulted in dose-dependent alterations in motor map expression using high resolution short duration intracortical microstimulation (SD-ICMS). THC at 0.2mg/kg decreased movement thresholds and increased motor map size, while 1.0mg/kg had the opposite effect, and 2.5mg/kg had an even more dramatic effect. Deriving complex movement maps using long duration (LD)-ICMS at 1.0mg/kg resulted in fewer complex movements. Dosages of 1.0mg/kg and 2.5mg/kg THC reduced the number of reach attempts but did not affect percentage of success or the kinetics of reaching on the single pellet skilled reaching task. Rats that received 2.5mg/kg THC did show an increase in latency of forelimb removal on the bar task, while dose-dependent effects of THC on unskilled locomotor activity using the rotorod and horizontal ladder tasks were not observed. Rats may be employing compensatory strategies after receiving THC, which may account for the robust changes in motor map expression but moderate effects on behavior.

High frequency stimulation alters motor maps, impairs skilled reaching performance and is accompanied by an upregulation of specific GABA, glutamate and NMDA receptor subunits.

High frequency stimulation (HFS) has the potential to interfere with learning and memory. HFS and motor skill training both lead to potentiation of the stimulated network and alter motor map expression. However, the extent to which HFS can interfere with the learning and performance of a skilled motor task and the resulting effect on the representation of movement has not been examined. Moreover, the molecular mechanisms associated with HFS and skilled motor training on the motor cortex are not known. We hypothesized that HFS would impair performance on a skilled reaching task, and would be associated with alterations in motor map expression and protein levels compared to non-stimulated and untrained controls. Long Evans Hooded rats were chronically implanted with stimulating and recording electrodes in the corpus callosum and frontal neocortex, respectively. High frequency theta burst stimulation or sham stimulation was applied once daily for 20 sessions. The rats were divided into five groups: control, HFS and assessed at 1 week post stimulation, HFS and assessed 3 weeks post stimulation, reach trained, and HFS and reach trained. A subset of rats from each group was assessed with either intracortical microstimulation (ICMS) to examine motor map expression or Western blot techniques to determine protein expression of several excitatory and inhibitory receptor subunits. Firstly, we found that HFS resulted in larger and reorganized motor maps, and lower movement thresholds compared to controls. This was associated with an up-regulation of the GABA(A)α1 and NR1 receptor subunits 3 weeks after the last stimulation session only. Stimulation affected skilled reaching performance in a subset of all stimulated rats. Rats that were poor performers had larger rostral forelimb areas, higher proximal and lower distal movement thresholds compared to rats that were good performers after stimulation. Reach training alone was associated with an up-regulation of GABA(A)α1, α2, GluR2, NR1 and NR2A compared to controls. HFS and reach-trained rats showed an up-regulation of GABA(A)α2 compared to stimulated rats that were not reach-trained. Therefore, we have shown that HFS induces significant plasticity in the motor cortex, and has the potential to disrupt performance on a skilled motor task.

Force-frequency and force-length properties in skeletal muscle following unilateral focal ischaemic insult in a rat model.

AIM: Our purpose was to quantify skeletal muscle properties following unilateral focal ischaemic insult (stroke) in a rat model. METHODS: Male rats were divided into two groups: stroke and 2 weeks recovery (n = 8) and control group (n = 7). Stroke was induced in the area of the motor neocortex containing hind limb corticospinal neurones. Contractile properties of the medial gastrocnemius muscle were measured in situ in both limbs. Force-length and force-frequency properties were measured before and 35 min after 5 min fatiguing stimulation. RESULTS: Stroke resulted in bilateral tetanic fade during 200 Hz stimulation. When normalized to 100 Hz contractions, force at 200 Hz was 95.4 +/- 0.9% for the paretic muscles, 96.7 +/- 1.7% for non-paretic muscles and 102.2 +/- 1.0% for muscles of control rats (P = 0.006). Prior to fatiguing contractions, there was no difference in the length dependence of force. During repetitive contractions, active force fell significantly to 19 +/- 4 and 25 +/- 5% of initial force in paretic and non-paretic muscles of animals with a stroke respectively. In control animals active force fell to 37 +/- 5%. During repetitive contractions, fusion index increased in muscles of stroke animals to 1.0 +/- 0 but in control animals it was 0.95 +/- 0.02. There was selective force depression at short lengths for fatigued paretic muscle (significant difference at muscle lengths less than reference length -2 mm). CONCLUSION: The tetanic fade at high stimulation frequencies indicates that there may be activation failure following focal ischaemic insult. The greater magnitude of fatigue and selective depression at short lengths following repetitive contractions should be investigated further.

One hertz stimulation to the corpus callosum quenches seizure development and attenuates motor map expansion.

Low-frequency stimulation applied through indwelling electrodes has been used to depress or depotentiate synaptic efficacy. Moreover it has been reported to inhibit seizure expression and progression when started either during or after seizures. We have recently shown that low-frequency stimulation can also reduce the size of seizure-enlarged movement representations (motor maps) when delivered after 30 afterdischarges that had propagated from the hippocampus to the neocortex. This study was designed to examine the effects of low-frequency stimulation delivered to the corpus callosum on motor map topography when applied during or after each elicited seizure. Specifically, 15 min of 1 Hz stimulation was applied to the corpus callosum either concurrent with or immediately following a neocortical afterdischarge that had propagated from the hippocampus. Long-Evans hooded rats were electrically stimulated twice daily in the right ventral hippocampus until the first neocortical afterdischarge was elicited. Rats then received low-frequency stimulation which began either with the afterdischarge or following each afterdischarge for 20 additional kindling sessions; a sham low-frequency stimulation group was also included. Afterdischarges were recorded from both hippocampal and neocortical sites, and seizure expression was documented. One to six days following the last stimulation session, forelimb movement representations were derived using high-resolution intracortical microstimulation in the left sensorimotor neocortex. Low-frequency stimulation following each kindled seizure, suppressed behavioral seizure severity and hippocampal afterdischarge duration, as well as attenuated kindling-induced motor map expansion.

Low-frequency stimulation reverses kindling-induced neocortical motor map expansion.

Repeated application of low-frequency stimulation can interrupt the development and progression of seizures. Low-frequency stimulation applied to the corpus callosum can also induce long-term depression in the neocortex of awake freely moving rats as well as reduce the size of neocortical movement representations (motor maps). We have previously shown that seizures induced through electrical stimulation of the corpus callosum, amygdala or hippocampus can expand the topographical expression of neocortical motor maps. The purpose of the present study was to determine if low-frequency stimulation administered to the corpus callosum could reverse the expansion of neocortical motor maps induced by seizures propagating from the hippocampus. Adult Long-Evans hooded rats were electrically stimulated in the right ventral hippocampus, twice daily until 30 neocortical seizures were recorded. Subsequently, low-frequency stimulation was administered to the corpus callosum once daily for 20 sessions. High-resolution intracortical microstimulation was then utilized to derive forelimb-movement representations in the left (un-implanted) sensorimotor neocortex. Our results show that hippocampal seizures result in expanded motor maps and that subsequent low-frequency application can reduce the size of the expanded motor maps. Low-frequency stimulation may be an effective treatment for reversing seizure-induced reorganization of brain function.

Reduction of seizure thresholds following electrical stimulation of sensorimotor cortex is dependent on stimulation intensity and is not related to synaptic potentiation.

Epilepsy is characterized as a chronic brain state with a very low seizure threshold, and the occurrence of repeated seizure activity. Currently, there is no animal model of induced epilepsy that allows for the exploration of the brain mechanisms underlying a low seizure threshold without the elicitation of seizures. In this study, we employed repeated application of different intensities of electrical stimulation in an attempt to reduce afterdischarge (seizure) thresholds without eliciting seizures. We utilized an in vivo model of neocortical activation via stimulation of the corpus callosum of the adult rat. The intensities were chosen to be subthreshold (20, 30, 40, 50 microA), near threshold (150 microA), and suprathreshold (250, 500 microA) relative to the mean initial afterdischarge threshold (ADT). We also examined changes in the evoked field responses of the transcallosal pathway to the sensorimotor cortex as a measure of synaptic efficacy. Our results indicated that stimulation at 50 microA was effective at reducing the ADT, while minimizing the number of seizures elicited. Stimulation at 150 microA resulted in the concomitant reduction of ADT and repeated seizures typical of most electrical kindling studies. Finally, the 500 microA group showed repeated seizures, but no reduction of afterdischarge threshold. These stimulation intensities (50 microA, 150 microA, 500 microA and 0 microA-control) can be used to independently determine the brain mechanisms responsible for 1) the acquisition of a low afterdischarge threshold independent of the reorganizing effect of repeated seizures, and 2) the elicitation of repeated seizures independent of stimulation induced reduction of afterdischarge threshold.

Skilled-learning-induced potentiation in rat sensorimotor cortex: a transient form of behavioural long-term potentiation.

The relation between the acquisition of a skilled motor task and synaptic plasticity in the sensorimotor cortex of the awake, freely behaving rat was examined. Skilled-motor training was previously found to induce a functional reorganization of the caudal forelimb area, and to induce an increase in synaptic efficacy, measured in vitro, on the side contralateral to the reaching forelimb. Here, we repeatedly measured neocortical evoked potential recordings in awake, freely behaving rats to examine whether skilled training would induce changes in polysynaptic efficacy on the side contralateral to the reaching forelimb. We found that the increase in task proficiency, but not the acquisition of task requirements or the maintenance of task proficiency, induced an increase in synaptic efficacy on the side contralateral to the reaching forelimb. We also tested the hypothesis that skilled learning induced potentiation shares similar mechanisms to long-term potentiation (LTP) and long-term depression by artificially manipulating polysynaptic efficacy in skilled rats with high- and low-frequency stimulation. We observed that, compared with the ipsilateral side, less potentiation but more depression could be induced on the side contralateral to the reaching forelimb. We conclude that a transient, network-based LTP-like mechanism operates during the learning of a skilled motor task.

Conventional anticonvulsant drugs in the guinea-pig kindling model of partial seizures: effects of acute phenytoin.

This study addressed some of the controversial issues surrounding the anticonvulsant effect of phenytoin, and the predictive validity of the guinea-pig kindling model for the screening of anticonvulsant drugs. Following an intraperitoneal injection of either 50 or 75 mg/kg phenytoin, we analysed plasma concentrations of phenytoin at various time intervals. Behavioural toxicity was assessed at 0.5 h postinjection using quantitative locomotor tests, as well as scores on a sedation/muscle relaxation rating index. The anticonvulsant efficacy of phenytoin was evaluated from measurements of afterdischarge threshold (ADT), afterdischarge duration (ADD) and behavioural seizure severity at three phases of kindling: non-kindled, kindling acquisition (early and late) and kindled (50+ ADs). ADD and seizure severity were also measured in response to both threshold and suprathreshold kindling stimulation. Plasma levels of phenytoin corresponded to the human therapeutic range at the time of behavioural testing and kindling. Phenytoin did not exert significant adverse effects in guinea-pigs on both the behavioural tests and rating index. Phenytoin increased ADT in non-kindled and kindled guinea-pigs and effectively reduced ADD and seizure severity, indicating that the guinea-pig model correctly predicted phenytoin's anticonvulsant effect. Phenytoin produced reliable anticonvulsant activity in the guinea-pig at threshold stimulation but a somewhat reduced efficacy on seizure severity at suprathreshold stimulation intensities. Kindling in the guinea-pig is a valid model of human partial seizures.

Conventional anticonvulsant drugs in the guinea-pig kindling model of partial seizures: effects of acute carbamazepine.

This study addressed the anticonvulsant effect of carbamazepine (CBZ) in the guinea-pig kindling model to further test this model for the screening of anticonvulsant drugs. We analysed plasma concentrations of CBZ at various time intervals after intraperitoneal injection of either 10, 25 or 40 mg/kg CBZ. Behavioural toxicity was assessed at 0.5 h postinjection using quantitative locomotor tests, as well as scores on a sedation/muscle relaxation rating index. The anticonvulsant efficacy of CBZ was evaluated from measurements of afterdischarge threshold (ADT), afterdischarge duration (ADD), and behavioural seizure severity at three phases of kindling: non-kindled, kindling acquisition (early and late) and kindled (50+ ADs). ADD and seizure severity were also measured in response to both threshold and suprathreshold kindling stimulation. Plasma levels of CBZ were within, or higher, than the human therapeutic range at the time of behavioural testing and kindling. CBZ exerted slight effects in guinea-pigs on the sedation rating index but not the behavioural tests. CBZ increased ADT and reduced ADD and seizure severity throughout all phases of kindling, indicating that the guinea-pig model correctly predicts CBZ's anticonvulsant effect. CBZ in the guinea-pig kindling model produced consistent anticonvulsant activity that did not appear to be dependent on stimulation intensity.

Cortical layer III pyramidal dendritic morphology normalizes within 3 weeks after kindling and is dissociated from kindling-induced potentiation.

This experiment examined the effect of a 3-week rest after electrical kindling on kindling-induced potentiation and the morphology of frontal (Fr1) neocortical layer III pyramidal cell dendrites in both male and female rats. Repeated elicitation of afterdischarge resulted in an increase in the severity of the behavioural seizures and afterdischarge duration. The late component of the transcallosal evoked responses was significantly larger 1 and 21 days following the last kindling session in both male and female rats. Analysis of the Golgi-Cox impregnated pyramidal cell dendrites indicated no significant difference in the amount of apical and basilar dendritic, branching, length, and spine density in both male and female rats, relative to their respective control groups, 21 days following the last kindling session. There was, however, one exception, the male group showed a significant increase in apical spine density. The persistent expression of kindling-induced potentiation appears to be dissociated from the renormalized pyramidal cell dendritic morphology.

Mossy fiber sprouting is dissociated from kindling of generalized seizures in the guinea-pig.

Controversy surrounds whether aberrant mossy fiber sprouting in the hippocampus is necessary for the establishment of seizure states. We investigated the association between mossy fiber sprouting and kindling in guinea-pigs, using either single-site or alternate-site stimulation. Kindling with single-site amygdaloid stimulation did not induce significant sprouting, despite the development of partial seizures. In contrast, single-site septal and alternating amygdaloid-septal stimulation produced moderate but significant sprouting in about 60% of animals that failed to develop stage 5 generalized seizures. Since the magnitude of sprouting was similar despite striking differences in the intensity of seizures that developed, we conclude that mossy sprouting is not causally associated with seizure development.

Sex differences in cortical plasticity and behavior following anterior cortical kindling in rats.

This experiment examined the effect of electrical kindling on the morphology of frontal (Fr1) neocortical layer III pyramidal cell dendrites in both male and female rats. Repeated elicitation of afterdischarge resulted in an increase in the severity of the behavioural seizures and an increase in afterdischarge duration, frequency and amplitude in all rats. The late component of the transcallosal evoked responses also increased following both 7 and 25 kindling sessions in male rats and following 25 kindling sessions in female rats. Analysis of the Golgi-Cox impregnated pyramidal cell dendrites indicated a significant decrease in the amount of apical and basilar dendritic spine density, length and branching in female rats following 7 days, but not 25 days, of kindling. Male rats had significantly lower apical and basilar dendritic spine density and branching measures following 25 days, but not 7 days, of kindling, as well as significantly lower apical and basilar dendritic length following 7 days of kindling. The differential gender effect suggests that males and females recruit similar plastic mechanisms although at different times in response to electrical kindling.

Alternate-site kindling in the guinea-pig results in accelerated seizure progression and generalization.

In rats, the concurrent alternate elicitation of epileptiform activity in two forebrain structures can result in both the rapid production of severe seizures and the development of fully generalized seizures in one (dominant) site, while arresting the progress of seizure activity at intermediate stages in the other (suppressed) site. The latter phenomenon is known as kindling antagonism. In this study, we examined alternate-site kindling in the guinea-pig as they fail to express fully generalized (stage 5) convulsions during single-site kindling. We assessed both seizure stage and afterdischarge duration following inter-hemispheric alternate-site kindling stimulation of the amygdala and medial septal areas. Alternating-site kindling of the medial septal and amygdaloid areas bypassed the normal inhibitory mechanisms in some guinea-pigs, enabling them to reach a stage 5 seizure. Furthermore, alternate-site kindled guinea-pigs demonstrated three (absolute, relative, and mutual) types of kindling antagonism. Guinea-pig kindling as a model of human partial epilepsy is discussed.

Effect of complete and partial bilateral lesions of the deep cerebellar nuclei on amygdaloid kindling in rats.

PURPOSE: The roles of the deep cerebellar nuclei in epileptogenesis and seizure expression are not well defined. To determine their properties, we examined the effects of lesions to the dentate, fastigial, and interpositus nuclei in adult rats that were electrically kindled in the amygdaloid complex. Changes in afterdischarge duration (ADD) as well as the expression and progression of behavioral seizures to fully generalized tonic-clonic convulsions (stage 5) were assessed. METHODS: Fifty rats first underwent bilateral electrolytic lesions of either the dentate, fastigial, or interpositus nuclei. After a 7-day recovery period, they were kindled daily until they manifested two stage 5 convulsions. Careful histological examination was used to determine lesion extent. RESULTS: When the dentate or fastigial nucleus was completely destroyed on the side contralateral to the stimulated amygdala, fewer stimulations were required to produce stage 5 seizures and latencies to the expression of forelimb clonus were shorter, as were ADD. On the other hand, when the dentate or fastigial nucleus was only partly obliterated on the contralateral side, more stimulations were required to produce stage 5 seizures and ADD was longer. Neither complete nor partial lesions of the interpositus nuclei had any effect on the number of stimulations to reach a stage 5 seizure, latency to the expression of clonus, or ADD. CONCLUSIONS: Our findings suggest that the dentate and fastigial nuclei, but not the interpositus nuclei, may normally retard epileptogenesis and inhibit clonic behaviors, but paradoxically may facilitate ADD.

Post-activation potentiation in the neocortex of awake freely moving rats.

The search for the cellular processes that underlie information storage within neuronal systems lead to the development of two models of post-activation potentiation, long-term potentiation (LTP) and kindling. Both models give rise to a long-lasting increase in synaptic strength and altered unit discharge patterns. The present paper reviews synaptic plasticity in the neocortex of awake freely moving rats following both single and multiple sessions of activation with high-frequency, tetanic electrical stimulation. The phenomenology of neocortical post-activation potentiation and some possible underlying mechanisms are discussed. We speculate that the functional significance of potentiated responses may reflect a reorganization of the neocortex in a manner similar to those that create and define receptive fields.

Individual differences in radial maze performance and locomotor activity in the meadow vole, Microtus pennsylvanicus.

Individual differences in the radial maze performance and locomotor activity of wild-caught and first-generation laboratory-born meadow voles are described. Based on their patterns of response in an eight-arm radial maze the essentially wild voles fell into three behavioral categories: 1) strict algorithmic (i.e., they systematically chose the next adjacent arm to their previous choice); 2) nonalgorithmic (i.e., they ran the maze without any consistent or definable pattern); and 3) nonrunners (i.e., nonperformers of the task who remained relatively immobile in the arms of the maze). The algorithmic and nonalgorithmic voles further differed in their responses to an interference manipulation of the radial maze task. Algorithmic individuals displayed a marked performance deficit, while the nonalgorithmic individuals showed minimal disruption to a 1-min delay interruption of the maze task. Measurements of several aspects of locomotor activity using the automated Digiscan activity monitoring system revealed that the algorithmic individuals also displayed significantly greater levels of activity than the nonalgorithmic or nonrunners, with no significant difference in activity between the latter two groups. These findings suggest that the algorithmic voles were relatively inflexible in their behavior, while the nonalgorithmic individuals were more flexible in their maze performance and likely in their use of spatial and nonspatial information. These individual differences in laboratory measures of learning behavior and locomotor activity in meadow voles are consistent with the polymorphism that is proposed to occur in the wild.

Use of fluoxetine for the treatment of stereotypical pacing behavior in a captive polar bear.

Stereotypical behaviors are common in captive animals, particularly captive polar bears. Effects of oral administration of fluoxetine on chronic stereotypical and typical behaviors in a captive polar bear were monitored. Fluoxetine treatment terminated stereotypic pacing behavior, facial tic, and huffing/coughing activity. The expression of typical polar bear behaviors was not affected by fluoxetine treatment, although the proportion of time spent engaged in typical behaviors changed during the course of the observation period. Response of the bear to fluoxetine treatment indicated that pharmacologic manipulation of the serotonergic system can safely eliminate stereotypical behaviors in captive polar bears.

Arrest of seizure progression during electrical kindling in guinea-pigs with prior pentylenetetrazol-induced convulsions.

A number of comparative differences in the kindling phenomenon have been observed between guinea-pigs and rats. These differences likely reflect different mechanisms underlying brain plasticity. In this study, guinea-pigs were used to examine the kindling transfer phenomenon between peripheral pentylenetetrazol injection and electrical kindling of the amygdala. The changes in afterdischarge characteristics and behavioural seizures during electrical kindling were compared between animals that had experienced three PTZ-induced convulsions and PTZ-naive controls. We report that on the first electrical kindling session the PTZ-convulsed guinea-pigs displayed lower AD thresholds, enhanced AD durations and seizures, but that their seizures did not progress with repeated daily kindling stimulation.