Quantitative autoradiography of [3H]muscimol binding in rat brain.

A simple quantitative autoradiographic technique for the study of neurotransmitter receptors that includes the use of a tritium-sensitive film permits saturation, kinetic, and competition studies of brain samples as small as 0.01 cubic millimeter. This technique was used to study [3H]muscimol binding in rat brain. Unilateral gamma-aminobutyric acid receptor supersensitivity was observed in the substantia nigra pars reticulata after production of localized lesions of the ipsilateral corpus striatum.

Chronic cerebellar stimulation in the monkey. Preliminary observations.

In a single monkey, the surface of the cerebellum was stimulated electrically for 205 hours with electrodes and parameter values similar to those currently used in humans for treatment of epilepsy. Im pedance of stimulating and nonstimulating control electrodes remained unchanged throughout an observation period of six months. Potentials evoked by cerebellar stimulation could be recorded from the cranium, providing a noninvasive technique of determining the level of current delivered to cerebellum. Examination of the implantation site showed marked meningeal thickening surrounding the stimulating electrodes. Such thickening was not observed surrounding a control set. Light and electron microscopical examination revealed severe loss of Purkinje cells in tissue near the stimulating electrodes. There was also a moderate loss in other parts of cerebellar cortex down to a depth of about 1 mm from the exposed surface. Biochemical analysis revealed metabolic abnormalities consistent with the morphologic evidence of widespread tissue damage.

Alteration of Purkinje cell activity from transfolial stimulation of the cerebellum in the cat.

The activity of single Purkinje cells and other single neural units was recorded extracellularly from the cerebellar cortex of anesthetized cats during surface cerebellar stimulation with parameters similar to those used in human beings with epilepsy. At a stimulation rate of 0.5 Hz, most Purkinje cells responded with a simple stimulus-locked suppression of activity, although some showed more complex sequences of suppression and activation. At a stimulus rate of 10 Hz, essentially all responsive Purkinje cells showed a suppression of activity. Most of the other units responded to stimulation in the same manner. These effects were observed immediately adjacent to the stimulating electrodes and as far away as 6.5 mm. Therefore, surface stimulation of the cerebellum with parameters equivalent to those used in epileptic patients suppresses rather than activates Purkinje cells. These findings do not support the hypothesis that Purkinje cell activation accounts for seizure suppression in epileptic patients.

Chronic cerebellar stimulation in the monkey. Electron microscopic and biochemical observations.

The effects of chronic electrical stimulation to the surface of cerebellum in the Macaca mulatta monkey were studied with morphologic and biochemical techniques. There was considerable damage and loss of Purkinje cells in all specimens examined, including an area without electrodes, but the greatest changes appeared in tissue beneath the cathode and anode. Despite the damage, normal appearing synapses persisted in the molecular layer of all specimens. Fibrous glial processes were more numerous beneath the cathode. There were abnormalities in gamma-amino butyric acid (GABA) and polyamine concentrations in virtually all specimens, consistent with the morphologic evidence of widespread tissue damage.

Glutamate or aspartate as a possible neurotransmitter of cerebral corticofugal fibers in the monkey.

We measured high affinity glutamate uptake in subcortical projection sites of monkey (Macaca fascicularis) 8 weeks after unilateral ablation of Brodmann's areas 4 and 6. Uptake decreased ipsilateral to the lesion in ventrolateral nucleus of thalamus, caudate nucleus, and pons. Uptake also decreased contralateral to the lesion in cervical and lumbar spinal cord. These results suggest that L-glutamate (or possibly L-aspartate) is a neurotransmitter of corticofugal fibers in primate.

Effects of radiographic contrast agents on spinal cord physiology.

Segmental reflexes in the spinal cords of cats anesthetized with chloralose were used to evaluate the neurophysiologic effects of radiographic contrast agents. The exposed lumbar spinal cord was bathed with concentrations of ionic and nonionic agents including saline, sodium meglumine diatrizoate, meglumine iothalamate, meglumine iocarmate, and metrizamide. The following responses were evaluated: flexor and extensor monosynaptic reflex; polysynaptic flexion reflexes; spontaneous ventral root activity. Hypertonic solutions generally produced a transient decrease in all reflex activity for up to 1 hour. Isotonic solutions produced no significant changes in the monosynaptic responses, but an increase in amplitude of polysynaptic responses, and increased spontaneous activity. The usual facilitory effects of flexion reflex on the flexor monosynaptic reflex were unchanged, but the expected inhibitory effect of flexion reflex on the extensor monosynaptic reflex was changed to excitatory. The relative ability to produced these effects was sodium meglumine diatrizoate greater than meglumine iothalamate greater than meglumine iocarmate greater than metrizamide.

A densitometer for quantitative autoradiography.

A low cost spot densitometer system is described. This system is useful for quantitative autoradiography of local cerebral glucose utilization, blood flow, receptor binding and other applications requiring densitometry on films. The densitometer can be used alone or interfaced to a microcomputer. The densitometer consists of a photographic enlarger, a digital multimeter, and the densitometer electronics. We have described how to construct, test and use the densitometer and how to interface the densitometer to a microcomputer. The advantages of this system are: (1) the ability to enlarge the image for accurate measurements from 'small' areas; (2) a completely unobscured image during measurement; (3) low cost and (4) ease of use.

Computer-assisted estimates of lesion sizes and shrinkage in denervated areas from receptor autoradiograms using a digitizing tablet.

Area measurements taken from receptor autoradiograms were employed to estimate the size of striatal kainate lesions and the amount of shrinkage in deafferented projection areas. There was no significant difference in the size of substantia nigra (SN) on the denervated side as compared to the intact side one week and one month after unilateral striatal lesions. Although there was no change in the size of globus pallidus (GP) on the lesioned side one week after the lesion, there was a 17% shrinkage one month after the lesion. At 3-4 months after the lesion, the amount of shrinkage was 19% in SN and 16% in GP.

Responses of fastigial single neural units to transfolial electrical stimulation of the cerebellar cortex in the cat.

In anesthetized cats, we recorded the responses of single neural units in the fastigial nucleus to electrical stimulation of the posterior vermal surface of the cerebellar cortex. Stimulus frequencies of 0.3 Hz, 10 Hz, and 200 Hz were used and the responses were similar at each frequency. Two principal responses were found: suppression-activation sequences and suppression followed by return to baseline. The former were found in the caudal portions of the nucleus and the latter along with the majority of nonresponsive units were found in the rostral portions. An area of overlap was found in the central portion of the nucleus.

Long-term surface stimulation of the cerebellum in the monkey. I. Light microscopic, electrophysiologic, and clinical observations.

The effects of chronic implantation of platinum electrodes over the surface of the cerebellum and of stimulation through these electrodes was studied in monkeys. Stimulation parameters were similar to those used in epileptic human beings; two animals received continous stimulation and two received intermittent stimulation in eight-minute on-off cycles. Both stimulating and non-stimulating electrodes became encapsulated in dense reactive fibrous tissue. As determined by light microscopy, continous stimulation resulted in widespread loss or damage to Purkinje cells. Intermittent stimulation led to damage similar in extent to mechanical damage caused by control electrodes. The impedance of the stimulating electrodes was higher after long-term than short-term implnatation and increased progressively in animals stimulated intermittently. There were no clinical neurological signs of cerebellar dysfunction, even in animals with extensive damage.

Long-term surface stimulation of the cerebellum in the monkey. II. Electron microscopic and biochemical observations.

Phase and electron microscopy were used to study the effects of chronic intermittent or continuous stimulation applied to the surface of the cerebellum with bipolar platinum electrodes in monkeys. Damage under the anodes consisted of swelling of neuropil, variable loss of Purkinje cells, and a variable increase in fibrous glial processes. Under the cathode there was marked loss of Purkinje cells, thinning of the molecular layer, moderate swelling, occasional phagocytes with lipofuscin granules, and often an increase of fibrous glial processes. Despite the damage, normal appearing synaptic terminals remained in all layers under both anode and cathode, though sparser in fibrotic areas Gamma-aminobutyric acid (GABA) concentrations were decreased beneath the cathodes, reflecting the loss of Purkinje cells and other GABA-containing cells, and spermidine concentrations were increased in areas with increased fibrous glial processes.

Basal ganglia glucose utilization after recent precentral ablation in the monkey.

In the macaque monkey, unilateral ablation of areas 4 and 6 of Brodmann results initially in a significant decrease of glucose metabolic activity in the ipsilateral caudate nucleus, putamen, globus pallidus, substantia nigra, and subthalamic nucleus. The contralateral hemisphere shows nonsignificant but consistently decreased activity in the caudate nucleus, putamen, and globus pallidus. Cerebral blood flow is decreased in the same pattern as the glucose metabolic activity. The change in glucose metabolic activity results from loss of neurons known to project directly from the cerebral cortex to the basal ganglia and also from indirect effects (diaschisis) in basal ganglia structures that do not receive connections from the cerebral cortex.

Development and evaluation of a new water-soluble iodinated myelographic contrast medium with markedly reduced convulsive effects.

A new water-soluble myelographic contrast agent with more hydrophilic properties than metrizamide was developed and tested in the primate (Macaca mulatta) and the cat. A new animal model that allows study of the convulsive effects of intrathecally administered contrast agents in the awake monkey was designed. With this sensitive model the new contrast agent was shown to have a remarkable reduced convulsive effect when compared with metrizamide and other media. Intrathecal hypertonic solutions in the cat depress the evoked cortico-spinal responses whereas the neurotoxic effects of the iodinated water-soluble contrast media increase them. The net effect is a combination of the two actions.

Effect of isotonic contrast agents on spinal reflexes in the cat.

The effects of isotonic solutions of three contrast agents, administered intrathecally, were studied in cats. A pronounced increase in the electrically evoked cortico-spinal responses and segmental spinal polysynaptic reflexes was recorded after administration of methylglucamine diatrizoate. Injection of methylglucamine iothalamate resulted in an increase of the amplitudes of these reflexes, while metrizamide did not show any consistent effect. The data suggest that water-soluble media have direct effects upon spinal gray matter.

Experimental hemiplegia in the monkey: basal ganglia glucose activity during recovery.

Unilateral ablation of cerebral cortical areas 4 and 6 of Brodmann in the macaque monkey results in a dense contralateral hemiplegia that recovers partially with time. During the phase of dense hemiplegia, the local cerebral metabolic rate for glucose (lCMRGlc) is decreased significantly in the caudate nucleus, putamen, globus pallidus, subthalamic nucleus, substantia nigra, and red nucleus of the hemisphere ipsilateral to the lesion. In the present study, lCMRGlc in the basal ganglia was studied during the phase of partial recovery of motor activity. lCMRGlc was partially restored, and the greatest degree of restoration occurred in structures with direct connections to the cerebral cortex (caudate nucleus, putamen, subthalamic nucleus, substantia nigra, and red nucleus). Restoration was least in structures that do not receive direct connections from the cerebral cortex (the internal and external segments of the globus pallidus). The findings support the hypothesis that corticofugal activity accounts for a substantial degree of functional recovery.

Thalamic, brainstem, and cerebellar glucose metabolism in the hemiplegic monkey.

Unilateral ablation of cerebral cortical areas 4 and 6 of Brodmann in the macaque monkey results in a contralateral hemiplegia that resolves partially with time. During the phase of dense hemiplegia, local cerebral metabolic rate for glucose (1CMRG1c) is decreased significantly in most of the thalamic nuclei ipsilateral to the ablation, and there are slight contralateral decreases. The lCMRGlc is reduced bilaterally in most of the brainstem nuclei and bilaterally in the deep cerebellar nuclei, but only in the contralateral cerebellar cortex. During the phase of partial motor recovery, lCMRGlc is incompletely restored in many of the thalamic nuclei ipsilateral to the ablation and completely restored in the contralateral nuclei. In the brainstem and deep cerebellar nuclei, poor to moderate recovery occurs bilaterally. Moderate recovery occurs in the contralateral cerebellar cortex. The findings demonstrate that a unilateral cerebral cortical lesion strongly affects lCMRGlc in the thalamus ipsilaterally and in the cerebellar cortex contralaterally, but in the brainstem bilaterally. Partial recovery of lCMRGlc accompanies the progressive motor recovery. The structures affected include those with direct, and also those with indirect, connections to the areas ablated.

Quisqualate- and NMDA-sensitive [3H]glutamate binding in primate brain.

Excitatory amino acids (EAA) such as glutamate and aspartate are probably the neurotransmitters of a majority of mammalian neurons. Only a few previous studies have been concerned with the distribution of the subtypes of EAA receptor binding in the primate brain. We examined NMDA- and quisqualate-sensitive [3H]glutamate binding using quantitative autoradiography in monkey brain (Macaca fascicularis). The two types of binding were differentially distributed. NMDA-sensitive binding was most dense in dentate gyrus of hippocampus, stratum pyramidale of hippocampus, and outer layers of cerebral cortex. Quisqualate-sensitive binding was most dense in dentate gyrus of hippocampus, inner and outer layers of cerebral cortex, and molecular layer of cerebellum. In caudate nucleus and putamen, quisqualate- and NMDA-sensitive binding sites were nearly equal in density. However, in globus pallidus, substantia nigra, and subthalamic nucleus, quisqualate-sensitive binding was several-fold greater than NMDA-sensitive binding. In thalamus, [3H]glutamate binding was generally low for both subtypes of binding except for the anterior ventral, lateral dorsal, and pulvinar nuclei. In the brainstem, low levels of binding were found, and strikingly the red nucleus and pons, which are thought to receive glutamatergic projections, had approximately 1/20 the binding observed in cerebral cortex. These results demonstrate that NMDA- and quisqualate-sensitive [3H]glutamate binding are observed in all regions of primate brain, but that in some regions one subtype predominates over the other. In addition, certain areas thought to receive glutamatergic projections have low levels of both types of binding.