- Email: [email protected]
Intracellular recording and HRP-staining of cortical neurons in feline ganglioside storage disease
446
Brain Research, 1~;1 i198U)446 449 :i: l£1sevier/North-Hollarld t3 0medical Presii
Intracellular recording and HRP-staining of cortical neurons in feline ganglioside storage disease
D. P. P U R P U R A . S. M. HIGHSTE1N. A. B. KARABELAS and S. U. WALKLE5
Department o f Neuroscience and the Rose F. Kennedy Center/or Research in Mental Retardation and Human Development, Albert Einstehz College o f Medichw, Bronx, N.Y. 10461 USA ~ (Accepted September 13th, 1979~
Key words: gangliosidosis - - HRP-staining cortical neurons
animal models
storage disease -
Inherited disorders of ganglioside metabolism are major causes of unrelenting neurobehavioral deterioration in infancy and childhood. The relation between faulty ganglioside metabolism and neuronal dysfunction has usually been sought in the pathognomonic features of lipid storage disease 2& i.e. the presence of large numbers of abnormal cytosomes in swollen neurons 10. In the gangliosidoses, storage of complex lipids occurs largely in membranous cytoplasmic bodies (MCBs) 11, which progressively increase in number as a consequence of specific lysosomal hydrolase deficiencies. According to traditional views 2,3, MCBs crowd out and eventually compromise cytoplasmic organelles necessary for normal cell metabolism: neuronal dysfunction and cell death ensue. Though eminently logical this cytotoxicity hypothesis of neuronal dysfunction in the gangliosidoses has not been subjected to experimental inquiry. An adequate test would require assessment of the physiological status of MCB-laden neurons from subjects with confirmed gangtiosidosis. While such studies are not possible in human subjects, they are feasible in mutant cats with ganglioside storage disease, Feline GM~-gangliosidosis is of particular interest as an animal model since it replicates human GMl-gangliosidosis in terms of the genetic-biochemical deficiency in /%galactosidase. neuropathological changes and the relative time course and clinical picture of neurobehavioral deterioration 1,4. Recent morphological studies have established that in terminal stages of feline GMt-gangliosidosis cortical neurons exhibit a variety of structural abnormalities and are swollen with massive accumulations of MCBs 7,8. The question posed in the present study is whether neurons containing large aggregates of MCBs exhibit overt abnormalities in basic electrophysiotogical properties. The assumption here is that any toxic action of MCB-accumulation on neuronal function is likely to be reflected in the capacity of neurons to generate normal membrane potentials and spontaneous and evoked spike discharges.
447
•
.
#
~
,
I
;3 % ~O
t
tl
!
I00 ~m Fig. 1. Non-pyramidal neuron of layer VI of precruciate cortex from a 9-month-old mutant cat with advanced G.~l-ganglioside storage disease. The neuron was injected with HRP after a ~eriod of intracellular recording. A: low magnification to show the character and extent of dendritic branches. Higher magnifications (B and C) reveal the distortion of the swollen cell body. Such distortions have never been observed in normal neurons studied with the HRP-injection method utilized here. Note balloon-like bulge of cytoplasm that outlines a compartment with fine granular elements, most likely membranous cytoplasmic bodies (MCBs). A bulbous dilatation is seen in the initial portion of the axon. Magnification bar applies only to B. Feline mutants in advanced stages of neurological deterioration were anesthetized with ketamine and prepared for intracellular recording from pericruciate cortex neurons. Micropipettes filled with 5 ~ horseradish peroxidase (HRP) in 0.5 M KCl.Tris buffer (pH 7.4) were employed for recording and cell marking. H R P injection was achieved by passing positive DC pulses, 10-15 nA, 600 msec duration, frequency I/sec. Concentrically bipolar stimulating electrodes were placed in the medullary pyramidal tract or cerebral peduncle and the ventrolateral thalamic nucleus. After systemic perfusion, whole brain fixation, sectioning and histochemical staining, neurons studied electrophysiologically were reconstructed from serial sections. The major objective sought here was to record from and identify MCB-laden neurons exhibiting prominent structural distortions limited to the cell body. This objective was satisfied in recording from neurons identified as stellate cells or modified star-pyramids. The neuron shown in Fig. 1 was recorded from a 9 month-old mutant cat with advanced GMl-ganglioside storage disease. HRP-injection revealed the cell body to be several times the diameter of normal star-pyramids. HRP-reaction product was clearly demonstrable t h r o u g h o u t the dendritic system which appeared to exhibit normal branching characteristics. The perikaryon was bizarrely swollen in several
448
A
.1//
s' j / !
B
\
/
/
\
\ i/
! 2 mm
\x.J
/
lOOu~
50mY
50 msec Fig. 2. A: location in precruciate cortex of HRP-injected neuron shown in Fig. I. B: camera tucida drawing of neuron reconstructed from serial sections. The main stem axon was traceable to postsigmoid gyrus. C: intraceltular recording during HRP injection. D and E: characteristics of spontaneous burst discharges superimposed on slow depolarizations• ways. A large mass of finely granular cytoplasm appeared to bulge f r o m the cell body. The origins o f several dendrites could be traced to the bulging c o m p a r t m e n t indicating continuity o f the aberrant structural c o m p a r t m e n t with the perikaryon. Since it is well established that swollen neurons in feline GMl-gangliosidosis are packed with M C B s (see ref. 8), the granularity o f the cytoplasm is accounted for by HRP-staining o f t h e m e m b r a n o u s bodies. The location o f the identified n e u r o n m the depths o f the cruciate sulcus and the U-fiber distribution of its axon and axon-collaterals are shown in Fig. 2A and B, respectively,
449 Despite the distorted geometry of the swollen cell body, intracellular recording revealed resting potential and spontaneous discharges similar to those reported for normal nonpyramidal neurons 5. Spontaneous burst discharges with normal interspike intervals were superimposed on spontaneous depolarizations (Fig. 2D and E). Additionally evoked excitatory and inhibitory postsynaptic potentials recorded from nonpyramidal cells in feline mutants exhibited patterns similar to those of normal animals. This preliminary study involving intracellular recording and HRP-staining of ganglioside-laden neurons indicates that the presence of accumulated MCBs sufficient to increase the size and distort the shape of the cell body, does not appreciably alter basic electrophysiological properties of nonpyramidal cells. In effect, the observations do not support the notion that lipid storage material is cytotoxic to neurons. The role which factors such as meganeurite formation6, 9 may play in the onset of neuronal dysfunction in the feline gangliosidoses is currently under investigation. Supported by N a t i o n a l Institutes of Health G r a n t s NS-07512 a n d HD-01799. This study is part of a collaborative project with Dr. H e n r y J. Baker who is supported by G r a n t s NS-10967 a n d T32-RR07003 (to S.U.W.).
1 Baker, H. J., Mole, J. A., Lindsey, J. R. and Creel, R. M., Animal models of human ganglioside storage diseases, Fed. Proc., 35 (1976) 1193 1201. 2 Blackwood, W., Meyer, A., McMenemey, W. H., Normal, R. M. and Russell, D. S., Greenfield's Neuropathology, Williams and Wilkins, Baltimore, 1963. 3 Desnick, R. J., Thorpe, S. R. and Fiddler, M. B., Toward enzyme therapy for lysosomal storage diseases, Physiol. Rev., 56 (1976) 57-59. 4 Farell, D. F., Baker, H. J., Herndon, R. M., Lindsey, J. R. and McKhann, G. M., Feline G~Igangliosidosis: biochemical and ultrastructural comparisons with the disease in man, J. Neuropath. exp. Neurol., 32 (1973) 1-18. 5 Purpura, D. P., lntraeellular studies of synaptic organizations in the mammalian brain. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Function of Synapses, Raven Press, New York, 1972, pp. 257 302. 6 Purpura, D. P., Pathobiology of cortical neurons in metabolic and unclassified amentias. In R. Katzman (Ed.), Congenital and Acquired Cogn#ive Disorders, Raven Press, New York, 1979, PF43-68. 7 Purpura, D. P. and Baker, H. J., Meganeurites and other aberrant processes of neurons in feline G.~tl-gangliosidosis: a Golgi study, Brain Research, 143 (1978) 13-26. 8 Purpura, D. P., Pappas, G. D. and Baker, H. J., Fine structure of meganeurites and secondary growth processes in feline G~l-gangliosidosis, Brain Research, 143 (1978) 1 12. 9 Purpura, D. P. and Suzuki, K., Distortion of neuronal geometry and formation of aberrant synapses in neuronal storage disease, Brain Research, 116 (1976) 1-21. 10 Suzuki, K., Neuronal storage disease: a review. In H. M. Zimmerman (Ed.), Progress in Neuropathology, Vol. 3, Grune and Stratton, New York, 1976, pp. 173-202. 11 Terry, R. D. and Weiss, M., Studies in Tay-Sachs disease. II. Ultrastructure of cerebrum, J. Neuropath. exp. Neurol., 22 (1963) 18 55.
Brain Research, 1~;1 i198U)446 449 :i: l£1sevier/North-Hollarld t3 0medical Presii
Intracellular recording and HRP-staining of cortical neurons in feline ganglioside storage disease
D. P. P U R P U R A . S. M. HIGHSTE1N. A. B. KARABELAS and S. U. WALKLE5
Department o f Neuroscience and the Rose F. Kennedy Center/or Research in Mental Retardation and Human Development, Albert Einstehz College o f Medichw, Bronx, N.Y. 10461 USA ~ (Accepted September 13th, 1979~
Key words: gangliosidosis - - HRP-staining cortical neurons
animal models
storage disease -
Inherited disorders of ganglioside metabolism are major causes of unrelenting neurobehavioral deterioration in infancy and childhood. The relation between faulty ganglioside metabolism and neuronal dysfunction has usually been sought in the pathognomonic features of lipid storage disease 2& i.e. the presence of large numbers of abnormal cytosomes in swollen neurons 10. In the gangliosidoses, storage of complex lipids occurs largely in membranous cytoplasmic bodies (MCBs) 11, which progressively increase in number as a consequence of specific lysosomal hydrolase deficiencies. According to traditional views 2,3, MCBs crowd out and eventually compromise cytoplasmic organelles necessary for normal cell metabolism: neuronal dysfunction and cell death ensue. Though eminently logical this cytotoxicity hypothesis of neuronal dysfunction in the gangliosidoses has not been subjected to experimental inquiry. An adequate test would require assessment of the physiological status of MCB-laden neurons from subjects with confirmed gangtiosidosis. While such studies are not possible in human subjects, they are feasible in mutant cats with ganglioside storage disease, Feline GM~-gangliosidosis is of particular interest as an animal model since it replicates human GMl-gangliosidosis in terms of the genetic-biochemical deficiency in /%galactosidase. neuropathological changes and the relative time course and clinical picture of neurobehavioral deterioration 1,4. Recent morphological studies have established that in terminal stages of feline GMt-gangliosidosis cortical neurons exhibit a variety of structural abnormalities and are swollen with massive accumulations of MCBs 7,8. The question posed in the present study is whether neurons containing large aggregates of MCBs exhibit overt abnormalities in basic electrophysiotogical properties. The assumption here is that any toxic action of MCB-accumulation on neuronal function is likely to be reflected in the capacity of neurons to generate normal membrane potentials and spontaneous and evoked spike discharges.
447
•
.
#
~
,
I
;3 % ~O
t
tl
!
I00 ~m Fig. 1. Non-pyramidal neuron of layer VI of precruciate cortex from a 9-month-old mutant cat with advanced G.~l-ganglioside storage disease. The neuron was injected with HRP after a ~eriod of intracellular recording. A: low magnification to show the character and extent of dendritic branches. Higher magnifications (B and C) reveal the distortion of the swollen cell body. Such distortions have never been observed in normal neurons studied with the HRP-injection method utilized here. Note balloon-like bulge of cytoplasm that outlines a compartment with fine granular elements, most likely membranous cytoplasmic bodies (MCBs). A bulbous dilatation is seen in the initial portion of the axon. Magnification bar applies only to B. Feline mutants in advanced stages of neurological deterioration were anesthetized with ketamine and prepared for intracellular recording from pericruciate cortex neurons. Micropipettes filled with 5 ~ horseradish peroxidase (HRP) in 0.5 M KCl.Tris buffer (pH 7.4) were employed for recording and cell marking. H R P injection was achieved by passing positive DC pulses, 10-15 nA, 600 msec duration, frequency I/sec. Concentrically bipolar stimulating electrodes were placed in the medullary pyramidal tract or cerebral peduncle and the ventrolateral thalamic nucleus. After systemic perfusion, whole brain fixation, sectioning and histochemical staining, neurons studied electrophysiologically were reconstructed from serial sections. The major objective sought here was to record from and identify MCB-laden neurons exhibiting prominent structural distortions limited to the cell body. This objective was satisfied in recording from neurons identified as stellate cells or modified star-pyramids. The neuron shown in Fig. 1 was recorded from a 9 month-old mutant cat with advanced GMl-ganglioside storage disease. HRP-injection revealed the cell body to be several times the diameter of normal star-pyramids. HRP-reaction product was clearly demonstrable t h r o u g h o u t the dendritic system which appeared to exhibit normal branching characteristics. The perikaryon was bizarrely swollen in several
448
A
.1//
s' j / !
B
\
/
/
\
\ i/
! 2 mm
\x.J
/
lOOu~
50mY
50 msec Fig. 2. A: location in precruciate cortex of HRP-injected neuron shown in Fig. I. B: camera tucida drawing of neuron reconstructed from serial sections. The main stem axon was traceable to postsigmoid gyrus. C: intraceltular recording during HRP injection. D and E: characteristics of spontaneous burst discharges superimposed on slow depolarizations• ways. A large mass of finely granular cytoplasm appeared to bulge f r o m the cell body. The origins o f several dendrites could be traced to the bulging c o m p a r t m e n t indicating continuity o f the aberrant structural c o m p a r t m e n t with the perikaryon. Since it is well established that swollen neurons in feline GMl-gangliosidosis are packed with M C B s (see ref. 8), the granularity o f the cytoplasm is accounted for by HRP-staining o f t h e m e m b r a n o u s bodies. The location o f the identified n e u r o n m the depths o f the cruciate sulcus and the U-fiber distribution of its axon and axon-collaterals are shown in Fig. 2A and B, respectively,
449 Despite the distorted geometry of the swollen cell body, intracellular recording revealed resting potential and spontaneous discharges similar to those reported for normal nonpyramidal neurons 5. Spontaneous burst discharges with normal interspike intervals were superimposed on spontaneous depolarizations (Fig. 2D and E). Additionally evoked excitatory and inhibitory postsynaptic potentials recorded from nonpyramidal cells in feline mutants exhibited patterns similar to those of normal animals. This preliminary study involving intracellular recording and HRP-staining of ganglioside-laden neurons indicates that the presence of accumulated MCBs sufficient to increase the size and distort the shape of the cell body, does not appreciably alter basic electrophysiological properties of nonpyramidal cells. In effect, the observations do not support the notion that lipid storage material is cytotoxic to neurons. The role which factors such as meganeurite formation6, 9 may play in the onset of neuronal dysfunction in the feline gangliosidoses is currently under investigation. Supported by N a t i o n a l Institutes of Health G r a n t s NS-07512 a n d HD-01799. This study is part of a collaborative project with Dr. H e n r y J. Baker who is supported by G r a n t s NS-10967 a n d T32-RR07003 (to S.U.W.).
1 Baker, H. J., Mole, J. A., Lindsey, J. R. and Creel, R. M., Animal models of human ganglioside storage diseases, Fed. Proc., 35 (1976) 1193 1201. 2 Blackwood, W., Meyer, A., McMenemey, W. H., Normal, R. M. and Russell, D. S., Greenfield's Neuropathology, Williams and Wilkins, Baltimore, 1963. 3 Desnick, R. J., Thorpe, S. R. and Fiddler, M. B., Toward enzyme therapy for lysosomal storage diseases, Physiol. Rev., 56 (1976) 57-59. 4 Farell, D. F., Baker, H. J., Herndon, R. M., Lindsey, J. R. and McKhann, G. M., Feline G~Igangliosidosis: biochemical and ultrastructural comparisons with the disease in man, J. Neuropath. exp. Neurol., 32 (1973) 1-18. 5 Purpura, D. P., lntraeellular studies of synaptic organizations in the mammalian brain. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Function of Synapses, Raven Press, New York, 1972, pp. 257 302. 6 Purpura, D. P., Pathobiology of cortical neurons in metabolic and unclassified amentias. In R. Katzman (Ed.), Congenital and Acquired Cogn#ive Disorders, Raven Press, New York, 1979, PF43-68. 7 Purpura, D. P. and Baker, H. J., Meganeurites and other aberrant processes of neurons in feline G.~tl-gangliosidosis: a Golgi study, Brain Research, 143 (1978) 13-26. 8 Purpura, D. P., Pappas, G. D. and Baker, H. J., Fine structure of meganeurites and secondary growth processes in feline G~l-gangliosidosis, Brain Research, 143 (1978) 1 12. 9 Purpura, D. P. and Suzuki, K., Distortion of neuronal geometry and formation of aberrant synapses in neuronal storage disease, Brain Research, 116 (1976) 1-21. 10 Suzuki, K., Neuronal storage disease: a review. In H. M. Zimmerman (Ed.), Progress in Neuropathology, Vol. 3, Grune and Stratton, New York, 1976, pp. 173-202. 11 Terry, R. D. and Weiss, M., Studies in Tay-Sachs disease. II. Ultrastructure of cerebrum, J. Neuropath. exp. Neurol., 22 (1963) 18 55.