Undercut Cortex Research Articles

Chapter Two - Neocortical Focus: Experimental View

All brain normal or pathological activities occur in one of the states of vigilance: wake, slow-wave sleep, or REM sleep. Neocortical seizures preferentially occur during slow-wave sleep. We provide a description of neuronal behavior and mechanisms mediating such a behavior within neocortex taking place in natural states of vigilance as well as during seizures pointing to similarities and differences exhibited during sleep and seizures. A concept of epileptic focus is described using a model of cortical undercut, because in that model, the borders of the focus are well defined. In this model, as in other models of acquired epilepsy, the main factor altering excitability is deafferentation, which upregulates neuronal excitability that promotes generation of seizures. Periods of disfacilitation recorded during slow-wave sleep further upregulate neuronal excitability. It appears that the state of neurons and neuronal network in the epileptic focus produced by deafferentation are such that seizures cannot be generated there. Instead, seizures always start around the perimeter of the undercut cortex. Therefore, we define these areas as the seizure focus. In this zone, neuronal connectivity and excitability are moderately enhanced, lowering the threshold for seizure generation.

Neocortical Post-Traumatic Epileptogenesis Is Associated with Loss of GABAergic Neurons

The subtle mechanisms of post-traumatic epileptogenesis remain unknown, although the incidence of chronic epilepsy after penetrating cortical wounds is high. Here, we investigated whether the increased frequency of seizures occurring within 6 weeks following partial deafferentation of the suprasylvian gyrus in cats is accompanied with a change in the ratio between the number of excitatory and inhibitory neurons. Immuno-histochemical labeling of all neurons with neuronal-specific nuclear protein (NeuN) antibody, and of the GABAergic inhibitory neurons with either gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD 65&67) antibodies, was performed on sections obtained from control and epileptic animals with chronically deafferented suprasylvian gyrus. Quantification of the labeled neurons was performed in control animals and at 2, 4, and 6 weeks following cortical deafferentation, in the suprasylvian and marginal gyri, both ipsi- and contra-lateral to the cortical trauma. In all epileptic animals, the neuronal loss was circumscribed to the deafferented suprasylvian gyrus. Inhibitory GABAergic neurons were particularly more sensitive to cortical deafferentation than excitatory ones, leading to a progressively increasing ratio between excitation and inhibition towards excitation, potentially explaining the increased propensity to seizures in chronic undercut cortex.

Is Modulation of Cortical Synapses after Brain Trauma Homeostatic? or, since When is Epilepsy Normal?

Synaptic Strength Modulation after Cortical Trauma: A Role in Epileptogenesis. Avramescu S, Timofeev I. J Neurosci. 2008;28(27):6760–6772. Traumatic brain injuries are often followed by abnormal hyperexcitability, leading to acute seizures and epilepsy. Previous studies documented the rewiring capacity of neocortical neurons in response to various cortical and subcortical lesions. However, little information is available on the functional consequences of these anatomical changes after cortical trauma and the adaptation of synaptic connectivity to a decreased input produced by chronic deafferentation. In this study, we recorded intracellular (IC) activities of cortical neurons simultaneously with extracellular (EC) unit activities and field potentials of neighboring cells in cat cortex, after a large transection of the white matter underneath the suprasylvian gyrus, in acute and chronic conditions (at 2, 4, and 6 weeks) in ketamine–xylazine-anesthetized cats. Using EC spikes to compute the spike-triggered averages of IC membrane potential, we found an increased connection probability and efficacy between cortical neurons weeks after cortical trauma. Inhibitory interactions showed no significant changes in the traumatized cortex compared with control. The increased synaptic efficacy was accompanied by enhanced input resistance and intrinsic excitability of cortical neurons, as well as by increased duration of silent network periods. Our electrophysiological data revealed functional consequences of previously reported anatomical changes in the injured cortex. We suggest that homeostatic synaptic plasticity compensating the decreased activity in the undercut cortex leads to an uncontrollable cortical hyperexcitability and seizure generation.

Synaptic Strength Modulation after Cortical Trauma: A Role in Epileptogenesis

Traumatic brain injuries are often followed by abnormal hyperexcitability, leading to acute seizures and epilepsy. Previous studies documented the rewiring capacity of neocortical neurons in response to various cortical and subcortical lesions. However, little information is available on the functional consequences of these anatomical changes after cortical trauma and the adaptation of synaptic connectivity to a decreased input produced by chronic deafferentation. In this study, we recorded intracellular (IC) activities of cortical neurons simultaneously with extracellular (EC) unit activities and field potentials of neighboring cells in cat cortex, after a large transection of the white matter underneath the suprasylvian gyrus, in acute and chronic conditions (at 2, 4, and 6 weeks) in ketamine-xylazine-anesthetized cats. Using EC spikes to compute the spike-triggered averages of IC membrane potential, we found an increased connection probability and efficacy between cortical neurons weeks after cortical trauma. Inhibitory interactions showed no significant changes in the traumatized cortex compared with control. The increased synaptic efficacy was accompanied by enhanced input resistance and intrinsic excitability of cortical neurons, as well as by increased duration of silent network periods. Our electrophysiological data revealed functional consequences of previously reported anatomical changes in the injured cortex. We suggest that homeostatic synaptic plasticity compensating the decreased activity in the undercut cortex leads to an uncontrollable cortical hyperexcitability and seizure generation.

Modulation of epileptiform activity by glutamine and system A transport in a model of post-traumatic epilepsy.

Epileptic activity arises from an imbalance in excitatory and inhibitory synaptic transmission. To determine if alterations in the metabolism of glutamate, the primary excitatory neurotransmitter, might contribute to epilepsy we directly and indirectly modified levels of glutamine, an immediate precursor of synaptically released glutamate, in the rat neocortical undercut model of hyperexcitability and epilepsy. We show that slices from injured cortex take up glutamine more readily than control slices, and an increased expression of the system A transporters SNAT1 and SNAT2 likely underlies this difference. We also examined the effect of exogenous glutamine on evoked and spontaneous activity and found that addition of physiological concentrations of glutamine to perfusate of slices isolated from injured cortex increased the incidence and decreased the refractory period of epileptiform potentials. By contrast, exogenous glutamine increased the amplitude of evoked potentials in normal cortex, but did not induce epileptiform potentials. Addition of physiological concentrations of glutamine to perfusate of slices isolated from injured cortex greatly increased abnormal spontaneous activity in the form of events resembling spreading depression, again while having no effect on slices from normal cortex. Interestingly, similar spreading depression like events were noted in control slices at supraphysiological levels of glutamine. In the undercut cortex addition of methylaminoisobutyric acid (MeAIB), an inhibitor of the system A glutamine transporters attenuated all physiological effects of added glutamine suggesting that uptake through these transporters is required for the effect of glutamine. Our findings support a role for glutamine transport through SNAT1 and/or SNAT2 in the maintenance of abnormal activity in this in vitro model of epileptogenesis and suggest that system A transport and glutamine metabolism are potential targets for pharmacological intervention in seizures and epilepsy.

Chronic Cortical Injury and Epileptogensis: A Possible Role for Intracellular Chloride Homeostasis

Impaired Cl– Extrusion in Layer V Pyramidal Neurons of Chronically Injured Epileptogenic Neocortex Jin X, Huguenard JR, Prince DA J Neurophysiol 2005;93:2117–2126 In the mature brain, the K+/Cl– cotransporter KCC2 is important in maintaining low [Cl–]i, resulting in hyperpolarizing GABA responses. Decreases in KCC2 after neuronal injuries result in increases in [Cl–]i and enhanced neuronal excitability due to depolarizing GABA responses. We used the gramicidin perforated-patch technique to measure ECl(∼ EGABA) in layer V pyramidal neurons in slices of partially isolated sensorimotor cortex of adult rats to explore the potential functional consequence of KCC2 downregulation in chronically injured cortex. EGABA was measured by recording currents evoked with brief GABA puffs at various membrane potentials. No significant difference was found in ECl between neurons in control and undercut animals (–71.2 ± 2.6 and −71.8 ± 2.8 mV, respectively). However, when loaded with Cl– by applying muscimol puffs at 0.2 Hz for 60 seconds, neurons in the undercut cortex had a significantly shorter time constant for the positive shift in ECl during the Cl– loading phase (4.3 ± 0.5 s for control and 2.2 ± 0.4 s for undercut; p < 0.01). The positive shift in ECl 3 seconds after the beginning of Cl– loading was also significantly larger in the undercut group than in the control, indicating that neurons in undercut cortex were less effective in maintaining low [Cl–]i during repetitive activation of GABAA receptors. Application of furosemide eliminated the difference between the control and undercut groups for both of these measures of [Cl–]i regulation. The results suggest an impairment in Cl– extrusion resulting from decreased KCC2 expression that may reduce the strength of GABAergic inhibition and contribute to epileptogenesis.

Impaired Cl−Extrusion in Layer V Pyramidal Neurons of Chronically Injured Epileptogenic Neocortex

In the mature brain, the K(+)/Cl- cotransporter KCC2 is important in maintaining low [Cl-]i, resulting in hyperpolarizing GABA responses. Decreases in KCC2 after neuronal injuries result in increases in [Cl-]i and enhanced neuronal excitability due to depolarizing GABA responses. We used the gramicidin perforated-patch technique to measure E(Cl) ( approximately E(GABA)) in layer V pyramidal neurons in slices of partially isolated sensorimotor cortex of adult rats to explore the potential functional consequence of KCC2 downregulation in chronically injured cortex. E(GABA) was measured by recording currents evoked with brief GABA puffs at various membrane potentials. There was no significant difference in E(Cl) between neurons in control and undercut animals (-71.2 +/- 2.6 and -71.8 +/- 2.8 mV, respectively). However, when loaded with Cl- by applying muscimol puffs at 0.2 Hz for 60 s, neurons in the undercut cortex had a significantly shorter time constant for the positive shift in E(Cl) during the Cl- loading phase (4.3 +/- 0.5 s for control and 2.2 +/- 0.4 s for undercut, P < 0.01). The positive shift in E(Cl) 3 s after the beginning of Cl- loading was also significantly larger in the undercut group than in the control, indicating that neurons in undercut cortex were less effective in maintaining low [Cl-]i during repetitive activation of GABA(A) receptors. Application of furosemide eliminated the difference between the control and undercut groups for both of these measures of [Cl-]i regulation. The results suggest an impairment in Cl- extrusion resulting from decreased KCC2 expression that may reduce the strength of GABAergic inhibition and contribute to epileptogenesis.

Partial cortical deafferentation promotes development of paroxysmal activity.

This study tested the hypothesis that early functional alterations in neuronal synchrony in the partially deafferented cortex may lead to spontaneously occurring electrographic seizures. In vivo experiments with partial deafferentation of cat suprasylvian gyrus after extensive undercut of the white matter were conducted using multi-site EEG, extracellular unit and intracellular recordings. The amplitudes of EEG waves were much higher in the areas surrounding deafferented cortical fields as compared with control and with undercut cortex. In 40% of animals with undercut cortex, paroxysmal activity occurred 2-3 h after the undercut and was initiated in the relatively intact cortex, adjacent to the more disconnected one. The seizures that followed the undercut consisted of spike-wave/polyspike-wave complexes and fast runs, resembling the electrographic patterns of some clinical epileptic syndromes. An increased local synchrony in the relatively intact cortex evolved into paroxysmal activity that ultimately spread to the deafferented cortex. The electrographic seizures were found only in animals that showed a propagation of the slow sleep-like oscillation in control conditions. The increase of long-range synchrony within a given seizure was associated with seizure termination. These results indicate that alterations in neuronal synchrony following neuronal trauma can be a critical factor triggering electrographic seizures.

Hyperexcitability of intact neurons underlies acute development of trauma-related electrographic seizures in cats in vivo.

Cortical trauma can lead to development of electrographic paroxysmal activities. Current views of trauma-induced epileptogenesis suggest that chronic neuronal hyperexcitability and extensive morphological reorganization of the traumatized cortex are required for the generation of electrographic seizures. However, the mechanisms responsible for the initiation of electrographic seizures shortly after cortical injury are poorly understood. Here we show that, in the experimental model of partially deafferented (undercut) cortex, an increase in intrinsic and synaptic excitability of neurons in areas adjacent to the undercut cortex is sufficient for the generation of electrographic paroxysmal activity within few hours after partial cortical deafferentation. Locally increased and spatially restricted neuronal excitability arose from the increased incidence of intrinsically bursting neurons, enhanced intrinsic and synaptic neuronal responsiveness, and slight disinhibition. These mechanisms only operate in neurons located in the vicinity of partially deafferented sites because, after the cortical injury, partially deafferented neurons are mostly silent and hypoexcitable. Our results suggest that trauma-induced electrographic seizures first arise in cortical fields that are closest to the site of injury and such seizures do not require long-term neuronal reorganization.

Acetylcholinesterase-containing neurons of layer VIb in immature neocortex: possible component of an early formed intrinsic cortical circuit.

In this ontogenetic study the neurons of layer VIb of rodent somatosensory cortex have been characterized using acetylcholinesterase (AchE) histochemistry, Golgi, and electron microscopic techniques. Already, at birth, the neurons were found to be AchE-rich. They contain presumed AchE reaction product within their granular endoplasmic reticulum. These cells send fine axons upwards towards the subpial layer where they terminate in a dense, AchE-rich, fiber plexus. In chronically undercut cortex, AchE staining persists in layer VIb neurons and in the subpial fiber plexus. These observations continue to support the view that there is an intrinsic neuronal circuit connecting layer VI with I in immature neocortex. The findings also raise the possibility that the putative circuit is cholinergic.

Choline acetyltransferase and acetylcholinesterase containing projections from the basal forebrain to the amygdaloid complex of the rat

The origin of the cholinergic innervation to the amygdaloid complex was investigated with the use of acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) assay of microdissected nuclei. Visualization of AChE-positive neurones in the ventral forebrain was facilitated by pretreatment of rats with 1.5 mg/kg di-isopropyl phosphofluoridate (DFP). The AChE-positive neurones in the ventral forebrain are distributed in a continuous system from the septum through the lateral preoptic area to the entopeduncular nucleus caudally. Knife cuts or kainic acid injections (1.5 μg/1 μl) placed in the lateral preoptic area resulted in substantial depletion of the AChE and ChAT content of the amygdala nuclei. Kainic acid injections (1.5 μ/1 μl) in the diagonal band area or cuts through the stria terminalis dorsally did not significantly modify the AChE staining or ChAT content of the amygdala (although diagonal band injections partially depleted the hippocampus of ChAT). Knife cuts severing both the so-called ventral pathway and the stria terminalis did not produce significantly greater ChAT depletion in the amygdala than those produced by the knife cuts or kainic acid injections in the lateral preoptic area. Parasagittal knife cuts undercutting the lateral pyriform cortex also failed to modify the AChE of ChAT content of the amygdala, but they depleted the undercut cortex of both ChAT and AChE; AChE-positive material accumulated ventrally and medially to the knife cut. It is suggested that the major source of the cholinergic innervation of the amygdala is the magnocellular AChE-positive neurones in the lateral preoptic area and adjacent regions of the ventral forebrain.

The effects of denervation and stimulation upon synaptic ultrastructure.

Quantitative studies of synaptic ultrastructure were made in the upper layers of cat cerebral cortex. Tissues were from intact cortex and from long-term (chronic) undercut cortex with or without electrical stimulation. The synaptic effects of chronic electrical stimulation of denervated cortex are most readily understood as growth and remodeling of synaptic elements. Associated with chronic stimulation were increases in: symmetric membrane contacts; areas of round and flat vesicle containing terminals; dendritic shaft contacts; and synaptic contact lengths. Even without stimulation there were indications of synaptic plasticity in denervated cortex; compared with intact cortex, synapses having symmetric membrane contacts showed an increase in bouton area and an increase in synaptic contacts on dendritic shafts. These data are consistent with the observations of others in which axonal terminal growth occurred after differentation. But it appears that chronic electrical stimulation in the adult nervous system promotes significantly more plasticity than occurs without stimulation. In a functional sense stimulation in the present experiments produced effective inhibition which did not occur with denervation alone. Thus the plasticity observed with stimulation had both structural and functional components.

Distribution of radioactivity from topically applied [H 3]acetylcholine in relation to seizure

A study was performed to determine if topically applied acetylcholine (ACh) could reach infragranular cortical layers by the time epileptiform activity appeared in intact and bilaterally chronically undercut cat cortex. At various times after bilateral application of neostigmine and [H 3]ACh, the brain was frozen with liquid nitrogen. The cortex was sectioned parallel to the surface, placed in liquid sicntillation vials, and curves of radioactivity estimated as ACh vs. cortical depth constructed. In 2 min activity is found in white matter with maximum amounts of activity in all layers of the cortex reached in 3 to 5 min, the time when seizure begins when it occurs. It is concluded that topically applied ACh is not restricted to supragranular layers and probably acts throughout the cortical depth to induce seizure. Seizure occurred in nine of 24 foci, eight of which were in chronically undercut preparations. With these bilateral foci, seizure began earlier ( x = 4.8 min ) than seizure from unilateral foci ( x = 8.7 min ). However, no difference in depth distribution was found between seizure and nonseizure foci or between intact and chronically undercut cortex. Therefore, depth distribution alone is not sufficient to explain the seizure propensity of chronically undercut cortex.

Altered neuronal excitability accompanying experimental prevention of supersensitivity in undercut cortex

Altered neuronal excitability accompanying experimental prevention of supersensitivity in undercut cortex

Enzyme changes after undercutting of cerebral cortex in the rat

Studies were undertaken to investigate changes in the activity of some neurotransmitter enzymes after deep surgical undercutting of the cortex in rat. The isolated regions were rostrocaudally limited by the bregma and the lambda. Homogenates of the undercut cortex were prepared after 1 wk and assayed for AChE, ChAc, MAO, and GAD. In the operated animals, a marked reduction in AChE activity (45%) was found, a moderated (10%) reduction in MAO and ChAc activities, and only a slight and reversible decrease in the level of GAD (4%).

Acetylcholine epilepsy: Modification of DC shift in chronically undercut cat cortex

1. 1. Cortical depth penetrations were made with DC recording during Ach induced epileptiform activity in a series of cats with undercut suprasylvian gyrus acute to 82 days chronic. 2. 2. This epileptiform acitivity consists of rhythmic oscillations (bursts) superimposed on a sudden DC shift with periods of electrocorticographic “silence” (suppression) in between. 3. 3. Whereas in acutely undercut preparations, the surface DC shift was negative, in the chronic preparations it was positive. 4. 4. Histologica reconstruction of electrode tracts showed that the bursts in acute and chronic preparations, and the DC shift in chronic preparations reversed phase in cortical layers II–III and became maximally negative near layer IV. 5. 5. The appearance of the superficial positive DC shift in the chronics has led to an hypothesis concerning a supragranular inhibitory system in the genesis of Ach induced epileptiform activity. It has not provided further explanation in our analysis for the seizure propensity of chronically undercut cortex. 6. 6. Current density analysis using the second derivative of voltage with respect to depth (− α 2V/ αZ 2) reveals that there is a superficial source and deep sink in both acute and chronic preparations which has a similar laminar depth distribution for both DC shift and burst components. Unit analysis would implicate neuronal depolarization in deeper layers in the genesis of the deep sink for both the DC shift and the bursts. The superficial source may be a passive phenomenon.

The effect of cortical undercutting and long-term electrical stimulation on synaptic acetylcholinesterase

(1) A centrifugal fractionation technique was used to study the acetylcholinesterase (AChE) activity in a number of subcellular membrane fractions of cat cerebral cortex. In two groups of animals a marginal gyrus of each cat was undercut for a long-term preparation (chronic). Cats in one of the groups received daily electrical stimulation over the period when supersensitivity would be developing in the other group. (2) The chronic, partial neuronal isolation of the cerebral cortex by undercutting and without long-term electrical stimulation led to a decrease of both the total AChE activity and the total protein in the subcellular fraction1.0 M, the fraction rich in ‘cholinergic’ synaptic membranes. The specific activity of AChE was decreased in fractions1.0 Mand1.2 M. (3) In parallel studies long-term electrical stimulation of the undercut cortex prevented the decreases in AChE activity and in the protein of the membrane fraction1.0 M and in specific AChE of fractions1.0 Mand1.2 M. (4) Supersensitivity and loss of AChE activity were related when the most supersensitive and the least supersensitive undercut cortices were compared. (5) The decrease in the activity of AChE and the supersensitivity in undercut cortex are likely the consequences of the loss of cholinergic innervation and of synaptic disuse following denervation.

Tubocurarine binding in undercut cerebral cortex and the effect of long-term electrical stimulation

(1) An in vitro technique was used to study the binding capacity of the acetylcholinesterase (AChE)-rich synaptic membrane fraction of cat cerebral cortex for dimethyl-[ 14C]- d-tubocurarine. Observations were made on the effect of undercutting and long-term electrical stimulation of the undercut cortex upon this binding. (2) Undercutting increased the binding of dimethyl-[ 14C]- d-tubocurarine by 63% as compared with the contralateral control cortex. In parallel studies long-term electrical stimulation of undercut cortex appreciably reduced this expected increase. (3) The binding of dimethyl-[ 14C]- d-tubocurarine to the membranes was more directly related to membrane protein levels than to the AChE activity associated with them. (4) Supersensitivity of undercut cerebral cortex is likely the result of deafferentation resulting in synaptic disuse and in a concomitantly increasing sensitivity of a post-synaptic receptor or receptors.

Laminar DC studies of acetylcholine-activated epileptiform discharge in cerebral cortex

Epileptiform paroxysms induced by local application of neostigmine and acetylcholine (ACh) to intact and undercut cerebral cortex of the cat have been studied by simultaneous DC recording of electrical activity from the cortical surface together with a penetrating micro-electrode used to plot the field potentials in depth as well as to obtain extracellular records of associated unit discharge. Each spontaneous paroxysm was initiated by a sudden surface negative DC shift upon which was superimposed regular rhythmic oscillating waves at 8–20/sec. The surface negative DC field potential did not show a phase reversal except deep to the Vth layer in the cortical depth, increased in negativity with a maximum in layer V in vicinity of large pyramidal cells. The rhythmic oscillating waves showed a phase reversal in most superficial cortical layers only approximately 200 μ beneath the surface. Rapid unit discharge of deep pyramidal cells accompanied the negative DC shift in intact cortex and was interrupted by gating or inhibitory action of rhythmic waves. ACh paroxysms induced in acutely undercut cortex were not accompanied by detectable unit discharge, raising a puzzling problem about their mode of generation. It is suggested that neuronal depolarization and possibly glial cells may play a role in the generation of the DC shifts of the ACh paroxysm as shown for direct cortical responses and evoked potentials by Castellucci and Goldring (1970).

A study of pyramidal cell axon collaterals in intact and partially isolated adult cerebral cortex

A quantitative study was made of pyramidal cell axon collaterals in Golgi impregnated adult cat cerebral cortex. Cellular samples were obtained from 3 groups, intact, undercut and long-term electrically stimulated undercut cortex. Undercut tissue had cells with significantly fewer axon collaterals and fewer branches than did intact. Long-term electrically stimulated undercut tissue contained neurons which were not different from intact in these two measures. There was, therefore, a preservation of axonal morphology in stimulated undercut, adult cortex. Results do not support a theory of axonal proliferation to explain supersensitivity in partially isolated mature cortex. The preservation of axonal components and the preservation of dendritic spines, previously reported, indicates the prevention of some degenerative changes in certain cortical neurons brought about by long-term electrical stimulation.