Barbiturate-induced glycogen accumulation in brain. An electron microscopic study

BRAIN RESEARCH

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BARBITURATE-INDUCED GLYCOGEN ACCUMULATION IN BRAIN. AN ELECTRON MICROSCOPIC STUDY

C R E I G H T O N H. PHELPS

Department of Anatomy, University of Connecticut Health Center, Farmington, Conn. 06032 (U.S.A.) (Accepted October 20th, 1971)

INTRODUCTION

There is much experimental evidence that barbiturates affect carbohydrate metabolism in the central nervous systemS,l°,2L For example, prolonged anesthesia with sodium phenobarbital causes a transient threefold increase in total brain glycogenZL Similar biochemical investigations have demonstrated regional differences in the accumulation of glycogen during barbiturate anesthesia, with the molecular layer of the cerebellum 1° and molecular layer I of the cerebral cortex s showing higher concentrations than other areas. Recordings of changes in brain electrical activity during barbiturate anesthesia also show regional specificity with a significant depression of activity occurring in the reticular activating system and in the cerebral cortex44. When normal adult mammalian brain is examined by electron microscopy, particulate glycogen is readily identifiable, primarily in astrocytes and only rarely in neurons 43. Conditions of fixation26 and external factors such as temperature changes5°, traumatic injury15, X-irradiation 26, and certain centrally acting drugs such as chlorpromazine21 and reserpine 39 have been shown to greatly influence the amount of particulate glycogen visible in astrocytes. These observations would seem to indicate that changes in the amount of glycogen present in astrocytes may reflect changes in activity of brain tissue in response to external factors. The present study was undertaken to determine the effects of prolonged barbiturate anesthesia on the ultrastructure of the brain. Particular emphasis was placed on identifying the regions of the brain and the specific cell types which showed changes in the appearance of particulate glycogen. The results of this study indicate that during prolonged barbiturate anesthesia the primary site of glycogen accumulation is the cytoplasm of astrocytes in regions of high synaptic density. MATERIALS AND METHODS

Adult C 57 BL/6J mice (25-30 g) were injected (i.p.) with sodium phenobarbital (250 mg/kg) in physiologic saline. Two groups of animals were studied: (1) animals Brain Research, 39 (1972) 225-234

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Fig. I. An astrocyte (A) in the area dentata from a mouse treated with sodium phenobarbital for 6 h ; beta-glycogen particles (G) both singly and in aggregates are present along with bundles of gliofilaments (F) in the astrocytic cytoplasm. A membrane-bound inclusion body also containing glycogen is present (M).

killed as soon as reflexes c o m p l e t e l y d i s a p p e a r e d (30-50 rain following injection) a n d (2) a n i m a l s anesthetized for 6 h. Both g r o u p s o f animals were killed a n d fixed by perfusion t h r o u g h the heart with a mixture o f 0.5~o g l u t a r a l d e h y d e a n d 4 ~ f o r m a l d e h y d e in M i l l o n i g ' s p h o s p h a t e buffer ( p H 7.2) 49. Selected areas o f the b r a i n Brain Research, 39 (1972) 225-234

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Fig. 2. An astrocyte (A) adjacent to a small neuron (N) in the area dentata from a mouse treated with sodium phenobarbital for 6 h. Single and aggregated beta-glycogen particles (G) are scattered throughout the astrocyte cytoplasm but no glycogen is present in the neuron.

Fig. 3. Pericapillary astrocytic processes (P) laden with single and aggregated beta-glycogen particles (G) bordering a capillary (C) in the area dentata from a mouse treated with sodium phenobarbital for 6 h .

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Fig. 4. Synaptic neuropil in the area dentata of the mouse hippocampus treated with sodium phenobarbital for 6 h; glycogen laden astrocytic processes (A) are interspersed among axo-dendritic synapses (S). In the large astrocyte process in the center is a collection of gliofilaments and tubules (F) and a membrane-bound body containing glycogen (M).

including h y p o t h a l a m u s , h i p p o c a m p u s (area dentata), m i d b r a i n reticular f o r m a t i o n , cerebellar, a n d cerebral (frontal) cortex were dissected out a n d small tissue blocks postfixed in 2 ~ OsO4, d e h y d r a t e d in ethanol, a n d e m b e d d e d in E p o n . Thin sections were stained with lead citrate a n d c o n t r o l sections were digested with a l p h a - a m y l a s e . Brain Research, 39 (1972) 225-234

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The sections were examined and photographed with a Philips EM300 electron microscope. RESULTS

All regions of the brain appeared normal by standard criteria a7 in those mice killed 30-50 min following injection of sodium phenobarbital. Individual betaglycogen particles 5 (20-30 nm in diameter) were seen diffusely scattered through the cytoplasm of the astrocytes and their processes. Glycogen was not observed in any other cell type. However, in the mice anesthetized with sodium phenobarbital 6 h before perfusion, particularly large amounts of particulate glycogen were visible in the cytoplasm of astrocytes located in 2 of the 5 areas of the brain examined. By far the greatest concentrations of glycogen appeared in the astrocytes of the area dentata of the hippocampus and of the frontal cortex (Figs. 14). In a few animals smaller accumulations of glycogen occurred in the astrocytes of the molecular layer of the cerebellum. The hypothalamus and the midbrain reticular formation appeared to have normal concentrations of glycogen. Glycogen was not observed in neurons, oligodendroglia or microglia. Not all of the astrocytes in the hippocampus and cortex showed increases in glycogen content, but no recognizable pattern of response was apparent. All portions of those astrocytes involved showed increases in particulate glycogen including: the perikaryon (Figs. 1, 2), pericapillary (Fig. 3) and subpial astrocytic endfeet and astrocytic processes scattered through the neuropil (Fig. 4). The response appeared greatest in areas of high synaptic density (Figs. 3, 4) and near neuronal perikarya (Fig. 2). The structural form of the glycogen varied from place to place with some astrocytes having mainly beta-particles (20-30 nm) while others had a mixture of beta-particles and aggregates of beta-particles (alpha or gamma particles) 5 sometimes as large as 0.3/~m in diameter (Figs. 1, 3, 4). These aggregates were rarely seen in normal tissue but were quite common in the phenobarbital treated animals. The aggregates resembled closely in size and conformation those usually seen in the liver 5. Small membrane-bound inclusion bodies containing glycogen and other dense material were often noted (Figs. 1, 4). These bodies resemble autophagic vacuoles and may be related to intracellular breakdown of glycogen. DISCUSSION

Glycogen accumulation in astrocytes has been observed under a number of different circumstances. These include (1) various pathological states such as trauma 14,15,17, X-irradiation26,27, and hereditary ataxia48; (2) hibernation and hypothermia31,41,51; (3) synaptic degeneration24; and (4) administration of depressant drugs including reserpine 39, chlorpromazine21, Haloperido122 and in a preliminary report of the present study, sodium phenobarbital 88. Phenobarbital and the other depressant drugs listed above all cause decreases in neuronal activity. Barbiturates markedly inhibit catecholamine release in certain Brain Research, 39 (1972) 225-234

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neurons of the brain (see discussion in Olson and Fuxe 32) and as pointed out in several biochemical studies 18,29 also stimulate accumulation of glycogen in the brain. The results of electron microscopic studies of brain after reserpine ~9 and our own results in the present study using phenobarbital indicate that glycogen accumulation occurs primarily in astrocytes. Astrocytes have often been mentioned as possible metabolic connecting links between the blood stream and neurons12,13, 23 and to be involved in glucose transport30, 31. If this is correct, glucose transported from the blood could be stored as glycogen in the astrocyte cytoplasm or released to the extracellular space to be available for neuronal metabolism. In periods of low neuronal activity the need for glucose would be lessened and if transport of glucose from the blood to the astrocyte remained steady, most of the glucose would be converted to glycogen and stored in the astrocytic cytoplasm. In order for such a mechanism to work some sort of feedback between neurons and astrocytes would be necessary so that the glucose needs of the neuron could be communicated to the astrocyte. Communication between neurons and neuroglia is known to occur as evidenced by glial responses to neuronal injury 42 and by changes in the membrane potential of glial cells during neuronal activity 33. A possible mechanism for interaction between neurons and neuroglia may be stimulation of astrocytes by neurotransmitters such as norepinephrine which are released into the extracellular space during neuronal activity. Astrocytic processes enclose synaptic areas a4 and sometimes even invaginate the presynaptic axon terminal 45. It is, therefore, highly likely that transmitter substances do come into contact with the membranes of astrocytic processes. There is, as yet, no evidence for or against the presence of transmitter receptor sites on astrocytic membranes*. Catecholamines, epinephrine for example, deplete glycogen in brain tissue 2s and drugs such as the amphetamines, which stimulate norepinephrine release 2, also deplete brain glycogen 7. On the other hand, drugs which decrease norepinephrine activity in brain including reserpine 11, barbiturates a~, and propranolol 6 stimulate glycogen accumulation18, 29,47. Chlorpromazine, which blocks the action of norepinephrine 19, also stimulates glycogen accumulation ls,47. Norepinephrine has been shown to increase adenyl cyclase activity in brain resulting in increased tissue levels of cyclic 3',5'-adenosine monophosphate (cyclic AMP)3,9,19,20,40; cyclic A M P stimulates phosphorylase activity and inhibits glycogen synthetase thus promoting breakdown and reducing synthesis of glycogenS6, 46. I f norepinephrine receptor sites were present in the astrocyte cell membrane, the astrocyte could respond to increased neuronal activity as reflected by the release of norepinephrine into the synaptic cleft or the extracellular space. The norepinephrine could stimulate adenyl cyclase activity in the astrocyte membrane leading to ac-

* Beta-adrenergic receptors have recently been demonstrated in clonal lines of rat and human glial cells (GILMAN,A. G., ANDNIRENBERG,M., Proc. nat. Acad. Sci. (Wash.), 68 (1971) 2165). Norepinephrine and isoproterenol produced striking elevations of intracellular cyclic 3',5'-AMP when added to the culture medium and the response was inhibited when beta-adrenergic blocking agents preceded the catecholamines.

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tivation of phosphorylase, breakdown of glycogen, and release of glucose. At the same time glycogen synthesis would be inhibited and glucose would be available to the neuron. On the other hand, inhibition of neuronal activity, such as caused by depressant drugs, would decrease the amount of norepinephrine released into the extracellular space. Lack of norepinephrine stimulation would bring about a reduction of activity in the adenyl cyclase system leading to glycogen accumulation in the astrocyte cytoplasm. This hypothesis is supported by our own observations and those of others that depressant drugs cause glycogen accumulation in astrocytes21,22, 89, that they depress the activity of catecholaminergic neurons H,25, and that under certain circumstances they either lower brain levels of cyclic AMP 36 or inhibit cyclic AMP formation in response to stimulation 19,a5. In the present study the most profound glycogen increases occurred in astrocytes in regions of high synaptic density in the area dentata of the hippocampus and in the frontal cerebral cortex. Biochemical studies of hippocampus and cerebral cortex have also indicated significant increases in glycogen concentration during barbi'urate anesthesia ~0. It is interesting to note that Laatsch and Cowan z4, in a study ofsynaptic degeneration in the area dentata, described striking accumulations of glycogen in astrocytic processes in areas of high synaptic density. This fits in well with the hypothesis that decreasing neuronal activity (in this case due to degeneration of the axon) leads to glycogen accumulation in astrocytes. Blackstad et al. 1 reported that in the hippocampus the highest concentration of fluorescent norepinephrine terminals occurred in the area dentata. Norepinephrine terminals have also been described in the frontal cerebral cortex 4. Since the electron microscopic techniques used did not allow identification of glycogen and norepinephrine in the same tissue, we were unable to determine in this study if the astrocytes demonstrating increased glycogen were located in the vicinity of norepinephrine terminals. This remains an important problem for future work.

SUMMARY

Glycogen accumulation in the mouse brain during prolonged barbiturate anesthesia (6 h with sodium phenobarbital, 250 mg/kg) was studied with the electron microscope. Areas examined included hypothalamus, hippocampus (area dentata), midbrain reticular formation, cerebellar cortex and frontal cerebral cortex. Large increases of particulate glycogen were noted in astrocytes of the area dentata and the frontal cortex. Smaller increases were sometimes seen in astrocytes of the cerebellar cortex. No changes were noted in the hypothalamus or midbrain reticular formation and glycogen was never seen in neurons, oligodendroglia or microglia. The greatest accumulation of astrocytic glycogen occurred in areas of high synaptic density and near neuronal perikarya. These results suggest that the fluctuations in brain glycogen resulting from barbiturates and other drugs, as described in numerous biochemical and pharmacologBrain Research,

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ical studies, are p r o b a b l y o c c u r r i n g p r i m a r i l y in a s t r o c y t e s a n d t h a t a s t r o c y t i c glyc o g e n levels m a y be i n f l u e n c e d by c h a n g e s in n e u r o n a l activity. ACKNOWLEDGEMENTS I a m i n d e b t e d to Mrs. I r e n e O s t a p i u k a n d M r . R a y m o n d D ' A m a t o f o r t e c h n i c a l assistance d u r i n g t h e c o u r s e o f this study. T h i s w o r k was s u p p o r t e d

by a g r a n t f r o m the U n i v e r s i t y o f C o n n e c t i c u t

Research Foundation.

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