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Subconvulsive dose of strychnine enhances the transneuronal effect of peripheral sensory nerve transection
320
Brain R~',~'~iri ~; ~2~i 19~4112!}
q. 5:
BRE 20513
Subconvulsive dose of strychnine enhances the transneuronal effect of peripheral sensory nerve transection TOMOSADA SUGIMOTO, MOTOHIDE TAKEMURA, JOHJI OKUBO and AKIRA SAKAI
2nd Department of Oral Anatomy, Osaka University Faculty o[' Dentistry, Osaka (Japan) (Accepted July 23rd, 1984)
Key words: peripheral nerve injury - - trigeminal nerve I subnucIeus caudalis I transneuronal degeneration
The effect of transection of the inferior alveolar nerve on the trigeminal subnucleus caudalis neuron was examined under the inlluence of systemic strychnine in the rat. Chronic intoxication with a subconvulsive dose of strychnine induced electron dense degeneration of somata and dendrites at 30 days following transection in the area of subnucleus caudalis which received primary projection from the transected nerve. Removal of the postsynaptic inhibition appears to enhance the transneuronal effect of peripheral nerve injury. Transection of a p e r i p h e r a l nerve has been reported to cause transneuronal d e g e n e r a t i o n in the spinal dorsal horn 7. The tooth pulp extirpation also caused transneuronal changes in the subnucleus caudalis 5. In addition we have recently shown that small dendrites involute as a result of transection of superficial radial nerve in the cat ~8. These transneuronal changes were all caused by the transection of p e r i p h e r a l axonal branches of p r i m a r y sensory neurons. Although primary neurons can survive and maintain their central axonal arbors and synaptic connections for up to 3 months following transection of their peripheral axonal branches ~7, it is certain that the altered quality and/or quantity of input from the peripherally axotomized primary neurons affect biological activity of second o r d e r neurons and cause transneuronal changes. T h e r e f o r e it is logical to assume that various neuroactive drugs, which do not affect neuronal m o r p h o l o g y by themselves, may prevent or enhance the transneuronal changes by compensating or exaggerating the functional changes. The present study investigates the effect of systemic strychnine on the transneuronal changes following transection of a sensory nerve, the inferior alveolar nerve.
On day 1, 3 adult S p r a g u e - D a w l e y rats (b.wt. ,~ 200 g) were anesthetized with p e n t o b a r b i t a l sodium (40 mg/kg i.p.). The buccal skin was incised and masseter muscle retracted to expose the buccal surface of the mandible on one side. The m a n d i b u l a r canal was o p e n e d with a dental burr and the inferior alveolar nerve was ligated near the m a n d i b u l a r foramen, Thereafter, the nerve was transected distally to the ligature and overlying muscle and skin were sutured. Starting on day 8 or 24, I mg/kg of strychnine (1 mg/ml in normal saline, i.p.) was given daily until day 30. For one animal, strychnine injection was totally omitted. On day 31, the animals were deeply anesthetized with p e n t o b a r b i t a l sodium and perfused through the left ventricle with 1~;~ f o r m a l d e h y d e and 1% glutaraldehyde in 0.12 M p h o s p h a t e buffer. The brainstem and the cervical e n l a r g e m e n t were osmicared, d e h y d r a t e d in a series of graded alcohols and subsequently e m b e d d e d in an epoxy resin. For two rats, the nerve transection was omitted but strychnine was given for 23 days. On the 24th day, they were sacrificed and the brainstem and cervical enlargement were processed as described above. Three rats were used to d e t e r m i n e the central projection of inferior alveolar nerve primary neurons.
Correspondence." T. Sugimoto, Osaka University, Faculty of Dentistry, 2nd Dept. of Oral Anatomy, 1-8 Yamadaoka, Suita city, Osaka 560, Japan. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
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Fig. 1. Camera lucida drawings of plastic embedded 1 *an sections of the medulla oblongata approximately 1 mm caudal to the obex. The strychnine treatment was started on day 8. A few degenerating neuronal cell bodies (dots) are seen on thc contralateral side to the unilateral transection of inferior alveolar nerve (A), while many can be seen on the ipsilateral side (B). Transgan~xlionic labeling of the primary neurons following HRP application to the nerve on the same side as the transection is schematically superimposed on the drawings (shaded areas). Some of degenerating neurons lie across laminae I - I V ventro-laterally to as welt as inside the HRP-labclcd terminal field ol the transccted nerve on both sides. Dotted lines indicate laminae I/[I and II/lll borders. ×32. Fig. 2. A photomicrograph illustrating an example of subnucleus caudalis area ipsilateral to the transection. The animal was treated with strychnine from day 8. Many neuronal cell bodies are degenerating and deeply stained with toluidine biuc (arrowheads). ×25t). lnscl: a high power m~lgnification of the degenerating cell body pointed with an arrow. ×3,101).
322
Figs. 3-6, Neuronal changes in the subnucleus caudalis ipsilateral to the transection with (Figs. 3-5) and without (Fig. 6) strychnine treatment. Fig. 3. A degenerating lamina 1I neuronal cell body. The electron density of cytoplasmic matrix is elevated and the nuclear chromatin is condensed. The nuclear envelope is irregularly infolded and Golgi cisternae are distended (arrows). Strychnine-treated from day 8, x 14,600. Fig. 4. This dendrite in lamina 1I shows elevated electron density and is filled with tightly packed microtubule~ lmt). Many ribosomes
323 The nerve was exposed as described above, transected and horseradish peroxidase ( H R P ) was applied to the proximal stump. On the next day, animals were sacrificed and the brainstem was processed according to Mesulam~2 for histochemical demonstration of transganglionically transported HRP. This part of the study will be published elsewhere in detail. One um thick sections of the brainstem of the nerve-transected strychnine-treated rats contained many degenerating neuronal cell bodies. The topographic location of these degenerating neurons roughly coincided with that of the projection of inferior alveolar nerve primary afferent neurons demonstrated with transganglionically transported HRP. They were found at all rostro-caudal levels of brainstem trigeminal sensory nuclear complex on the ipsilateral side to the transected nerve and occupied the dorsal parts of the main sensory nucleus and each subnucleus of the nucleus of spinal trigeminal tract. Some degenerating neurons were also found in the subnucleus caudalis contralateral to the transection. The transganglionic H R P also showed some contralateral projection of inferior alveolar nerve primary neurons in the subnucleus caudalis (Fig. 1). However, the dorsal horn of the cervical enlargement did not show neuronal degeneration. In the rostral part of subnucleus caudalis, where degenerating neurons were most frequently encountered, neuronal degeneration was seen across hnninae I - V . Although most of these degenerating neurons were located within the area which received projection from the inferior alveolar nerve primary neurons as shown by the H R P label, some degenerating neurons were observed in laminae I - I V beyond the ventral b o r d e r of this area (Fig. 1). Although the topographic distribution of degenerating neurons was comparable in animals with different periods of strychnine administration, the longer strychnine administration p r o d u c e d morc degenerating neurons. Degenerating neuron was not found in the brainstem or cervical enlargement of
rats which received only one of either nerve transection or strychnine treatment. Light microscopically, the cytoplasm of degenerating neuron was deeply stained with toluidine blue and the nuclear chromatin was condensed (Fig. 2). Except for the nucleole, degenerating neurons had homogeneously stained nucleoplasm and were easily distinguished from glial cells with conspicuous heterochromatin. Electron microscopically, degenerating neurons had electron dense cytoplasm and cisternae of the Golgi apparatus were distended. Many free ribosomes were homogeneously scattered over the perikaryal cytoplasm. Nuclei of these cells showed chromatin condensation and were ruffled with many irregular infoldings of nuclear envelope (Fig. 3). Synaptic contacts of electron dense degenerating neurons were most frequently seen on their dendrites (Fig. 4). Although many of these electron dense dendrites were relatively small in diameter (<1 urn). they a p p e a r e d to be shrivelled proximal dendrites. They almost invariably contained at least some ribosomes and occasional rough endoplasmic reticulum. In addition electron dense dendrites were usually filled with tightly packed microtubules (Fig. 4). Although electron density of many neurons was more or less elevated in the vicinity of the above degenerating neurons, some neurons remained pale and a p p e a r e d relatively health~. A considerable number of these pale neurons, however, contained swollen mitochondria. Matrix of swollen mitochondria was pale and their cristae had disappeared (Fig. 5). When a swollen mitochondrion was found in a neuronal cell body, many of mitochondria in the cell were affected and cisternae of the Golgi apparatus were often found distended. Degeneration of neuronal cell body was not seen in the animal without strychnine treatment at the light level. However, electron microscopic examination of that area of the subnucleus caudalis, which received the primary input from the transected nerve on the
arc also found in the cytoplasm and mitochondria are swollen (arrowheads). Two axonal endings h)rm synapses on this degenerating dendrite (arrows). Strychnine-treated from day 8. ×29.100. Fig. 5. Although the cytoplasm and nucleus of this lamina II neuron appear relatively healthy, mitochondria are swollen and their cristac have disappeared. Strychnine-treated from day 8. ×25,900. Fig. 6. A lamina II dendrite containing membrane-bounded cavities which arc connected to the agranular rcticulum at arrowheads. Strychnine-untreated. x 36,3(10.
324 ipsilateral side, revealed some neuronal cell bodies whose mitochondria were swollen. In these neurons, some distention of Golgi cisternae was also noted. In addition small caliber dendrites contained memb r a n e - b o u n d e d cavities. These dendritic cavities were often connected with agranular reticulum and might have been indeed distended cisternae of agranular reticulum (Fig. 6) lg. Neuronal d e g e n e r a t i o n observed in this study was not a non-specific effect of strychnine because it was not seen in the brainstem or cervical e n l a r g e m e n t of strychnine-treated rats without nerve transection. It was seen in those areas of the brainstem trigeminal sensory nuclei which receive input from the inferior alveolar nerve but not in the cervical e n l a r g e m e n t of rats which received both nerve transection and strychnine treatment. Therefore, it is likely that neuronal d e g e n e r a t i o n was caused by the transneuronal effect of transection of the nerve. Some neuronal cell bodies degenerating outside the area of inferior alveolar nerve p r o j e c t i o n d e m o n s t r a t e d with H R P might have had dendrites extending into the area and received monosynaptic input from the nerve. In other instances, they might have received polysynaptic input from distant terminals of the nerve's primary neurons. In addition, the possibility cannot be excluded that the terminal field area of inferior alveolar nerve primaries was u n d e r e s t i m a t e d because of a possible limitation of T M B histochemistry such as fading. Transneuronat damage of the second o r d e r neurons may be caused by the pathophysiological activity of injured p r i m a r y neurons. Primary neurons comprising n e u r o m a t a are known to show ongoing spontaneous activity, mechanosensitivity 6,13,19,> and sensitivity to chemical substances such as norepinephrine and epinephrine<13,2o, and establish ephaptic connections between themselves-~,~-~,lL These
Ben-Ari, Y., Trembley, E., Ottersen, O. P. and Meldrum. B. S., The role of epileptic activity in hippocampal and 'remote' cerebral lesions induced by kainic acid, Brain Research, 191 (1980) 79-97. 2 Blumberg, H. and Janig, W., Activation of fibers via experimentally produced stump neuromas of skin nerves: ectopic transmission or retrograde sprouting? Exp. NeuroL, 76 (1982) 468-482. 3 Collins, R. C. and Olney, J. W., Focal cortical seizures cause distant thalamic lesions, Science. 218 (1982) 177-179. 1
hyperactivity of injured primary nettrons may cause sustained depolarization of the .~econd o r d e r net~ron's m e m b r a n e . Such depolarization will maintain the ionic imbalance across the m e m b r a n e and damage the cell's viability. Transneuronal degeneration of neurons along the seizure pathway has been reported to follow experimental o~rtical seizures reduced by application of chemical convulsants to and electrical stimulation of cerebral c~rtcx~.~, 14.~, Mitochondrial damage in the strychnine-treated and -untreated animals is probably the initial change induced by transneuronal effect and the electron dense d e g e n e r a t i o n is the advanced stage of the same pathology. Since strychnine is a potent antagonist of an inhibitory neurotransmitter glycmc, it is conceivable that strychnine enhanced the neuronal d a m a g e by removing the inhibition on the second o r d e r neurons postsynaptic to the peripherally axotomized primary neuron. The present study showed that systemic application of strychnine e n h a n c e d the transneuronal effect of transection of a peripheral nerve and induced electron dense degeneration of the second o r d e r neurons. If this p h e n o m e n o n was caused by the elctrophysiological hyperactivity of injured primary neurons and enhanced by the antagonism of the postsynaptic inhibition of second o r d e r neurons, it will be interesting to examine the effect of different neuroactive drugs in combination with peripheral nerve transection. Such experiments will potentially serve for understanding synaptic circuitry of the sensory pathways. In addition, the strychnine-enhanced transsynaptic d e g e n e r a t i o n may m a k e a conventional silver impregnation for degenerating axon a highly selective and sensitive tool for detecting simultaneously the projection of primary and secondary neurons of discretionary nerves.
4 Devor, M. and Janig, W., Activation of myelinated afferents ending in a neuroma by stimulation of the sympathetic supply in the rat, Neurosci. Lett., 24 (1981) 43-47. 5 Gobel, S. and Binck, J. M., Degenerative changes in primary trigeminal axons and in neurons in nucleus caudalis following tooth pulp extirpations in the cat, Brain Research, 132 (1977) 347-354. 6 Govrin-Lippman, R. and Devor, M,, Ongoing activity in severed nerves: source and variation with time, Brain Research, 159 (1978) 406-410. 7 Knyihar-Csillik, E. and Csillik, B., Selective qabellingr by
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transsynaptic degeneration of substantia gelatinosal cells: an attempt to decipher intrinsic wiring in the Rolando substance of primates, Neurosci. Lett., 23 ( 1981 ) 131-136. Korenman, E. M. D. and Devor, M., Eetopic adrenergic sensitivity in damaged peripheral nerve axons in the rat, Exp. Neurol., 72 (1981) 63-81. Lisney, S. J. W. and Pover, C. M., Coupling between fibres in sensory nerve neuromas in cats, J. Physiol. (Lond. j, 327 (1982) 6-7P. Lisney, S. J. W. and Pover, C. M., Coupling between regenerated sensory nerve fibres in the cat, J. Physiol. (Lond.), 334 (1983) 71-72P. Lisney, S. J. W. and Pover, C. M., Coupling between fibres involved in sensory nerve neuromata in cats, J. Neurol. Sci., 59 (1983) 255-264. Mesulam, M.-M. and Brushart, T. M., Transganglionic and anterograde transport of horseradish peroxidase across dorsal root ganglia: a tetramethylbenzidine method for tracing central sensory connections of muscles and peripheral nerves, Neuroscience, 4 (1979) 1107-1117. Scadding, J. W., Development of ongoing activity, mechanosensitivity, and adrenaline sensitivity in severed peripheral nerve axons, Exp. Neurol., 73 (198l) 345-364. Schwob, J. E., Fuller, T., Price, J. L. and Olney, J. W.,
Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience, 5 (1980) 991 - 1014. 15 Seltzer, Z. and Devor, M., Ephaptic transmission in chronically damaged peripheral nerves, Neurolog 3, 29 (1979) 1061-1064. 16 Sloviter, R. S. and Damiano. B. P.. Sustained electrical stimulation of the perforant path duplicates kainate-mduced electrophysiological effects and hippocampal damage in rats, Neurosci. Lett., 24 (1981) 279-284. 17 Sugimoto, T. and Gobel, S., Primary neurons maintain their central axonal arbors in the spinal dorsal horn following peripheral nerve injury: an anatomical analysis using transganglionic transport of horseradish pcroxidase, Brairl Research, 248 (1982) 377-38I. 18 Sugimoto, T. and Gobel, S.. Dendritic changes in the spinal dorsal horn following transection of a peripheral nerve. Brain Research, in press. 19 Wall, P. D. and Gutnick, M., Properties of afferent nerve impulses originating from a neuroma. Nalure ¢l.ond. l, 24~ (1974) 740-743. 2(I Wall, P. D. and Gutnick, M., Ongoing activity in peripheral nerves: thc physiology and pharmacology of impulses originating from a neuroma, Exp. Neurol., 43 (1974) 58ll-593.
Brain R~',~'~iri ~; ~2~i 19~4112!}
q. 5:
BRE 20513
Subconvulsive dose of strychnine enhances the transneuronal effect of peripheral sensory nerve transection TOMOSADA SUGIMOTO, MOTOHIDE TAKEMURA, JOHJI OKUBO and AKIRA SAKAI
2nd Department of Oral Anatomy, Osaka University Faculty o[' Dentistry, Osaka (Japan) (Accepted July 23rd, 1984)
Key words: peripheral nerve injury - - trigeminal nerve I subnucIeus caudalis I transneuronal degeneration
The effect of transection of the inferior alveolar nerve on the trigeminal subnucleus caudalis neuron was examined under the inlluence of systemic strychnine in the rat. Chronic intoxication with a subconvulsive dose of strychnine induced electron dense degeneration of somata and dendrites at 30 days following transection in the area of subnucleus caudalis which received primary projection from the transected nerve. Removal of the postsynaptic inhibition appears to enhance the transneuronal effect of peripheral nerve injury. Transection of a p e r i p h e r a l nerve has been reported to cause transneuronal d e g e n e r a t i o n in the spinal dorsal horn 7. The tooth pulp extirpation also caused transneuronal changes in the subnucleus caudalis 5. In addition we have recently shown that small dendrites involute as a result of transection of superficial radial nerve in the cat ~8. These transneuronal changes were all caused by the transection of p e r i p h e r a l axonal branches of p r i m a r y sensory neurons. Although primary neurons can survive and maintain their central axonal arbors and synaptic connections for up to 3 months following transection of their peripheral axonal branches ~7, it is certain that the altered quality and/or quantity of input from the peripherally axotomized primary neurons affect biological activity of second o r d e r neurons and cause transneuronal changes. T h e r e f o r e it is logical to assume that various neuroactive drugs, which do not affect neuronal m o r p h o l o g y by themselves, may prevent or enhance the transneuronal changes by compensating or exaggerating the functional changes. The present study investigates the effect of systemic strychnine on the transneuronal changes following transection of a sensory nerve, the inferior alveolar nerve.
On day 1, 3 adult S p r a g u e - D a w l e y rats (b.wt. ,~ 200 g) were anesthetized with p e n t o b a r b i t a l sodium (40 mg/kg i.p.). The buccal skin was incised and masseter muscle retracted to expose the buccal surface of the mandible on one side. The m a n d i b u l a r canal was o p e n e d with a dental burr and the inferior alveolar nerve was ligated near the m a n d i b u l a r foramen, Thereafter, the nerve was transected distally to the ligature and overlying muscle and skin were sutured. Starting on day 8 or 24, I mg/kg of strychnine (1 mg/ml in normal saline, i.p.) was given daily until day 30. For one animal, strychnine injection was totally omitted. On day 31, the animals were deeply anesthetized with p e n t o b a r b i t a l sodium and perfused through the left ventricle with 1~;~ f o r m a l d e h y d e and 1% glutaraldehyde in 0.12 M p h o s p h a t e buffer. The brainstem and the cervical e n l a r g e m e n t were osmicared, d e h y d r a t e d in a series of graded alcohols and subsequently e m b e d d e d in an epoxy resin. For two rats, the nerve transection was omitted but strychnine was given for 23 days. On the 24th day, they were sacrificed and the brainstem and cervical enlargement were processed as described above. Three rats were used to d e t e r m i n e the central projection of inferior alveolar nerve primary neurons.
Correspondence." T. Sugimoto, Osaka University, Faculty of Dentistry, 2nd Dept. of Oral Anatomy, 1-8 Yamadaoka, Suita city, Osaka 560, Japan. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
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Fig. 1. Camera lucida drawings of plastic embedded 1 *an sections of the medulla oblongata approximately 1 mm caudal to the obex. The strychnine treatment was started on day 8. A few degenerating neuronal cell bodies (dots) are seen on thc contralateral side to the unilateral transection of inferior alveolar nerve (A), while many can be seen on the ipsilateral side (B). Transgan~xlionic labeling of the primary neurons following HRP application to the nerve on the same side as the transection is schematically superimposed on the drawings (shaded areas). Some of degenerating neurons lie across laminae I - I V ventro-laterally to as welt as inside the HRP-labclcd terminal field ol the transccted nerve on both sides. Dotted lines indicate laminae I/[I and II/lll borders. ×32. Fig. 2. A photomicrograph illustrating an example of subnucleus caudalis area ipsilateral to the transection. The animal was treated with strychnine from day 8. Many neuronal cell bodies are degenerating and deeply stained with toluidine biuc (arrowheads). ×25t). lnscl: a high power m~lgnification of the degenerating cell body pointed with an arrow. ×3,101).
322
Figs. 3-6, Neuronal changes in the subnucleus caudalis ipsilateral to the transection with (Figs. 3-5) and without (Fig. 6) strychnine treatment. Fig. 3. A degenerating lamina 1I neuronal cell body. The electron density of cytoplasmic matrix is elevated and the nuclear chromatin is condensed. The nuclear envelope is irregularly infolded and Golgi cisternae are distended (arrows). Strychnine-treated from day 8, x 14,600. Fig. 4. This dendrite in lamina 1I shows elevated electron density and is filled with tightly packed microtubule~ lmt). Many ribosomes
323 The nerve was exposed as described above, transected and horseradish peroxidase ( H R P ) was applied to the proximal stump. On the next day, animals were sacrificed and the brainstem was processed according to Mesulam~2 for histochemical demonstration of transganglionically transported HRP. This part of the study will be published elsewhere in detail. One um thick sections of the brainstem of the nerve-transected strychnine-treated rats contained many degenerating neuronal cell bodies. The topographic location of these degenerating neurons roughly coincided with that of the projection of inferior alveolar nerve primary afferent neurons demonstrated with transganglionically transported HRP. They were found at all rostro-caudal levels of brainstem trigeminal sensory nuclear complex on the ipsilateral side to the transected nerve and occupied the dorsal parts of the main sensory nucleus and each subnucleus of the nucleus of spinal trigeminal tract. Some degenerating neurons were also found in the subnucleus caudalis contralateral to the transection. The transganglionic H R P also showed some contralateral projection of inferior alveolar nerve primary neurons in the subnucleus caudalis (Fig. 1). However, the dorsal horn of the cervical enlargement did not show neuronal degeneration. In the rostral part of subnucleus caudalis, where degenerating neurons were most frequently encountered, neuronal degeneration was seen across hnninae I - V . Although most of these degenerating neurons were located within the area which received projection from the inferior alveolar nerve primary neurons as shown by the H R P label, some degenerating neurons were observed in laminae I - I V beyond the ventral b o r d e r of this area (Fig. 1). Although the topographic distribution of degenerating neurons was comparable in animals with different periods of strychnine administration, the longer strychnine administration p r o d u c e d morc degenerating neurons. Degenerating neuron was not found in the brainstem or cervical enlargement of
rats which received only one of either nerve transection or strychnine treatment. Light microscopically, the cytoplasm of degenerating neuron was deeply stained with toluidine blue and the nuclear chromatin was condensed (Fig. 2). Except for the nucleole, degenerating neurons had homogeneously stained nucleoplasm and were easily distinguished from glial cells with conspicuous heterochromatin. Electron microscopically, degenerating neurons had electron dense cytoplasm and cisternae of the Golgi apparatus were distended. Many free ribosomes were homogeneously scattered over the perikaryal cytoplasm. Nuclei of these cells showed chromatin condensation and were ruffled with many irregular infoldings of nuclear envelope (Fig. 3). Synaptic contacts of electron dense degenerating neurons were most frequently seen on their dendrites (Fig. 4). Although many of these electron dense dendrites were relatively small in diameter (<1 urn). they a p p e a r e d to be shrivelled proximal dendrites. They almost invariably contained at least some ribosomes and occasional rough endoplasmic reticulum. In addition electron dense dendrites were usually filled with tightly packed microtubules (Fig. 4). Although electron density of many neurons was more or less elevated in the vicinity of the above degenerating neurons, some neurons remained pale and a p p e a r e d relatively health~. A considerable number of these pale neurons, however, contained swollen mitochondria. Matrix of swollen mitochondria was pale and their cristae had disappeared (Fig. 5). When a swollen mitochondrion was found in a neuronal cell body, many of mitochondria in the cell were affected and cisternae of the Golgi apparatus were often found distended. Degeneration of neuronal cell body was not seen in the animal without strychnine treatment at the light level. However, electron microscopic examination of that area of the subnucleus caudalis, which received the primary input from the transected nerve on the
arc also found in the cytoplasm and mitochondria are swollen (arrowheads). Two axonal endings h)rm synapses on this degenerating dendrite (arrows). Strychnine-treated from day 8. ×29.100. Fig. 5. Although the cytoplasm and nucleus of this lamina II neuron appear relatively healthy, mitochondria are swollen and their cristac have disappeared. Strychnine-treated from day 8. ×25,900. Fig. 6. A lamina II dendrite containing membrane-bounded cavities which arc connected to the agranular rcticulum at arrowheads. Strychnine-untreated. x 36,3(10.
324 ipsilateral side, revealed some neuronal cell bodies whose mitochondria were swollen. In these neurons, some distention of Golgi cisternae was also noted. In addition small caliber dendrites contained memb r a n e - b o u n d e d cavities. These dendritic cavities were often connected with agranular reticulum and might have been indeed distended cisternae of agranular reticulum (Fig. 6) lg. Neuronal d e g e n e r a t i o n observed in this study was not a non-specific effect of strychnine because it was not seen in the brainstem or cervical e n l a r g e m e n t of strychnine-treated rats without nerve transection. It was seen in those areas of the brainstem trigeminal sensory nuclei which receive input from the inferior alveolar nerve but not in the cervical e n l a r g e m e n t of rats which received both nerve transection and strychnine treatment. Therefore, it is likely that neuronal d e g e n e r a t i o n was caused by the transneuronal effect of transection of the nerve. Some neuronal cell bodies degenerating outside the area of inferior alveolar nerve p r o j e c t i o n d e m o n s t r a t e d with H R P might have had dendrites extending into the area and received monosynaptic input from the nerve. In other instances, they might have received polysynaptic input from distant terminals of the nerve's primary neurons. In addition, the possibility cannot be excluded that the terminal field area of inferior alveolar nerve primaries was u n d e r e s t i m a t e d because of a possible limitation of T M B histochemistry such as fading. Transneuronat damage of the second o r d e r neurons may be caused by the pathophysiological activity of injured p r i m a r y neurons. Primary neurons comprising n e u r o m a t a are known to show ongoing spontaneous activity, mechanosensitivity 6,13,19,> and sensitivity to chemical substances such as norepinephrine and epinephrine<13,2o, and establish ephaptic connections between themselves-~,~-~,lL These
Ben-Ari, Y., Trembley, E., Ottersen, O. P. and Meldrum. B. S., The role of epileptic activity in hippocampal and 'remote' cerebral lesions induced by kainic acid, Brain Research, 191 (1980) 79-97. 2 Blumberg, H. and Janig, W., Activation of fibers via experimentally produced stump neuromas of skin nerves: ectopic transmission or retrograde sprouting? Exp. NeuroL, 76 (1982) 468-482. 3 Collins, R. C. and Olney, J. W., Focal cortical seizures cause distant thalamic lesions, Science. 218 (1982) 177-179. 1
hyperactivity of injured primary nettrons may cause sustained depolarization of the .~econd o r d e r net~ron's m e m b r a n e . Such depolarization will maintain the ionic imbalance across the m e m b r a n e and damage the cell's viability. Transneuronal degeneration of neurons along the seizure pathway has been reported to follow experimental o~rtical seizures reduced by application of chemical convulsants to and electrical stimulation of cerebral c~rtcx~.~, 14.~, Mitochondrial damage in the strychnine-treated and -untreated animals is probably the initial change induced by transneuronal effect and the electron dense d e g e n e r a t i o n is the advanced stage of the same pathology. Since strychnine is a potent antagonist of an inhibitory neurotransmitter glycmc, it is conceivable that strychnine enhanced the neuronal d a m a g e by removing the inhibition on the second o r d e r neurons postsynaptic to the peripherally axotomized primary neuron. The present study showed that systemic application of strychnine e n h a n c e d the transneuronal effect of transection of a peripheral nerve and induced electron dense degeneration of the second o r d e r neurons. If this p h e n o m e n o n was caused by the elctrophysiological hyperactivity of injured primary neurons and enhanced by the antagonism of the postsynaptic inhibition of second o r d e r neurons, it will be interesting to examine the effect of different neuroactive drugs in combination with peripheral nerve transection. Such experiments will potentially serve for understanding synaptic circuitry of the sensory pathways. In addition, the strychnine-enhanced transsynaptic d e g e n e r a t i o n may m a k e a conventional silver impregnation for degenerating axon a highly selective and sensitive tool for detecting simultaneously the projection of primary and secondary neurons of discretionary nerves.
4 Devor, M. and Janig, W., Activation of myelinated afferents ending in a neuroma by stimulation of the sympathetic supply in the rat, Neurosci. Lett., 24 (1981) 43-47. 5 Gobel, S. and Binck, J. M., Degenerative changes in primary trigeminal axons and in neurons in nucleus caudalis following tooth pulp extirpations in the cat, Brain Research, 132 (1977) 347-354. 6 Govrin-Lippman, R. and Devor, M,, Ongoing activity in severed nerves: source and variation with time, Brain Research, 159 (1978) 406-410. 7 Knyihar-Csillik, E. and Csillik, B., Selective qabellingr by
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