Cordula Nitsch

Effect of Motor and Premotor Cortex Ablation on Concentrations of Amino Acids, Monoamines, and Acetylcholine and on the Ultrastructure in Rat Striatum. A Confirmation of Glutamate as the Specific Cortico‐Striatal Transmitter

Abstract: At 1, 2, and 4 weeks after unilateral premotor and motor cortex ablation in rats, a significant and lasting decrease in glutamate levels in the ipsilateral versus contralateral striatum was observed. A significant corresponding fall in aspartate was seen only after 1 week. In contrast, there was a large increase in the striatal concentrations of lysine, threonine, alanine, and glutamine 1 week after the cortical ablation. This correlates with the extensive glial proliferation in the deafferented ipsilateral striatum. Four weeks after cortical ablation the GABA concentration was significantly increased. There was no decrease in other putative transmitters (dopamine, serotonin, acetyl‐choline, glycine and taurine), nor was a glutamate decrease observed in the hippocampus or in the hypothalamus, which do not receive direct premotor and motor cortical inputs. Both biochemical and morphological evidence for a minor contralateral cortico‐striatal projection was obtained. Correlating with the fall in glutamate, ultrastructural observations indicated the degeneration of two types of striatal synapses, i.e., those of the axo‐spinous type III and of the axo‐dendritic type VII. Frontal cortex ablation clearly affects, in opposite directions, the metabolism of various striatal amino acids but not that of acetylcholine and the monoamine transmitters. The results strongly support the view that glutamate is the transmitter of the cortico‐striatal fibers.

Differential decrease of GABA in the substantia nigra and other discrete regions of the rabbit brain during the preictal period of methoxypyridoxine-induced seizures

It is well known that during generalized seizures induced by antivitamin B6 agents, such as hydrazides, as well as by direct antagonists of pyridoxine, such as desoxypyridoxine, the GABA concentration is reduced in the whole brain2, 23. The same is true in the vicinity of the human epileptic focus z2. However, previous studies have not established if the GABA reduction is the primary cause of the seizure, i.e. whether the decrease in GABA content occurs prior to the onset of the seizure or in the course of the seizure. Comparing the changes taking place during the preictal period of drug induced seizures with those of the ictal period may answer this problem. A thorough study of the preictal alterations necessitates a regional investigation for two reasons. First, the GABA level differs among brain regions 6,16 in this respect the substantia nigra with its high GABA content is very interesting and second, the susceptibility towards seizure discharges differs among brain regions in this respect the hippocampus, one of the most seizure prone regions of the brain 1°,2°, earns special regard. This led us to determine the GABA content in 11 different regions of the rabbit brain during the preictal and ictal period of generalized seizure discharges. For this purpose it is necessary to use a convulsant which induces seizures after a constant latency, so that the moment at which the seizure starts can be predicted. This requirement is fulfilled by the pyridoxine-analog methoxypiridoxine (MP), a convulsant agent described by Purpura and Gonzales-Monteagudo in 195919, which reduces GABA content in cat cortex after systemic application 18. An intravenous administration of 100 mg/kg MP to immobilized rabbits induced generalized seizure discharges starting constantly after 22-28 min as checked by EEG recordings 7. This made it possible to analyze precisely changes occurring during the preictal period. The MP-induced sustained epileptic discharges lasted throughout the experiment for 2 h and longer. To avoid side-effects such as dyspnoea and anoxia, which may also alter the GABA content, all rabbits were artificially respirated and immobili-

Regulation of GABA Metabolism in Discrete Rabbit Brain Regions under Methoxypyridoxine?Regional Differences in Cofactor Saturation and the Preictal Activation of Glutamate Decarboxylase Activity

Abstract: The activities of the enzymes of the GABA system, glutamate decarboxylase (GAD) and GABA‐transaminase, were measured in discrete regions of the rabbit brain before the onset and during the course of sustained epileptiform seizures induced by the vitamin B6, analogue methoxypyridoxine (MP). GAD activities were measured in a reaction mixture alternatively containing the cofactor pyridoxal‐5′‐phosphate (PLP) in excess or containing no PLP (holoenzyme of GAD). A comparison between these two estimations showed that the apoenzyme of GAD is only partially saturated with cofactor and that the degree of saturation varied from brain area to brain area, being highest in cerebellar cortex and lowest in substantia nigra. Holoenzyme activity fell steeply after administration of 100 mg/kg MP. The regional degree of enzyme inhibition by MP was a function of the saturation of the apoenzyme with cofactor; i.e., a low rate of saturation resulted in a high degree of inhibition, and vice versa. That GAD from the regio inferior of the hippocampus did not fit into the scheme (strong inhibition is present although the degree of saturation is high) is discussed in view of the role of the hippocampus in seizure genesis and generalization. Inhibition of GAD activity by MP was completely reversible in vitro by excess PLP. Before the onset of seizures but not during their course, apoenzyme activity surpassed control levels. This preictal activation is significant in regio inferior of hippocampus, in superior colliculus, and in cerebellar cortex. GABA‐transaminase activities were not significantly altered. The present study demonstrates that only investigation during the preictal period and in regional brain areas can reveal changes specific for the drug and perhaps representing the cause for seizure development, without being masked by additional alterations resulting from the severe functional and metabolic derangement during the ictal events. Thereby, it was disclosed that a decrease in vivo in the level of the enzyme product, GABA, is able to activate GAD.

Large dense-core vesicle exocytosis and membrane recycling in the mossy fibre synapses of the rabbit hippocampus during epileptiform seizures

SummaryThe ultrastructure of the hippocampal mossy fibre layer was studied in ultrathin sections and freeze-fracture preparations of rabbits under deep Nembutal anaesthesia, after recovery from ether anaesthesia, and 40 min after a single injection of methoxypyridoxine, that is, during the second generalized seizure discharge. The giant mossy fibre boutons contain two types of vesicles: evenly distributed, small round clear vesicles (50 nm) and a few scattered large dense-core vesicles (100 nm). In rare instances fusion of dense-core vesicles with the presynaptic membrane was observed. No differences in the morphology of the mossy fibre synapses were found between anaesthetized and unanaesthetized animals. During epileptiform seizures, however, the size and shape of clear and dense-core vesicles varied greatly. The active synaptic zones were covered with large, core-containing omega profiles or bumps and indentations. Only dense-core vesicles seem to undergo exocytosis. A fusion of clear vesicles with the presynaptic membrane was not observed.Various explanations for the fact that only dense-core vesicles seem to undergo exocytosis are discussed. The hypothesis is put forward that in the mossy fibre bouton two morphologically and functionally distinct populations of synaptic vesicles exist and that only one of them undergoes visible irreversible exocytosis, whereas the majority, that is, the small vesicles discharge their transmitter by reversible fusion.After MP injection features of membrane retrieval were also prominent. Frequently, at the borders of the active synaptic zones coated membrane convolutes of both pre- and postsynaptic membranes had invaded the terminals as well as the postsynaptic spine. Thus, in contrast to electrical stimulation, the self-sustained seizures allow energy-expensive processes such as extensive membrane internalization to take place during the interictal pauses.

Effect of hippocampus extirpation in the rat on glutamate levels in target structures of hippocampal efferents

Twenty days after complete uni- or bilateral hippocampus extirpation in rats, a 25% decrease in glutamate concentration was observed in the septum. Glutamate content also decreased in other terminal structures of the hippocampo-subicular system, i.e. entorhinal cortex, nucleus accumbens septi, mammillary bodies and contralateral hippocampus. It is concluded that the fall in glutamate content which is absent in caudate nucleus is specific for target regions of the hippocampal efferents, adding further support to the suggested transmitter role of glutamate in the limbic system.

Alterations in the Content of Amino Acid Neurotransmitters Before the Onset and During the Course of Methoxypyridoxine-Induced Seizures in Individual Rabbit Brain Regions

In rabbits, generalized seizures were induced by methoxypyridoxine, and changes in amino acid concentrations of 15 brain regions were investigated before seizure onset and during the course of sustained epileptiform activity. As previously reported, γ‐aminobutyric acid (GABA) concentration decreased preictally in most regions. At the same time, taurine level was elevated in the hypothalamus, thalamus, hippocampus, caudatum, and frontal cortex. After 90 min of seizures, it was significantly decreased in the hypothalamus, periaqueductal grey, substantia nigra, frontal cortex, and cerebellum. Glycine content was reduced preictally only in the substantia nigra; after seizure onset its concentration rose in all brain areas. Glutamate content in the frontal cortex decreased before seizure onset; after 1.5 h of seizures, its concentration in cerebellum, caudatum, and hippocampus was reduced. Aspartate level was decreased in most areas after sustained seizures; in putamen, however, it was elevated. In contrast, glutamine content increased preictally in the superior colliculus and in all brain areas by ˜200% after 90 min of seizures. Alanine and valine content also rose markedly in most brain areas after prolonged seizures, and threonine showed the same tendency. The single brain regions were observed to respond to methoxypyridoxine in highly individualistic ways. For example, the glycine content of the substantia nigra, which is believed to utilize this amino acid as a neurotransmitter, decreased preictally. The potential importance of the superior colliculus in seizure induction is considered in view of the early rise in glutamine level. The antagonistic preictal behavior of taurine and GABA is discussed with respect to synthesis, uptake from the blood, and antiepileptic properties.

Aspartate, glutamate and GABA levels in pallidum, substantia nigra, center median and dorsal raphe nuclei after cylindric lesion of caudate nucleus in cat

Aspartate, glutamate and GABA levels were determined in afferent and efferent projection nuclei of the striatum after unilateral cylindric lesion in the head of the caudate nucleus in cats. Two and four weeks after operation, GABA content was significantly reduced in substantia nigra and pallidum ipsilateral to the lesion. Glutamate (GLU) level was decreased in substantia nigra and pallidum only 4 weeks after lesion, whereas aspartate content in substantia nigra decreased significantly already after 2 weeks. No changes in the contents of these amino acids were detected in the dorsal raphe nucleus, which receives a projection from the caudate, as well as in the center median nucleus, which projects to the striatum. These experiments using longer survival times substantiate the role of GABA in caudato-pallidal and caudatonigral projections. The possibility is discussed that aspartate (ASP) could function as transmitter of cortico-nigral fibers.

Synaptic reorganization in the rabbit hippocampus after lesion of commissural afferents

SummaryThe degeneration of commissural afferents to the hippocampus in the rabbit was studied by using the Fink-Heimer degeneration method, electron microscopy, and the combined Golgi/EM technique. The stratum oriens (CA3) was selected for quantitative electron microscopic evaluation of postlesional changes since the degeneration of commissural fibers as seen in Fink-Heimer preparations was dense throughout the width of that layer. Accordingly, in electron micrographs of stratum oriens many electron-dense degenerating boutons were found after short survival times (3 and 6 days, respectively), most of them (96%) in synaptic contact with dendritic spines. In the fine structural analysis of Golgi-impregnated CA3 pyramidal cells, spines of basal dendrites were identified as postsynaptic elements of degenerating commissural afferents in stratum oriens.Three days after the lesion, the number of intact synapses/unit area was reduced in stratum oriens of CA3 to 64% of the control; 20% of the synapses were degenerating. Thus, part of the degenerated synapses had disappeared. Evidence is provided that phagocytosis of degenerated boutons still attached to fragments of dendritic spines played a role in this process.Seven weeks after the lesion, the number of intact synapses had returned to control level, suggesting reactive growth of synaptic structures. When the ratio of spine synapses versus shaft synapses was compared with controls, no change had occurred. Thus, after an initial loss of spine synapses after short survival times, new spines have been formed in parallel with ingrowth (sprouting) of neighbouring nonlesioned afferents.

Effects of Frontal Motor Cortex Ablation on the Ultrastructure of Cat Substantia Nigra

3,4 and 5 days after the removal by suction of the left motor and premotor cortex in cats, the presence of ultrastructural changes in the substantia nigra was investigated. Whereas after 3 days dark bouton degeneration was rare, after 4- and 5-day survival it was regularly found for a distinct type of synapse containing in its bouton densely packed small round vesicles and possessing an asymmetric synaptic junction with a dendrite. Often these darkly degenerated boutons contained dense bodies which were also observed in the same type of synapse not yet exhibiting a dark axoplasm. Various inclusions, especially glycogen depots, were present in these boutons suggesting that they were in the process of degeneration. The glial reaction was comparatively severe. In addition, darkly shrunken dendrites contacted both, by intact and by altered boutons were frequently encountered as well as single degenerated neuronal perikarya. The nature of this effect, i.e. whether transneuronal or retrograde, could not be clarified. All these alterations were found bilaterally after the unilateral cortex ablation, and were confined to the substantia nigra pars compacta along its whole anterior-posterior extent. In the pars reticulata, solely traversing myelinated axons in the process of degeneration were observed. Thus, the results are in agreement with the older studies and with evidence from primates demonstrating that the substantia nigra receives a bilateral projection from the motor and premotor cortex.

The commissural fibers in rabbit hippocampus: synapses and their transmitter.

Publisher Summary This chapter presents an experiment for a complete unilateral hippocampus extirpation in rabbits. It investigates the degree of commissural deafferentation in the contralateral hippocampus by electron microscopy. Glutamate (Glu) levels were determined in single layers of the hippocampal fields, regio superior, regio inferior, and area dentata, 20 days after the operation and compared to those found in sham-operated controls. After commissural deafferentation, Glu content decreased significantly in stratum oriens, but no decreases were observed in the other layers receiving commissural endings. Additionally, Glu level increased in layers containing the supposed Glu-ergic terminals of the entorhinal perforant path fibers and the Schaffers collaterals. To explain these findings, the chapter proposes that Glu is located in the commissural nerve endings, but that as a result of the sprouting of neighboring Glu-ergic terminals of other origin, Glu content may normalize in some of the denervated zones.