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Vasoactive intestinal polypeptide (VIP)-containing neurons in the spinal cord of the rat and their projections
5!
N e u m ~ / e n ~ Letters, 37 (1983) 51-55 Elsevier Scientific Publishers Ireland Ltd.
VASOACI2WE INTESTINAL POLYPEPTIDE (VlP)..CONTAINING
ov+
ASh
m
+
KIKUO FUJI, EMIKO SENBA*. YUTAKA UEDA and MASAYA TOHYAMA*
Department of Anesthaiology, Osaka Dental University, 1-47 Kyobashi, Higashiku. Osaka (540) and *Department of Neuroanatomy, Institute of Higher NervousActivity. Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka (530) (Japan) (Received February Ist. 1983; Revised version received and accepted February 28th. 1983)
Key words: vasoactive intestinal polypcptide - immunohistochemistry - spinal cord - lateral spinal nucleus - rat
The distriFdtion of vasoactive intestinal polypeptide (VIP)-Iike immunoreactive structures in the rat ~pinal cora and their projections were investigated by means of an immunofluorescent method. In the ,ormal rat. a small number of VIP-positive fibers were observed in the superficial layer of the dorsal Ptorn and in the lateral funiculus. With colchicine pretreatment, VIP-positive neurons were demonstrated in the lateral spinal nucleus (Isn) and in the lamina X (Rexed). Transections of the spinal cord at vario:ts levels revealed that some of the VIP neurons in the Isn might project to supraspinal areas via I~f.etal funiculus.
Vasoactive intestinal polypeptide (riP) was originally isolated from porcine gastrointestinal tract by Said and Mutt [17]. Subsequent radioimmunoassay and iramunohistochemical studies have revealed the wide spread but uneven distribution of this peptide in the central nervous system [6, 8, 13, 18]. In the spinal cord, this peptide was also detected by radioimmunoassay and immunohistochemistry [7, 9, 10]. These previous studies reported that no VIP neurons were detectable in the spinal cord, though several groups of VIP fibers were seen. It has been believed, therefore, that these fibers may originate from supraspinal VIP neurons or VIP neurons in the dorsal root ganglia (DRG) [7, 10, 14]. in the present study, we report recently demonstrated VIP neurons in the spinal cord and present iramunohistochemical evidence concerning their projections. Male albino rats (100-150 g body weight) were divided into three groups. The first group was used for the immunohistochemical study of the normal distribution of VIP in the spinal cord. The second group of animals was injected with a few microliters of colchicine (7 mg/ml saline) directly into the spinal cord 48 h prior to perfusion. Transection of the spinal cord at various levels was performed off the 0304-3940/83/0000-0000/S 0J.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
~2 third group of animals anesthetized with sodium pentobarbitone (Nembutal, 20 mg/kg, i.p.) and tho" were kept alive for 5-7 days after the operation. Under pentobarbital anesthesia, the rats were perfused through the heart with saline followed by Zamboni's solution [20]. Spinal cords were removed immediately, postfixed overnight, and stored in 0.1 M phosphate buffer containing 30*/0 sucro~ till they sank. VIP was detected using the inditer: immunofluorescent technique of Coons [4] on 15-20 #m frozen sections on gelatin+~:oated slide glasses. Serum raised against porcine VIP (MILAB)was used at a dilution of 1:750, and control experiments were performed on adjacent sections using serum previously absorbed by excess of antigen (100 #g/ml diluted anti-serumL Distribution o f vIP-positive structures, in the spinal cord of the normal rats, ~¢~crai groups of ViP fibers were distributed (Fig. Id): these were located in the ~uperficial layer of the dorsal horn, lamina X and lateral funiculus (Fig. la); ho~vcver no VIP-positiv¢ neurons were detected in these animals. On the other hand, in ~h¢ animals which received colchicinc treatment, several groups of VIP-positi~e neurens were observed in the dorsolaterai part of the lateral funiculus, defined as the lateral spinal nucleus (lsn) by Gwyn and Waldron [12], and in lamina X (Fig. ld). The former group of cells ~erc multipolar and had long dendritic processes, most of which passed obliquely in a medial direction towards the base of the dorsal horn (Fig. I b)+ Most of these neurons were found laterally near the tip of the dorsal hor;t, but some of th~.m ~¢re found medially near the base of the dorsal horn. The latter grotlp of cells ~vere oval with a few relatively short dendrites located near the central canal (Fig. Ic)+ Similar findings were encountered throughout the entire level of the spinal cord. It should be noted that, even in animals pretreated with colchicin¢, no VIP°positive neurons could be detected in the DRG, although Lundberg c! al. [14J reported the existence of such cells in the DRG and VIP fibers in the dorsal horn were supposed to be supplied from the DRG [101. .l::ff~erimeuta! group. Transcctions of the spinal cord at the level of C I - 2 resulted in an accumulation of immunoreactive materials in the axons caudal to the lesion, while no accumulation was observed in the axons rostral to the lesion. Interestingly, most of the VIP accumulating fibers were located in the lateral funiculus and a few of them were observed in the anterior and posterior funiculi. These findings suggest that supraspinal VIP-containing neurons do not project to the spinal cord, but that the spinal VIP system may project to the supraspinal area. Furthermore, though transection of the spinal cord at the level of C I - 2 resulted in demonstrating VIPpositive neurons in the lsn which could not be detected in the animals without col¢hicinc pretrcatment, it failed to demonstrate VIP-positive neurons in the lamina X. When the spinal cord was transected at the level of the lower cervical, thoracic or lumbar cord, a large number of axons which accumulate immunoreactive materials could be seen in the area caudal to the lesion; these were concentrated in the lateral funiculus and scattered also in the anterior and posterior funiculi. On the
53
d Fig. I. a: fluorescent photomicrograph showing VIP-positive fibers in the lateral funiculus of the rat. × ~ . b: fluorescent photomicrograph showing a VIP-positive neuron in the lateral spinal nucleus (Isn) detected in a colchicine treated rat. × 190. c: fluorescent photomicrograph showing VIP-positive neuron~ in the lamina X (Rexed). x 240. d: schematic representation of the coronal section of the rat cervical cord, showing the distribution of VIP-immunoreactive structures. Full circles indicate VIPcontaining ~ell bodies, and dots, fibers and terminals. Abbreviations: DH, dorsal horn; VH, ventral horn; If. lateral funiculus; Isn, lateral spinal nucleus; X, lamina X (Rexed); CC, central canal.
contrary, in the lateral funiculus of the rostral part to the lesion, only a small number of accumJlating fibers could be observc~d. Furthermore, VIP-containing cells were identified in the lsn on both sides of the lesion, showing predominancy in the caudal part to the lesion, while VIP-positive neurons in the lamina X, though they were very few in number, were identified in the rostral par~ to the lesion. No significant change was observed in the distribution of VIP-positive fibers irJ the grey
54
matter of the spinal cord on both sides of the lesion. Although it has been suggestedthat VIP-positive fibers in the spinal cord are supplied by supraspinal VIP-containing neurons or VIP neurons in the DRG, we could
funiculus, whereas VIP cells in the ~ X have short axous which terminate within the spinal cord. The role of the lsn in the rat is still not fully understood, althoush the function of the lateral cervical nucleus in the cat or other species, which is homologous to the lsn in the rat [12], has been investigated by many authors 115, 16] (for review see ref$. 3 and I I). The lateral cervical nucleus is believed to receive cutaneous impulses and its axons ascend the dorsolateral fum'culns to the medial lemniscus finally reaching the thalamus [I, 2, 5, 15]. The VIP-containing neurons in the lsn revealed in the present stud)" possibly have similar functions as the lateral cervical nucleus in the cat or other species. Furthermore, the lsn has been shown to have various kinds of peptid¢-containing structures, such as substance P, ¢nkephalin, neuroteusin and CCK-8 containing nerve fibres, and substance P-positive cell bodies [9, 19]. Not only these newly found VIP-containing neurons in the lsn, but also the peptidecontaining structures in this region, might play an important role in the sensory transmission system.
l Boivic, J., Termination of the cervicoahalamic tract in she cal. An experimental study with silver impregnalion methods. Brain Rest, 19 (1970) 3J3-360. 2 Blomqvist. A.. Rink, R., Bowsher, b., Griph, S. and Westman, J.. Tectal and thaiamic proje~ion~ of dorsal column and lateral cervical nuclei: a quantitative study in the cat, Brain Res., 141 (1978) 3J5-341. 3 Brown. A.G., Ascending and long spinal pathws~ ~- dorsal columns, spinocervical tract and spinothalamic tract. In A. lggo (Ed.). Handbook of Sensor- Physiology. Vol. II, Springer-Verlag, 1973, pp. 3 i 5-.t38. 4 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochcmical Methods, Academic Press, New York, 1958, pp. 399-422. 5 Craig, A,D., Jr, and Burton. H., The lateral cervical nucleus in the cat: Anatomic organization of cervicothalamic neurons, J. comp. Neurol., 185 (1979) 329-346. 6 Eiden, L.E., Nilaver, G. and Palkovits, M., Distribution of vasoactive intestinal polypeptide (VIP) in the rat brain slem nuclei, Brain Res.. 231 (1982) 472-477, 7 Emson, P,C., Gilbert, R.F.T., Loren, !, Fahrenkrug, J., Sundler, F. and Schaffalitzky de Muckadcll, O,B,, Development of vasoactive intestinal polypeptide (VIP) containing neurons in the rat brain, Brain Res., 177 (1979) 437-444. 8 Fuxe, K,, Fi6kfelt, T,, Said, S.I. and Mutt, V., Vasoactiv¢ intestinal polypeptide and the nervous system: immunohistochemical evidence for localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex, Neurosci. Let[.. 5 (1977) 241-246, 9 Gibson, S,J,, Polak, J.M., Bloom, S.R. and Patrick, D., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. comp. Neurol., 201 (1981) 65-79.
55 10 Go, V.L.W. and Yaksh, T.L., Vasoactive intestinal peptide (VIP) and cholecystokinin (CCK-8) in cat spinal cord and dorsal root ganglion: Release from cord by'peripheral nerve stimulation, Regulat. Peptides, Suppl. I (1980) $43. I ! Gordon, G, The concept of relay nuclei. In A. lggo (Ed.), Handbook of Senso~ Physiology, Vol. !!, Springer-Verlag, 1973, pp. 137-150. 12 Gwyn, D.G. and Waldron, H.A., A nucleus in the dorsolaterai funiculus of the spinal cord of the rat, Brain Res., 10(1968) 342-351. 13 Larsson, L.-I., Fahreukrug, J., Schaffalitzky de Muckadell, O., Sundler, F., HAkanson, R. and Rehfeld, J'F., ~ i o n of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. nat. Acad. SCi. U.S.A,, 73 (1976) 3197-3200. 14 Lundberg, J.M., H0kfelt, T., Nilson, G., Terenius, L., Rehfeld, J., Elde, R. and Said, S., Peptide neurons in the vagus, splanchnic and sciatic nerves, Acta physiol, scand., 104 (1978) 499-501. 15 Morin, F. and Catalano, J.F., Central connections of a cervical nucleus (nucleus cervicalis lateralis of the cat), J. comp. Neurol., 103 (1955) 17-32. 16 Rexed, B. and StrOm G., Afferent nervous connections of the lateral cervical nucleus, Acta physiol. scand., 25 (1952) 219-229. 17 Said, S.I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science. 169 (1970) 1217-1218. 18 Sims. K.B., Hoffman, D.L., Said, S.I. and Zimmerman, E.A., Vasoactive intestinal polypeptide (VIP) in mouse and rat brain: An immunocytochemical study, Brain Res., 186 (1980) 165-183. 19 Scnba, E., Shiosaka, S., Hara, Y., Inagaki, S., Sakanaka, M., Takatsuki, K., Kawai, Y. and Tohyama, M., Ontogeny of the peptider~c system in the rat spinal cord: immunohistochemical analysis, J. comp. Neurol., 208 (1982) 54-66. 20 Zamboni, L. and De Martino, C., Buffered picric acid formaldehyde: a new rapid fixative for electron microscopy, J. Cell Biol, 35 (1967) 148A.
N e u m ~ / e n ~ Letters, 37 (1983) 51-55 Elsevier Scientific Publishers Ireland Ltd.
VASOACI2WE INTESTINAL POLYPEPTIDE (VlP)..CONTAINING
ov+
ASh
m
+
KIKUO FUJI, EMIKO SENBA*. YUTAKA UEDA and MASAYA TOHYAMA*
Department of Anesthaiology, Osaka Dental University, 1-47 Kyobashi, Higashiku. Osaka (540) and *Department of Neuroanatomy, Institute of Higher NervousActivity. Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka (530) (Japan) (Received February Ist. 1983; Revised version received and accepted February 28th. 1983)
Key words: vasoactive intestinal polypcptide - immunohistochemistry - spinal cord - lateral spinal nucleus - rat
The distriFdtion of vasoactive intestinal polypeptide (VIP)-Iike immunoreactive structures in the rat ~pinal cora and their projections were investigated by means of an immunofluorescent method. In the ,ormal rat. a small number of VIP-positive fibers were observed in the superficial layer of the dorsal Ptorn and in the lateral funiculus. With colchicine pretreatment, VIP-positive neurons were demonstrated in the lateral spinal nucleus (Isn) and in the lamina X (Rexed). Transections of the spinal cord at vario:ts levels revealed that some of the VIP neurons in the Isn might project to supraspinal areas via I~f.etal funiculus.
Vasoactive intestinal polypeptide (riP) was originally isolated from porcine gastrointestinal tract by Said and Mutt [17]. Subsequent radioimmunoassay and iramunohistochemical studies have revealed the wide spread but uneven distribution of this peptide in the central nervous system [6, 8, 13, 18]. In the spinal cord, this peptide was also detected by radioimmunoassay and immunohistochemistry [7, 9, 10]. These previous studies reported that no VIP neurons were detectable in the spinal cord, though several groups of VIP fibers were seen. It has been believed, therefore, that these fibers may originate from supraspinal VIP neurons or VIP neurons in the dorsal root ganglia (DRG) [7, 10, 14]. in the present study, we report recently demonstrated VIP neurons in the spinal cord and present iramunohistochemical evidence concerning their projections. Male albino rats (100-150 g body weight) were divided into three groups. The first group was used for the immunohistochemical study of the normal distribution of VIP in the spinal cord. The second group of animals was injected with a few microliters of colchicine (7 mg/ml saline) directly into the spinal cord 48 h prior to perfusion. Transection of the spinal cord at various levels was performed off the 0304-3940/83/0000-0000/S 0J.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
~2 third group of animals anesthetized with sodium pentobarbitone (Nembutal, 20 mg/kg, i.p.) and tho" were kept alive for 5-7 days after the operation. Under pentobarbital anesthesia, the rats were perfused through the heart with saline followed by Zamboni's solution [20]. Spinal cords were removed immediately, postfixed overnight, and stored in 0.1 M phosphate buffer containing 30*/0 sucro~ till they sank. VIP was detected using the inditer: immunofluorescent technique of Coons [4] on 15-20 #m frozen sections on gelatin+~:oated slide glasses. Serum raised against porcine VIP (MILAB)was used at a dilution of 1:750, and control experiments were performed on adjacent sections using serum previously absorbed by excess of antigen (100 #g/ml diluted anti-serumL Distribution o f vIP-positive structures, in the spinal cord of the normal rats, ~¢~crai groups of ViP fibers were distributed (Fig. Id): these were located in the ~uperficial layer of the dorsal horn, lamina X and lateral funiculus (Fig. la); ho~vcver no VIP-positiv¢ neurons were detected in these animals. On the other hand, in ~h¢ animals which received colchicinc treatment, several groups of VIP-positi~e neurens were observed in the dorsolaterai part of the lateral funiculus, defined as the lateral spinal nucleus (lsn) by Gwyn and Waldron [12], and in lamina X (Fig. ld). The former group of cells ~erc multipolar and had long dendritic processes, most of which passed obliquely in a medial direction towards the base of the dorsal horn (Fig. I b)+ Most of these neurons were found laterally near the tip of the dorsal hor;t, but some of th~.m ~¢re found medially near the base of the dorsal horn. The latter grotlp of cells ~vere oval with a few relatively short dendrites located near the central canal (Fig. Ic)+ Similar findings were encountered throughout the entire level of the spinal cord. It should be noted that, even in animals pretreated with colchicin¢, no VIP°positive neurons could be detected in the DRG, although Lundberg c! al. [14J reported the existence of such cells in the DRG and VIP fibers in the dorsal horn were supposed to be supplied from the DRG [101. .l::ff~erimeuta! group. Transcctions of the spinal cord at the level of C I - 2 resulted in an accumulation of immunoreactive materials in the axons caudal to the lesion, while no accumulation was observed in the axons rostral to the lesion. Interestingly, most of the VIP accumulating fibers were located in the lateral funiculus and a few of them were observed in the anterior and posterior funiculi. These findings suggest that supraspinal VIP-containing neurons do not project to the spinal cord, but that the spinal VIP system may project to the supraspinal area. Furthermore, though transection of the spinal cord at the level of C I - 2 resulted in demonstrating VIPpositive neurons in the lsn which could not be detected in the animals without col¢hicinc pretrcatment, it failed to demonstrate VIP-positive neurons in the lamina X. When the spinal cord was transected at the level of the lower cervical, thoracic or lumbar cord, a large number of axons which accumulate immunoreactive materials could be seen in the area caudal to the lesion; these were concentrated in the lateral funiculus and scattered also in the anterior and posterior funiculi. On the
53
d Fig. I. a: fluorescent photomicrograph showing VIP-positive fibers in the lateral funiculus of the rat. × ~ . b: fluorescent photomicrograph showing a VIP-positive neuron in the lateral spinal nucleus (Isn) detected in a colchicine treated rat. × 190. c: fluorescent photomicrograph showing VIP-positive neuron~ in the lamina X (Rexed). x 240. d: schematic representation of the coronal section of the rat cervical cord, showing the distribution of VIP-immunoreactive structures. Full circles indicate VIPcontaining ~ell bodies, and dots, fibers and terminals. Abbreviations: DH, dorsal horn; VH, ventral horn; If. lateral funiculus; Isn, lateral spinal nucleus; X, lamina X (Rexed); CC, central canal.
contrary, in the lateral funiculus of the rostral part to the lesion, only a small number of accumJlating fibers could be observc~d. Furthermore, VIP-containing cells were identified in the lsn on both sides of the lesion, showing predominancy in the caudal part to the lesion, while VIP-positive neurons in the lamina X, though they were very few in number, were identified in the rostral par~ to the lesion. No significant change was observed in the distribution of VIP-positive fibers irJ the grey
54
matter of the spinal cord on both sides of the lesion. Although it has been suggestedthat VIP-positive fibers in the spinal cord are supplied by supraspinal VIP-containing neurons or VIP neurons in the DRG, we could
funiculus, whereas VIP cells in the ~ X have short axous which terminate within the spinal cord. The role of the lsn in the rat is still not fully understood, althoush the function of the lateral cervical nucleus in the cat or other species, which is homologous to the lsn in the rat [12], has been investigated by many authors 115, 16] (for review see ref$. 3 and I I). The lateral cervical nucleus is believed to receive cutaneous impulses and its axons ascend the dorsolateral fum'culns to the medial lemniscus finally reaching the thalamus [I, 2, 5, 15]. The VIP-containing neurons in the lsn revealed in the present stud)" possibly have similar functions as the lateral cervical nucleus in the cat or other species. Furthermore, the lsn has been shown to have various kinds of peptid¢-containing structures, such as substance P, ¢nkephalin, neuroteusin and CCK-8 containing nerve fibres, and substance P-positive cell bodies [9, 19]. Not only these newly found VIP-containing neurons in the lsn, but also the peptidecontaining structures in this region, might play an important role in the sensory transmission system.
l Boivic, J., Termination of the cervicoahalamic tract in she cal. An experimental study with silver impregnalion methods. Brain Rest, 19 (1970) 3J3-360. 2 Blomqvist. A.. Rink, R., Bowsher, b., Griph, S. and Westman, J.. Tectal and thaiamic proje~ion~ of dorsal column and lateral cervical nuclei: a quantitative study in the cat, Brain Res., 141 (1978) 3J5-341. 3 Brown. A.G., Ascending and long spinal pathws~ ~- dorsal columns, spinocervical tract and spinothalamic tract. In A. lggo (Ed.). Handbook of Sensor- Physiology. Vol. II, Springer-Verlag, 1973, pp. 3 i 5-.t38. 4 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochcmical Methods, Academic Press, New York, 1958, pp. 399-422. 5 Craig, A,D., Jr, and Burton. H., The lateral cervical nucleus in the cat: Anatomic organization of cervicothalamic neurons, J. comp. Neurol., 185 (1979) 329-346. 6 Eiden, L.E., Nilaver, G. and Palkovits, M., Distribution of vasoactive intestinal polypeptide (VIP) in the rat brain slem nuclei, Brain Res.. 231 (1982) 472-477, 7 Emson, P,C., Gilbert, R.F.T., Loren, !, Fahrenkrug, J., Sundler, F. and Schaffalitzky de Muckadcll, O,B,, Development of vasoactive intestinal polypeptide (VIP) containing neurons in the rat brain, Brain Res., 177 (1979) 437-444. 8 Fuxe, K,, Fi6kfelt, T,, Said, S.I. and Mutt, V., Vasoactiv¢ intestinal polypeptide and the nervous system: immunohistochemical evidence for localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex, Neurosci. Let[.. 5 (1977) 241-246, 9 Gibson, S,J,, Polak, J.M., Bloom, S.R. and Patrick, D., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. comp. Neurol., 201 (1981) 65-79.
55 10 Go, V.L.W. and Yaksh, T.L., Vasoactive intestinal peptide (VIP) and cholecystokinin (CCK-8) in cat spinal cord and dorsal root ganglion: Release from cord by'peripheral nerve stimulation, Regulat. Peptides, Suppl. I (1980) $43. I ! Gordon, G, The concept of relay nuclei. In A. lggo (Ed.), Handbook of Senso~ Physiology, Vol. !!, Springer-Verlag, 1973, pp. 137-150. 12 Gwyn, D.G. and Waldron, H.A., A nucleus in the dorsolaterai funiculus of the spinal cord of the rat, Brain Res., 10(1968) 342-351. 13 Larsson, L.-I., Fahreukrug, J., Schaffalitzky de Muckadell, O., Sundler, F., HAkanson, R. and Rehfeld, J'F., ~ i o n of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. nat. Acad. SCi. U.S.A,, 73 (1976) 3197-3200. 14 Lundberg, J.M., H0kfelt, T., Nilson, G., Terenius, L., Rehfeld, J., Elde, R. and Said, S., Peptide neurons in the vagus, splanchnic and sciatic nerves, Acta physiol, scand., 104 (1978) 499-501. 15 Morin, F. and Catalano, J.F., Central connections of a cervical nucleus (nucleus cervicalis lateralis of the cat), J. comp. Neurol., 103 (1955) 17-32. 16 Rexed, B. and StrOm G., Afferent nervous connections of the lateral cervical nucleus, Acta physiol. scand., 25 (1952) 219-229. 17 Said, S.I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science. 169 (1970) 1217-1218. 18 Sims. K.B., Hoffman, D.L., Said, S.I. and Zimmerman, E.A., Vasoactive intestinal polypeptide (VIP) in mouse and rat brain: An immunocytochemical study, Brain Res., 186 (1980) 165-183. 19 Scnba, E., Shiosaka, S., Hara, Y., Inagaki, S., Sakanaka, M., Takatsuki, K., Kawai, Y. and Tohyama, M., Ontogeny of the peptider~c system in the rat spinal cord: immunohistochemical analysis, J. comp. Neurol., 208 (1982) 54-66. 20 Zamboni, L. and De Martino, C., Buffered picric acid formaldehyde: a new rapid fixative for electron microscopy, J. Cell Biol, 35 (1967) 148A.