Localized transmeningeal muscimol prevents neocortical seizures in rats and nonhuman primates: therapeutic implications.

PURPOSE: To determine whether muscimol delivered epidurally or into the subarachnoid space can prevent and/or terminate acetylcholine (Ach)-induced focal neocortical seizures at concentrations not affecting behavior and background electroencephalography (EEG) activity. METHODS: Rats (n = 12) and squirrel monkeys (n = 3) were chronically implanted with an epidural or subarachnoid drug delivery device, respectively, over the right frontal/parietal cortex, with adjacent EEG electrodes. Recordings were performed in behaving rats and chaired monkeys. Via the implants, either a control solution (artificial cerebrospinal fluid, ACSF) or muscimol (0.25-12.5 mm) was delivered locally as a "pretreatment," followed by the similar delivery of a seizure-inducing concentration of Ach. In five additional rats, the quantities of food-pellets consumed during epidural ACSF and muscimol (2.5 mm) exposures were measured. In a last group of four rats, muscimol (0.8-2.5 mm) was delivered epidurally during the ongoing, Ach-induced EEG seizure. RESULTS: In contrast to ACSF pretreatments, epidural muscimol pretreatment in rats completely prevented the seizures at and above 2.5 mm. In the monkeys, subarachnoid muscimol pretreatments at 2.5 mm completely prevented the focal-seizure-inducing effect of Ach, whereas similar deliveries of ACSF did not affect the seizures. Furthermore, 2.5 mm epidural muscimol left the eating behavior of rats intact and caused only slight changes in the EEG power spectra. Finally, muscimol delivery during Ach-induced EEG seizures terminated the seizure activity within 1-3 min. CONCLUSIONS: The results of this study suggest that muscimol is a viable candidate for the transmeningeal pharmacotherapy of intractable focal epilepsy.

Histological evidence for drug diffusion across the cerebral meninges into the underlying neocortex in rats.

Transmeningeal pharmacotherapy has been proposed to treat neurological disorders with localized pathology, such as intractable focal epilepsy. As a step toward understanding the diffusion and intracortical spread of transmeningeally delivered drugs, the present study used histological methods to determine the extent to which a marker compound, N-methyl-D-aspartate (NMDA), can diffuse into the neocortex through the meninges. Rats were implanted with bilateral parietal cortical epidural cups filled with 50 mM NMDA on the right side and artificial cerebrospinal fluid (ACSF) in the contralateral side. After 24 h, the histological effects of these treatments were evaluated using cresyl violet (Nissl) staining. The epidural NMDA exposure caused neuronal loss that in most animals extended from the pial surface through layer V. The area indicated by this neuronal loss was localized to the neocortical region underlying the epidural cup. These results suggest that NMDA-like, water soluble, small molecules can diffuse through the subdural/subarachnoid space into the underlying neocortex and spread in a limited fashion, close to the meningeal penetration site.

Spatial memory in nonhuman primates implanted with the subdural pharmacotherapy device.

This study investigated the possible influence of the Subdural Pharmacotherapy Device (SPD) on spatial memory in 3 adult, male bonnet macaques (Macaca radiata). The device was implanted in and above the subdural/subarachnoid space and cranium overlaying the right parietal/frontal cortex: a circuitry involved in spatial memory processing. A large test chamber, equipped with four baited and four non-baited food-ports at different locations, was used: reaches into empty food ports were counted as spatial memory errors. In this study of within-subject design, before SPD implantation (control) the animals made mean 373.3 ± 114.9 (mean ± SEM) errors in the first spatial memory test session. This value dropped to 47.7 ± 18.4 by the 8th session. After SPD implantation and alternating cycles of transmeningeal saline delivery and local cerebrospinal fluid (CSF) drainage in the implanted cortex the spatial memory error count, with the same port locations, was 33.0 ± 12.2 during the first spatial memory test session, further decreasing to 5.7 ± 3.5 by the 8th post-implantation session (P<0.001 for trend). Replacing transmeningeal saline delivery with similar delivery of the GABAA receptor agonist muscimol (1.0mM) by the SPD did not affect the animals' spatial memory performance, which in fact included at least one completely error-free session per animal over time. The study showed that complication-free implantation and use of the SPD over the parietal and frontal cortices for months leave spatial memory processes intact in nonhuman primates.

Spatial memory performance of freely-moving squirrel monkeys.

Few experiments have addressed the problem of cognitive map formation in non-human primates. Therefore, a paradigm was developed to assess spatial memory formation in squirrel monkeys (Saimiri sciureus) moving freely in three dimensions. While moving on the walls and floor of a large test chamber, the animals learned to collect pieces of cereal from baited food-ports interspersed among non-baited ports. The cereal-pellets were not visible to the monkeys, so the animals needed to develop spatial memory to visit only the baited ports for food and avoid the non-baited ones. A session consisted of ten consecutive trials, and 3 successive sessions were conducted on each day for a 5-day period. For each trial, correct choices (CC; number of visited baited-ports) and incorrect choices (IC; number of visited non-baited ports) were registered, and spatial memory performance index (SMPI; ranging from 0.00 to 1.00) was calculated as follows: SMPI=(CC-IC)/CC. For each session, mean SMPI, session duration, total reaches into the non-baited ports, and total reaches into the baited ports were documented. In an 8-port task, where 4 food-ports were baited and 4 were non-baited, the mean SMPI was higher than 0 in the first session (day 1), indicating the development of short-term spatial memory. By the fifth session (day 2), this index was significantly higher than in the first session, indicating the build-up of long-term spatial memory. These changes were related to a significant decrease in the total reaches into the non-baited ports. At the same time, the duration of the sessions and the total reaches into the baited ports did not change significantly. This paradigm can be used for (1) studying cognitive map formation in primates, (2) examining the underlying cellular and molecular mechanisms in integrative neurobiological experiments, and (3) screening cognition-enhancer drugs in a monkey model.

Detecting location-specific neuronal firing rate increases in the hippocampus of freely-moving monkeys.

The spatial properties of the firing of hippocampal neurons have mainly been studied in (a) freely moving rodents, (b) non-human primates seated in a moveable primate chair with head fixed, and (c) epileptic patients subjected to virtual navigation. Although these studies have all revealed the ability of hippocampal neurons to generate spatially selective discharges, the detected firing patterns have been found to be considerably different, even conflicting, in many respects. The present cellular electrophysiological study employed squirrel monkeys (Saimiri sciureus), which moved freely on the walls and floor of a large test chamber. This permitted the examination of the spatial firing of hippocampal neurons in nearly ideal conditions, similar to those used in rodents, yet in a species that belongs to the primate Suborder Anthropoidea. The major findings were that: (1) a group of slow-firing complex-spike cells increased their basal, awake firing rate more than 20-fold, often above 30 spikes/s, when the monkey was in a particular location in the chamber, (2) these location-specific discharges occurred consistently, forming 4-25 s action potential volleys, and (3) fast-firing cells displayed no such electrical activity. Thus, during free movement in three dimensions, primate hippocampal complex-spike cells do generate high-frequency, location-specific action potential volleys. Since these cells are components of the medial temporal lobe memory system, their uncovered firing pattern may well be involved in the formation of declarative memories on places.

The use of a remote-controlled minivalve, carried by freely moving animals on their head, to achieve instant pharmacological effects in intracerebral drug-perfusion studies.

Intracerebral drug-perfusion studies in animals can be very efficiently performed with the 'reverse-dialysis' procedure. In this procedure, drugs are delivered into the brain via an intracerebrally implanted microdialysis probe. Traditionally, in reverse-dialysis studies the flow of control and drug solutions in the microdialysis site is alternated by large and heavy valves placed far from the experimental animal. In this arrangement, the drugs travel from the fluid-alternating device for a long (20--60 min) period before reaching the brain. This can obscure the onset of drug action, makes it difficult to deliver drugs into the extracellular space during short-lasting behavioral episodes, and considerably limits the number of drug solutions that can be perfused within an experimental session. This report describes the use of a miniature (15 mm long and 8 mm diameter), lightweight (1.4 g) minivalve (patent pending) for combined neuronal recording--intracerebral microdialysis studies in freely moving rats. The device is activated remotely and carried by the animals on their head. This allows the experimenter to alternate the control and drug solutions in the intracerebral recording/dialysis site rapidly and to detect the drug-induced neuronal firing pattern changes instantly, without interfering with the animal's behavior. It is demonstrated that with this novel device the onset of drug actions on hippocampal neurons can be clearly defined and that these actions occur within 2 min after minivalve activation. Furthermore, it is demonstrated that the minivalve allows one to test a large number of drug solutions, successively, within the same experimental session. The described protocol offers a high-throughput method for testing the neuron-specific pharmacological effects of intracerebrally perfused drugs during various behaviors.

Autoradiographic evidence for the transmeningeal diffusion of muscimol into the neocortex in rats.

Electrophysiological and behavioral studies have demonstrated that muscimol administered through the cranial meninges can prevent focal neocortical seizures. It was proposed that transmeningeal muscimol delivery can be used for the treatment of intractable focal neocortical epilepsy. However, it has not been proved that muscimol administered via the transmeningeal route can penetrate into the neocortex. The purpose of the present study was to solve this problem by using combined autoradiography-histology methods. Four rats were implanted with epidural cups over the parietal cortices. A 50 µL mixture of [³H] muscimol and unlabeled muscimol with a final concentration of 1.0mM was delivered through each cup on the dura mater. After a 1-hour exposure, the muscimol solution was removed and replaced with formalin to trap the transmeningeally diffused molecules. Then the whole brain was fixed transcardially, sectioned, with the sections subjected to autoradiography and thionine counterstaining. Results showed that (1) [³H] muscimol diffused through the meninges into the cortical tissue underlying the epidural cup in all rats. (2) [³H] muscimol-related autoradiography grains were distributed in all six neocortical layers. (3) [³H] muscimol-related autoradiography grains were localized to the cortical area underneath the epidural delivery site and were absent in the cerebral cortical white matter and other brain structures. This study provided evidence that muscimol can be delivered via the transmeningeal route into the neocortical tissue in a spatially controlled manner. The finding further supports the rationale of using transmeningeal muscimol for the treatment of intractable focal neocortical epilepsy.

Long-term behavioral, electrophysiological, and neurochemical monitoring of the safety of an experimental antiepileptic implant, the muscimol-delivering Subdural Pharmacotherapy Device in monkeys.

OBJECT: The authors evaluated the extent to which the Subdural Pharmacotherapy Device (SPD), chronically implanted over the frontal cortex to perform periodic, localized muscimol-delivery/CSF removal cycles, affects overall behavior, motor performance, electroencephalography (EEG) activity, and blood and CSF neurochemistry in macaque monkeys. METHODS: Two monkeys were used to adjust methodology and 4 monkeys were subjected to comprehensive testing. Prior to surgery, the animals' behavior in a large test chamber was monitored, and the motor skills required to remove food pellets from food ports located on the walls of the chamber were determined. The monkeys underwent implantation of the subdural and extracranial SPD units. The subdural unit, a silicone strip integrating EEG electrodes and fluid-exchange ports, was positioned over the right frontal cortex. The control unit included a battery-powered, microprocessor-regulated dual minipump and radiofrequency module secured to the cranium. After implantation, the SPD automatically performed periodic saline or muscimol (1.0 mM) deliveries at 12-hour intervals, alternating with local CSF removals at 6-hour intervals. The antiepileptic efficacy of this muscimol concentration was verified by demonstrating its ability to prevent focal acetylcholine-induced seizures. During SPD treatment, the monkeys' behavior and motor performance were again monitored, and the power spectrum of their radiofrequency-transmitted EEG recordings was analyzed. Serum and CSF muscimol levels were measured with high-performance liquid chromatography electrochemical detection, and CSF protein levels were measured with turbidimetry. RESULTS: The SPD was well tolerated in all monkeys for up to 11 months. The behavioral study revealed that during both saline and muscimol SPD treatment, the monkeys could achieve the maximum motor performance of 40 food-pellet removals per session, as before surgery. The EEG study showed that local EEG power spectra were not affected by muscimol treatment with SPD. The neurochemical study demonstrated that the administration of 1.0 mM muscimol into the neocortical subarachnoid space led to no detectable levels of this compound in the blood and cisternal CSF, as measured 1-125 minutes after delivery. Total protein levels were within the normal range in the cisternal CSF, but protein levels in the cortical-site CSF were significantly higher than normal: 361 ± 81.6 mg/dl. Abrupt discontinuation of 3-month, periodic, subdural muscimol treatments induced withdrawal seizures, which could be completely prevented by gradually tapering off the subdural muscimol concentration from 1.0 mM to 0.12-0.03 mM over a period of 2 weeks. The monkeys' general health and weight were maintained. Infection occurred only in one monkey 9 months after surgery. CONCLUSIONS: Long-term, periodic, transmeningeal muscimol delivery with the SPD is essentially a safe procedure. If further improved and successfully adapted for use in humans, the SPD can be used for the treatment of intractable focal neocortical epilepsy affecting approximately 150,000 patients in the US.

An implantable triple-function device for local drug delivery, cerebrospinal fluid removal and EEG recording in the cranial subdural/subarachnoid space of primates.

Transmeningeal pharmacotherapy for cerebral cortical disorders requires drug delivery through the subdural/subarachnoid space, ideally with a feedback controlled mechanism. We have developed a device suitable for this function. The first novel component of the apparatus is a silicone rubber strip equipped with (a) fluid-exchange ports for both drug delivery and local cerebrospinal fluid (CSF) removal, and (b) EEG recording electrode contacts. This strip can be positioned between the dura and pia maters. The second novel component is an implantable dual minipump that directs fluid movement to and from the silicone strip and is accessible for refilling and emptying the drug and CSF reservoirs, respectively. This minipump is regulated by a battery-powered microcontroller integrating a bi-directional radiofrequency (RF) communication module. The entire apparatus was implanted in 5 macaque monkeys, with the subdural strip positioned over the frontal cortex and the minipump assembly secured to the cranium under a protective cap. The system was successfully tested for up to 8 months for (1) transmeningeal drug delivery using acetylcholine (ACh) and muscimol as test compounds, (2) RF-transmission of neocortical EEG data to assess the efficacy of drug delivery, and (3) local CSF removal for subsequent diagnostic analyses. The device can be used for (a) monitoring neocortical electrophysiology and neurochemistry in freely behaving nonhuman primates for more than 6 months, (b) determining the neurobiological impact of subdural/subarachnoid drug delivery interfaces, (c) obtaining novel neuropharmacological data on the effects of central nervous system (CNS) drugs, and (d) performing translational studies to develop subdural pharmacotherapy devices.

Periodic transmeningeal muscimol maintains its antiepileptic efficacy over three weeks without inducing tolerance, in rats.

Periodic transmeningeal administration of muscimol into the neocortical epileptogenic zone via a subdurally implanted device has been proposed for the treatment of intractable focal neocortical epilepsy. It is unknown whether such muscimol applications induce tolerance. The purpose of this study was to determine whether daily transmeningeal (epidural) muscimol applications into the rat parietal cortex induce tolerance to the antiepileptic effect of this drug. Rats were chronically implanted with an epidural cup and adjacent epidural EEG electrodes over the right parietal cortex. After recovery 1.0 mM muscimol was delivered into the implanted cortical area through the cup while the animal behaved freely, once per day for 4 consecutive days in each week, with each delivery followed within 3 min by the delivery of a seizure-inducing concentration of acetylcholine (Ach) into the same area. The study lasted for 3 weeks. In each week, one day was used to test the epileptogenicity of the examined cortical site by replacing muscimol with saline prior to Ach delivery. The duration of Ach-induced EEG seizures was measured in each experimental session to assess the antiepileptic efficacy of muscimol, while the rat's behavior was also monitored. The daily epidural muscimol pretreatments prevented Ach-induced EEG and behavioral seizures in all rats. This antiepileptic action did not diminish over time and was maintained throughout the 3-week test period. When muscimol was replaced with saline, the subsequent Ach administrations induced EEG and behavioral seizures. These results suggest that periodic transmeningeal administrations of a relatively low concentration of muscimol into the neocortex over three weeks do not induce tolerance to the localized antiepileptic effects of this drug.

Transmeningeal muscimol can prevent focal EEG seizures in the rat neocortex without stopping multineuronal activity in the treated area.

Muscimol has potent antiepileptic efficacy after transmeningeal administration in animals. However, it is unknown whether this compound stops local neuronal firing at concentrations that prevent seizures. The purpose of this study was to test the hypothesis that epidurally administered muscimol can prevent acetylcholine (Ach)-induced focal seizures in the rat neocortex without causing cessation of multineuronal activity. Rats were chronically implanted with a modified epidural cup over the right frontal cortex, with microelectrodes positioned underneath the cup. In each postsurgical experimental day, either saline or 0.005-, 0.05-, 0.5- or 5.0-mM muscimol was delivered through the cup, followed by a 20-min monitoring of the multineuronal activity and the subsequent delivery of Ach in the same way. Saline and muscimol pretreatment in the concentration range of 0.005-0.05 mM did not prevent EEG seizures. In contrast, 0.5-mM muscimol reduced the average EEG Seizure Duration Ratio value from 0.30±0.04 to 0. At this muscimol concentration, the average baseline multineuronal firing rate of 10.9±4.4 spikes/s did not change significantly throughout the 20-min pretreatment. Muscimol at 5.0mM also prevented seizures, but decreased significantly the baseline multineuronal firing rate of 7.0±1.8 to 3.7±0.9 spikes/s in the last 10 min of pretreatment. These data indicate that transmeningeal muscimol in a submillimolar concentration range can prevent focal neocortical seizures without stopping multineuronal activity in the treated area, and thus this treatment is unlikely to interrupt local physiological functions.

Comparison of the antiepileptic properties of transmeningeally delivered muscimol, lidocaine, midazolam, pentobarbital and GABA, in rats.

This study compared the potencies of epidurally delivered muscimol, lidocaine, midazolam, pentobarbital and gamma-aminobutyric acid (GABA) to prevent focal neocortical seizures induced by locally applied acetylcholine (Ach), in rats (n=5). An epidural cup was chronically implanted over the right somatosensory cortex in each animal, with epidural EEG electrodes placed posterior to the edge of the cup. After recovery, either artificial cerebrospinal fluid (ACSF; control solution) or one of the five drugs was delivered into epidural cup, followed by Ach administration into the cup to induce seizures. EEG seizure duration ratio was calculated for each drug delivery/seizure induction session to determine the potency of ACSF and the drugs to prevent the focal Ach-seizures. The concentration of all examined drug solutions was 1.0mM. ACSF, lidocaine, midazolam, pentobarbital and GABA all failed to prevent the Ach-induced neocortical EEG seizures, yielding EEG seizure duration ratios ranging from 0.41 to 0.80. In contrast, muscimol pretreatment fully prevented the development of ictal EEG in all animals. These results suggest that when used at low concentration muscimol was the best of the five drugs for transmeningeal pharmacotherapy trials for focal neocortical epilepsy.