Chronic epileptic foci in monkeys: correlation between seizure frequency and proportion of pacemaker epileptic neurons.

A total of 1,802 neurons from 15 alert, undrugged Macaca mulatta monkeys were studied. Thirteen monkeys had chronic epilepsy induced by subpial alumina injections in precentral cortex. Precentral neurons were judged epileptic by the magnitude and variability of the percentage of interspike intervals less than 5 msec during periods when the monkeys were awake. This method of quantifying epileptic single neuron activity appears highly reliable in distinguishing epileptic neurons from precentral neurons in either normal cortex, cortex contralateral to, or within the focus. For the 13 epileptic monkeys, the relative proportion of strongly epileptic neurons found within foci was logarithmically correlated with the mean number of daily seizures. Because of the similarity between the physiology of the alumina focus in monkeys and epileptic foci in humans, these data imply that the severity of focal human epilepsy is a function of epileptic neuronal mass.     

Antidromic and orthodromic activation of epileptic neurons in neocortex of awake monkey

One hundred forty normal and epileptic precentral neurons recorded from two awake monkeys with chronic experimental (alumina gel) epileptic foci were orthodromically and antidromically activated by thalamic and pyramidal tract stimulation. The response of normal neurons to single stimuli from either site was a single action potential, whereas epileptic neurons responded with a burst. Epileptic neurons firing in long-first-interval bursts responded antidromically to pyramidal tract stimulation with a long-first-interval burst, and orthodromically to thalamic stimulation with a burst whose timing coincided with the afterburst of the long-first-interval burst. Repetitive thalamic stimulation at critical frequencies of 4–5 Hz were particularly effective in evoking synchronized burst activity.     

A decrease in the number of GABAergic somata is associated with the preferential loss of GABAergic terminals at epileptic foci

Previous studies have indicated that a loss of GABAergic terminals occurs at epileptic foci. The present study was undertaken to investigate if this loss is associated with a loss of GABAergic neuronal somata. Seven juvenile monkeys (M. mulatta) received alumina gel injections to the pre-central gyrus of the left cerebral hemisphere to produce epileptic foci. Four of these monkeys were chosen for further quantitative study. One was sacrificed prior to seizure onset ('pre-seizure'), one had seizures for 3 days ('acute'), and two had a seizure record of one month ('chronic'). Sections of tissue from the epileptic cortex and from the contralateral, non-epileptic cortex were processed for glutamate decarboxylase (GAD) immunocytochemistry at the light microscopic level. Quantitative analysis revealed that a loss of GAD-positive neuronal somata ranging from 24 to 52% occurred at epileptic foci for all monkeys. This decrease was significant (P less than 0.01) for the two chronic monkeys. There was also a slight decrease in GAD-positive neurons 1 cm distal to the focus ('parafocus') in the chronic monkeys, but not in the acute or pre-seizure animals. In addition, small GAD-positive somata (50-150 micron2) were more severely decreased in number at epileptic foci than larger ones (200-250 micron2). As an experimental control, an additional monkey was given a surgical lesion in area 4 of one cerebral hemisphere. It did not display seizure activity prior to sacrifice and did not show a loss of GAD-positive neurons proximal to the control lesions. The results of this study indicate that a loss of GABAergic neuronal somata is associated with a loss of GABAergic terminals at epileptic foci, and that this loss may be more specific for the small GABAergic neurons.     

Effects of Chronic Epileptic Foci on Control of Pyramidal Tract Neurons in Monkeys

Five Macaca mulatta monkeys were operantly conditioned to control the firing patterns of single precentral pyramidal tract neurons. The accuracy with which the monkeys could control normal PTNs from within the focus was significantly poorer than PTNs from contralateral, homotopic cortex. In comparison to nonepileptic monkeys, there was no significant difference in the accuracy with which PTNs from cortex contralateral to interictal foci were controlled. By contrast, comparison of the time necessary to gain accurate control over individual PTNs from contralateral cortex showed the epileptic monkeys to be significantly encumbered when compared to nonepileptic monkeys. These data suggest that interictal foci produce "noise" in remote regions of brain that are involved in an operant task requiring a high degree of discrimination.     

Dendritic morphology, local circuitry, and intrinsic electrophysiology of principal neurons in the entorhinal cortex of macaque monkeys

Little is known about the neuroanatomical or electrophysiological properties of individual neurons in the primate entorhinal cortex. We have used intracellular recording and biocytin-labeling techniques in the entorhinal slice preparation from macaque monkeys to investigate the morphology and intrinsic electrophysiology of principal neurons. These neurons have previously been studied most extensively in rats. In monkeys, layer II neurons are usually stellate cells, as in rats, but they occasionally have a pyramidal shape. They tend to discharge trains, not bursts, of action potentials, and some display subthreshold membrane potential oscillations. Layer III neurons are pyramidal, and they do not appear to display membrane potential oscillations. The distribution of dendrites and of axon collaterals suggests that neurons in layers II and III are interconnected by a network of associational fibers. Layer V and VI neurons are pyramidal and tend to discharge trains of action potentials. The distribution of dendrites and axon collaterals suggests that there is an associative network of principal neurons in layers V and VI, and they also project axon collaterals toward superficial layers. Importantly, entorhinal cortical neurons in monkeys appear to exhibit significant differences from those in rats. Morphologically, neurons in monkey entorhinal layers II and III have more primary dendrites, more dendritic branches, and greater total dendritic length than in rats. Electrophysiologically, layer II neurons in monkeys exhibit less sag, and subthreshold oscillations are less robust and slower. Some monkey layer III neurons discharge bursts of action potentials that are not found in rats. The interspecies differences revealed by this study may influence information processing and pathophysiological processes in the primate entorhinal cortex. J. Comp. Neurol. 470:317-329, 2004.     

Operantly conditioned firing patterns of epileptic neurons in the monkey motor cortex

In an awake rhesus monkey we operantly conditioned the activity of single precentral pyramidal tract cells near a chronic alumina-induced epileptic focus. Units chosen for conditioning fired predominantly in stereotyped high-frequency long-first-interval bursts. Most units also exhibited brief periods of tonic regular firing, typical of normal precentral cells. The proportion of spikes occurring as long-first-interval bursts was determined on the basis of interspike intervals and defined as the epileptic index. Operantly reinforcing transient increases in unit activity with applesauce produced increases in average rates in all nine cells, with no consistent change in the mean epileptic index. Reinforcing transient decreases in firing rate produced a clear decrease in average rate for two cells, no sustained rate changes in six, and an increase in one; the average epileptic index did not change consistently, although transient pauses in cell activity were invariably preceded and followed by long-first-interval bursts. Reinforcing decreases in the epileptic index produced a sustained drop in the number of long-first-interval bursts/min and a concomitant increase in both regular firing and total rate. Reinforcing an increase in epileptic index produced no consistent changes. These results suggest that firing patterns of epileptic cells may be synaptically modified in awake animals. Analysis of reinforced responses suggest that transient increases in synaptic drive generating higher rates may also decrease the proportion of long-first-interval bursts.     

Operant control of precentral neurons: Control of modal interspike intervals

The objects of these expirements were: (a) to determine modal interspike intervals (ISIs) of precentral cells involved in repetitious, gross motor movements; (b) to compare those modal ISIs to the modal ISIs of similar neurons under operant control; and (c) to determine if monkeys could change the modal ISIs of operantly controlled precentral neurons. Data were obtained from 4 monkeys conditioned to produce tonic firing of precentral neurons and one monkey trained to produce repetitious movements of the neck and contralateral limbs. Results are: (a) the modal ISIs from operantly controlled precentral units do not differ significantly from precentral neurons involved in repetitive gross motor movements; and (b) while under operant control, the monkeys cannot modify significantly the modal ISI of the majority of precentral neurons.     

Chronic recording of neurons in epileptogenic foci of monkey during seizures

Activity of single neurons was recorded in the region of chronic alumina cream epileptogenic foci of an awake unanesthetized monkey, using chronically implanted microelectrodes. Continuous neuronal discharge patterns were obtained before, during, and after a number of clinical seizures. The firing patterns of cells from normal cortices of two monkeys were compared with the bursting activity found in the epileptogenic foci. Classification of discharge patterns as being normal or epileptic was made in terms of a burst index (i.e., the percentage of interspike intervals less than 5 msec in a record segment). A cell with a burst index of 10% or less was considered “normal”. Usually after each seizure an “epileptic” cell fired in a “normal” pattern for a short time before returning to a bursting pattern.     

Neuronal Activity in Chronic Ferric Chloride Epileptic Foci in Cats and Monkey

The firing patterns of neurons surrounding sites of ferric chloride (FeCl3) injection were studied in 6 cats and 1 Macaca mulatta monkey. Although a few neurons fired in poorly structured bursts similar to what has been described for some neurons in alumina foci in monkeys, no well-formed bursts were recorded. In addition, EEG spikes were not recorded chronically in two cats, nor were EEG spikes recorded during corticography. We could not confirm the reliability of this preparation in cats as a model of chronic epilepsy.     

Operant control of precentral neurons in monkeys: Evidence against open loop control

Four normal monkeys were operantly conditioned to change the firing pattern of 111 precentral neurons from phasic to tonic using an operant paradigm which quantifies the control of single neurons. Two monkeys then had their contralateral pyramidal tract (PT) sectioned and one monkey had C5-7 ventral rhizotomies. Postlesion data were: (1) contralateral C1-2PT lesions did not encumber the monkeys' control of precentral PTNs: (2) contralateral C5-7 ventral rhizotomies completely abolished accurate control of precentral neurons which received proprioceptive feedback from flaccid arm regions. These results indicate that precentral neurons are operantly controlled through proprioceptive feedback from peripheral mechanoreceptors. The output of the mechanoreceptors is probably dependent upon discrete joint angles and/or muscle tension which is maintained through non-PT pathways. These data do not support the concept that precentral neurons are operantly controlled directly from a central; 'open loop', pathway.     

Neuronal activity in the chronic and acute epileptogenic focus

Activity of single neurons was recorded in the chronic (alumina) epileptogenic focus of unanesthetized monkeys with micropipettes. In some instances, intracellular recordings were obtained. Most of the bursting units had long interspike intervals between the first spikes in the burst. The following part of the burst consisted of a high-frequency train of spikes riding on a small membrane-depolarizing shift in many cases. Some units did not show the long initial intervals and differed in other respects as well. The activity in the chronic focus of the monkey was compared with that in the acute penicillin focus of the cat. A distinct difference between the two types of foci was found in the interspike interval patterns of bursting units. In some instances, movement-related trains of bursts separated by quiet periods, occurred in the the chronic focus. These cyclically occurring groups of bursts had a repetition rate of 25–35 per min. They were regarded as drug-induced (Sernylan). Within the trains, interburst intervals and the number of spikes in each burst were significantly reduced. In one cycling neuron, there was a negative correlation between trains of bursts and paroxysmal discharges in the EEG. The different types of activity in the chronic and acute focus are seen in parallel with some presumed structural differences.     

Operant control of precentral neurons: The role of audio and visual feedback

Three monkeys were operantly conditioned to fire precentral neurons within a 30- to 60-ms interspike interval range. After being trained in this paradigm, the monkeys could demonstrate convincing control of newly encountered neurons in the absence of audio and visual feedback that was coincident with the units' action potentials. These data indirectly support previous findings implying that in this paradigm, subtle peripheral movements are primarily controlled by the monkey rather than by the firing of the precentral unit.     

Glutamate decarboxylase-immunoreactive neurons are preserved in human epileptic hippocampus

The present study was designed to determine whether inhibitory neurons in human epileptic hippocampus are reduced in number, which could reduce inhibition on principal cells and thereby be a basis for seizure susceptibility. We studied the distribution of GABA neurons and puncta by using glutamate decarboxylase (GAD) immunocytochemistry (ICC) together with Nissl stains. Using quantitative comparisons of GAD-immunoreactive (GAD-IR) neurons and puncta in human epileptic hippocampus and in the normal monkey hippocampus, we found that GAD-IR neurons and puncta are relatively unaffected by the hippocampal sclerosis typical of hippocampal epilepsy where 50-90% of principal (non-GAD-IR) cells are lost. GAD-IR neurons and puncta were not significantly decreased compared with normal monkey. In 6 patients, prior in vivo electrophysiology demonstrated that the anterior hippocampus generated all seizures. The anterior and posterior hippocampus were processed simultaneously, and the counts of hippocampal GAD-IR neurons were numerically greater in anterior than in the posterior hippocampus, where no seizures were initiated. These results indicate that GABA neurons are intact in sclerotic and epileptogenic hippocampus. Computerized image analysis of puncta densities in fascia dentata, Ammon's horn, and subicular complex in epileptic hippocampi (n = 7) were not different from puncta densities in the same regions in normal monkey (n = 2). Hence, despite the significant loss of principal cells (50-90% loss) GABA terminals (GAD-IR puncta) were normal, which suggests GABA hyperinnervation of the remnant pyramidal cells and/or dendrites in human epileptic hippocampus. The apparent increase in puncta ranged from 2 (fascia dentata) to 3.3 (CA1) times normal puncta densities. These findings would suggest increased inhibition and less excitability; however, those regions were epileptogenic. We suggest that GABA terminal sprouting or hyperinnervation of the few remnant projection cells may serve to synchronize their membrane potentials so that subsequent excitatory inputs will trigger a larger population of neurons for seizure onset in the hippocampus and propagation out to undamaged regions of subiculum and neocortex.     

Neuronal activity during seizures in monkeys

From six monkeys with chronic alumina gel-induced epileptogenic foci a total of 44 neurons was recorded extracellularly before, during, and after spontaneous seizures. In all cases the unit activity was recorded when the monkeys were undrugged and awake. In six experiments two units were recorded simultaneously through the same electrode. Neurons near the periphery of the focus often ceased firing at the initiation of the ictal event, whereas units (both normal and epileptic) within the focus synchronized with both multiunit background activity and local field potential spikes just preceding the onset of clinical seizures. (The epileptic neurons appeared to synchronize before the normal cells.) During the clonic phase and/or generalization of the seizure, units both within and on the periphery of the focus fired in synchrony with local field potential spikes. These data indirectly support two concepts of focal epileptogenesis: (a) that an epileptogenic neuronal aggregate and (b) “pacemaker” epileptic neurons sustain the epileptogenicity of chronic foci.     

Modulation during learning of the responses of neurons in the lateral hypothalamus to the sight of food

Recordings were made from single neurons in the lateral hypothalamus and substantia innominata of the rhesus and squirrel monkey during feeding. A population of these neurons which altered their firing rates while the monkeys looked at food but not at nonfood objects was investigated. Because the responses of these neurons must have been affected by the previous experience of the animals, the activity of the neurons was measured during tasks in which the monkeys learned whether or not objects which they saw were associated with food. During visual discrimination tests these neurons came to respond when the monkey saw one stimulus associated with food (e.g., a black syringe from which the animal was fed glucose), but not when the monkey saw a different stimulus which was not associated with food (e.g., a white syringe from which the animal was offered saline). During extinction tests these units ceased to respond when the monkey saw a visual stimulus such as a peanut if the peanut was repeatedly not given to the monkey to eat. The learning or extinction behavior approximately paralleled the response of the neurons.The findings that the neurons in the lateral hypothalamus and substantia innominata respond when a monkey is shown food only if he is hungry, and as shown here, if as a result of learning the visual stimulus signifies food, provide information on a part of the brain which may be involved in feeding. The findings are consistent with other data which suggest that the responses of these neurons are involved in the autonomic and/or behavioral reactions of the animal to the sight of food.     

A selective decrease in the number of GABAergic somata occurs in pre-seizing monkeys with alumina gel granuloma

Previous studies have shown that a loss of GABAergic neuronal somata is associated with a loss of GABAergic terminals at chronic cortical epileptic foci in monkeys. The present study was undertaken to determine whether GABAergic neuronal loss occurs prior to the onset of clinical seizures in monkeys that were treated with alumina gel but did not display seizures. Seven adolescent (Macaca mulatta) monkeys received alumina gel implants into the left pre- and post-central gyri, specifically centered in hand-face regions of sensorimotor cortex. Three other monkeys were used as controls. Two of these were surgical controls and the third was a normal animal. Three monkeys (pre-seizing) were sacrificed 2-4 weeks after the alumina gel implant but prior to clinically active seizures. Three other monkeys with chronic seizure activity (chronically seizing) were sacrificed 3-6 months after the implant. Tissue sections were taken from an area adjacent to the alumina gel granuloma (focus), from a site distal to it (parafocus) and from the non-epileptic contralateral side. Sections from all monkeys were processed for glutamate decarboxylase (GAD) immunocytochemistry and then examined with a light microscope. In addition, adjacent sections were stained with a Nissl stain and the total number of neurons was counted in these sections. Statistical analysis showed a significant decrease in the number of GAD-positive cells in the pre-seizing and chronic animals. The pre-seizing monkeys showed a significant loss of 23-44% at the focus in contrast to the total number of neurons which did not change significantly. The loss of GAD-positive cells was greater in the chronic animals that showed significant losses at both the focus and parafocus, 42-61% and 15-26%, respectively. It is important to note that the chronic monkeys displayed an 11-61% significant loss of total neurons at the epileptic focus. The surgical control animals showed no seizure activity and no significant loss of total neurons or GAD-positive cells. The main finding of this study indicates that a selective loss of GAD-positive neuronal somata occurs in pre-seizing monkeys with alumina gel implants. This finding is consistent with the previously reported loss of GABAergic terminals in pre-seizing monkeys. Since virtually all monkeys treated with alumina gel develop seizures, the results of this study add further support to the hypothesis that GABA neuronal loss plays a causal role in focal epilepsy.     

Spontaneous Rhythmic Synchronous Activity in Epileptic Human and Normal Monkey Temporal Lobe

Intracellular recordings were obtained from neurons in tissue taken from human epileptic temporal lobe and normal monkey hippocampus. Using the in vitro slice preparation, we confirmed that spontaneous rhythmic synchronous events (SRSEs) were predominantly found in cells of mesial temporal lobe. These synaptic-like events appeared to be mediated by a GABAergic mechanism, since they were blocked by bicuculline. An interneuron-like cell type was found which discharged in a burst pattern in parallel with SRSE occurrence in pyramidal neurons. Burst discharges were graded and excitatory postsynaptic potential (EPSP)-triggered; all-or-none paroxysmal depolarizations were extremely rare. These features of SRSE activity suggest that population synchrony in this tissue is largely dependent on local inhibitory interneuronal circuitry. SRSEs were found in normal monkey hippocampus as well as in mesial tissue from human epileptic temporal lobe. This result indicates that SRSEs are not a direct reflection of tissue epileptogenicity. However, the circuitry underlying SRSEs may be important in the determination of tissue seizure susceptibility, since it provides a substrate for cell synchronization.     

Behavioral control of firing patterns of normal and abnormal neurons in chronic epileptic cortex

One hundred ninety-eight cells in chronic alumina-induced foci of three monkeys were operantly conditioned for increased and decreased rates. One hundred seven cells exhibited entirely normal firing patterns. Of the abnormal cells, two groups were distinguished on the basis of the variability of their burst index (percentage of cell spikes occurring in bursts). Group I cells fired in structured bursts with high, invariant burst indices and could not be successfully bidirectionally conditioned; Group 2 cells had lower and more variable burst indices and were as easily conditioned as normal cells. These observations provide additional evidence that activity of the majority of epileptic cells may be modified synaptically and suggests therapeutic potentials for biofeedback conditioning in epileptic patients.     

Operant control of pyramidal tract neurons: the role of spinal dorsal columns.

A monkey was trained to control the firing patterns of precentral pyramidal tract neurons. The operant task was for the monkey to produce consecutive interspike intervals (ISI) within a requisite range, or target. The mean time off-target (error) is used to quantify the accuracy of control the monkey could assert over each PTN. Following partial destruction of the dorsal funiculi the number of PTNs driven by peripheral stimuli greatly decreased. Those PTNs which remained responsive to peripheral stimuli were as accurately controlled as those tested before column section, whereas, those PTNs unresponsive to peripheral stimuli were significantly less accurately controlled. The conclusion is that the monkey relies heavily upon proprioceptive feedback to operantly control precentral PTNs.