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Cutaneous receptive field alterations induced by 4-aminopyridine
Brain Research, 232 (1982) 177-180 Elsevier Biomedical Press
177
Cutaneous receptive field alterations induced by 4-aminopyridine
NAYEF E. SAAD15, NABIL R. BANNA, ARLETTE KHOURY, SUHAYL J. JABBUR and PATRICK D. WALL* Faculty of Sciences, Lebanese University, Hadath.Beirut and ( S.J,J.) Faculty of Medicine, American University, Beirut (Lebanon) and (P.D.W.) Cerebral Functions Research Group, Department of Anatomy, University College London, WC1E 6BT (U.K.)
(Accepted October 15th, 1981) Key words: receptive fields -- dorsal column nuclei -- 4-aminopyridine
The dorsal column nuclei contain a precise somatotopic map of the body surface (ref. 8). This map is formed by the existence of small discrete receptive fields (cRFs) evoked by natural cutaneous stimuli in the cells of the nuclei. However, if percutaneous electrical stimuli are applied, there are signs of a very extensive subliminal fringe beyond the cRF since responses, often apparently monosynaptic, are evoked by stimuli distant from the cRFL This subliminal fringe might provide a basis for the observed plasticity of connections to dorsal column nuclei la. It has been shown that cold block of the normal input to the gracile nucleus results in the immediate appearance of novel RFs in some cells 8. If dorsal roots supplying the gracile nucleus are cut, there is a slow appearance of new inputs to cells which have lost their normal afferent drive 9 in addition to the immediate change 3. It is therefore of interest to find if the application of excitatory chemicals would reveal the existence of ineffective inputs. In this study, the convulsant 4-aminopyridine (4-AP) has been applied to test for the existence of dormant inputs. Its mode of action seems to be presynaptic, synaptic and postsynapticS,6,11,1L The complexity of this action does not concern us here since the intention was only to raise the level of excitability to reveal normally ineffective inputs. The action is non-specific, facilitating both inhibitory as well as excitatory connections 5,6. Systemic 4-AP has been shown to unmask dormant connections in visual cortex 4. Adult cats were anaesthetized with Nembutal (30 mg/kg, i.p.) with additional doses administered as needed i.v. during the experiment. They were paralyzed with Flaxedil and CO2, temperature and blood pressure were monitored. Holes were drilled in the parietal bone to place stimulating electrodes stereotactically in the 2 medial lemnisci (co-ordinates A5, L4.5, V1.8). The dorsal column nuclei were exposed by removing the atlanto-occipital membrane and a portion of the occipital bone. Extra* To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © Elsevier BiomedicalPress
178 cellular spike potentials were recorded through double-barrelled electrodes. The barrel used for recording was one of 2 types: 3 M NaCl-filled glass or platinum-coated tungsten microelectrodes 7. The drug was applied in three ways. Electrophoresis from the second microelectrode barrel filled with a 0.025-0.5 g/litre 4-AP solution in distilled water using currents of 100-300 nA for 10-30 s (WPI 160E Electrophoresis Programmer). Pressure ejection of 10-30 nl quantities of the same strength 4-AP in saline with a WPI 1400 Nanoliter pump. In 3 experiments (12 cells) a microdrop of 4AP (0.025-0.25 g/litre) was applied on the surface of the dorsal column nuclei close to the recording glass micropipette as previous described1,10. The responses to natural stimuli were examined in 60 cells in the gracile and cuneate nuclei. Forty-two of the 60 responded antidromically with short latency and high frequency following to contralateral medial lemniscus stimulation. The cells responded to hair movement (n - : 21), to touch (n ~-- 22), to pressure (n -~ 6), and to hair and touch (n -- 1 i). These cRFs remained stable if repeatedly examined for periods up to 1 h. Once the c R F had been established and found to be stable, the drug was applied and effects were seen within 1-2 rain (2-3 rain after surface microdrop application). These effects persisted for 10-20 min. On occasions a second dose was applied at 5 min with a further increase of effect but subsequent applications produced Touch c! n
4-AP 0.025 mg.lml. A
50 nI.115sec. ._..~
C
I ~ , . ~ ~....~lt J~..~lk.
k
L[..
iiitdiI 1 tll I lqlIIll'11111 i i.ii I 0.2mV. t
20 msec.
L Fig. 1. Expansion of the cRF of a cuneate neurone on the underside of the forelimb, induced by baroejection of t2.5 nl of 0.025 g/litre solution of 4-AP. The upper trace shows the evoked single unit response to natural stimulation (brushing) of the area marked below. Dark areas represent the control cRF, speckled areas the increase in cRF 3 min after 4-AP application. A" control extracellular record of the response evoked by brushing the cRF before 4-AP application. B: response evoked by brushing the control cRF region after 4-AP application. C: response evoked by brushing the new area included in the cRF after 4-AP application. Prior to 4-AP, brushing this (speckled) area failed to evoke any discharge in this neurone.
179 TABLE I Alterations in RFs of cuneate and gracile neurones induced by 4-AP application Modality
Number Method of 4-AP application Surface Baroejection
Hair
1ontophoresis
Field changes Expansion Shrinkage
No change
21
4
13
4
16
2
3
Touch 22 Hair + touch 11 Pressure 6 Total 60
6 1 1 12
5 8 4 30
11 2 1 18
19 9 5 49
1 0 1 4
2 2 0 7
Relay cells
16 13 9 4 42
no additional increase. F o r 49 of the 60 units, the cRF expanded after 4-AP, in 4 units it contracted and 7 were unaffected. A typical example of expanded cRF is shown in Fig. 1. An example of a smaller expansion was a cell responding to touch stimuli on 2 toes before and to 3 toes after 4-AP. Much larger expansions were seen, particularly of proximal cRFs, including 3 examples of extension to the contralateral body surface. Table I shows that cells responding to all 4 categories of stimulus showed a similar reaction. The mode of application did not effect the results other than the latency of onset. The cells with axons projecting to the thalamus responded in the same manner as the cells which could not be driven from the medial lemniscus. Some cells increased their ongoing activity with 4-AP but this was not a necessary correlate of cRF expansion since expansion was observed without an increase of spontaneous activity. Where inhibitory fields were observed, they persisted and increased, except for one cell where the inhibitory zone disappeared. F o r 10 cells, the response to electrical stimulation of the skin was tested. Before the drug, threshold stimuli in the cRF produced a typical short latency burst response 2, while outside the cRF the same stimulus produced either no response or delayed variable responses. After the 4-AP, the response to stimulation within the original cRF had a faster latency (0.2-0.3 ms) and with less latency variation. The stimuli in the area outside the original cRF but within the expanded RF now produced brisk responses with the same latency as those evoked after the 4-AP from the original cRF. These results support the suggestion that there exists a subliminal fringe around the normal cutaneous R F and that some of these inputs can become effective in exciting cells with natural and electrical stimuli when 4-AP is applied. Furthermore, the results of electrical stimulation suggest that some of these novel inputs are in monosynaptic contact with the cells. The appearance of these newly effective afferents cannot be due to the disappearance of normal inhibitions since, as in the spinal cordS, 6, 4-AP increases the strength of inhibition as well as of excitation. The work was supported by two grants from the Lebanese National Research Council. We wish to thank Mr. M. Shakarji for technical assistance.
180 1 Chanelet, J. and Lonchampt, P., Etude microregionale de Faction du flaxedil sur la moelle fombaire, C.R. Soc. Biol., 161 (1967) 1934-1938. 2 Dostrovsky, J. O., Jabbur, S. and Millar, J., Neurones in cat gracile nucleus with both local and widefield inputs, J. Physiol. (Lond.), 278 (1978) 365-375. 3 Dostrovsky, J. O., Millar, J. and Wall, P. D., The immediate shift of afferent drive of dorsal column cells following deafferentation, Exp. Neurol., 52 (1976) 480~195. 4 Glowinski, A. J., Kennedy, H., Martin, K. A. C. and Whitteridge, D., Rapid changes in ocular dominance produced by 4-aminopyridine in monocularly deprived lambs, J. Physiol. (Lond.), 296 (1979) 64-65P. 5 Lemeignan, M., Analysis of the action of 4-aminopyridineon the cat lumbar spinal cord. I. Modification of the afferent volley, the monosynaptic discharge and the polysynaptic evoked responses, Neuropharmacology, 11 (1972) 551-558. 6 Lemeignan, M., Analysis of the effects of 4-aminopyridineon the lumbar spinal cord of the cat. II. Modification of certain spinal inhibitory phenomena, post-tetanic potentiation and dorsal root potentials, Neuropharmaeology, 12 (1973)641-651. 7 Merrill, E. G. and Ainsworth, A., Glass coated platinum plated tungsten microelectrodes, Med. Biol. Engng, 10 (1972) 662-672. 8 Millar, J. and Basbaum, A. I., Topography of the projection of the body surface of the cat to the gracile and cuneate nuclei, Exp. Neurol., 50 (1976) 658-672. 9 Millar, J., Basbaum, A. I. and Walt, P. D., Restructuring of the somatotopic map and the appearance of abnormal neuronal activity in the gracile nucleus after partial deafferentation, Exp. Neurol., 50 (1976) 658-672. 10 Saad6, N. E., Chanelet, J. and Lonchampt, P., Action facilitatrice de microinjections de 4-aminopyridine sur les activit6s m6dullaire r6flexes de la GrenouiUe, C.R. Soc. Biol., 165 (1971) 20692077. 11 Sherratt, R. M., Bostock, H. and Sears, T. A., Effects of 4-aminopyridine on normal and demyelinated mammalian nerve fibres, Nature (Lond.), 283 (1980) 570-572. 12 Thesleff, S., Aminopyridines and synaptic transmission, Neuroscienee, 5 (1980) 1413-1419. 13 Wall, P. D., The presence of ineffective synapses and the circumstances which unmask them, Phil. Trans. B, 278 (1977) 361-372.
177
Cutaneous receptive field alterations induced by 4-aminopyridine
NAYEF E. SAAD15, NABIL R. BANNA, ARLETTE KHOURY, SUHAYL J. JABBUR and PATRICK D. WALL* Faculty of Sciences, Lebanese University, Hadath.Beirut and ( S.J,J.) Faculty of Medicine, American University, Beirut (Lebanon) and (P.D.W.) Cerebral Functions Research Group, Department of Anatomy, University College London, WC1E 6BT (U.K.)
(Accepted October 15th, 1981) Key words: receptive fields -- dorsal column nuclei -- 4-aminopyridine
The dorsal column nuclei contain a precise somatotopic map of the body surface (ref. 8). This map is formed by the existence of small discrete receptive fields (cRFs) evoked by natural cutaneous stimuli in the cells of the nuclei. However, if percutaneous electrical stimuli are applied, there are signs of a very extensive subliminal fringe beyond the cRF since responses, often apparently monosynaptic, are evoked by stimuli distant from the cRFL This subliminal fringe might provide a basis for the observed plasticity of connections to dorsal column nuclei la. It has been shown that cold block of the normal input to the gracile nucleus results in the immediate appearance of novel RFs in some cells 8. If dorsal roots supplying the gracile nucleus are cut, there is a slow appearance of new inputs to cells which have lost their normal afferent drive 9 in addition to the immediate change 3. It is therefore of interest to find if the application of excitatory chemicals would reveal the existence of ineffective inputs. In this study, the convulsant 4-aminopyridine (4-AP) has been applied to test for the existence of dormant inputs. Its mode of action seems to be presynaptic, synaptic and postsynapticS,6,11,1L The complexity of this action does not concern us here since the intention was only to raise the level of excitability to reveal normally ineffective inputs. The action is non-specific, facilitating both inhibitory as well as excitatory connections 5,6. Systemic 4-AP has been shown to unmask dormant connections in visual cortex 4. Adult cats were anaesthetized with Nembutal (30 mg/kg, i.p.) with additional doses administered as needed i.v. during the experiment. They were paralyzed with Flaxedil and CO2, temperature and blood pressure were monitored. Holes were drilled in the parietal bone to place stimulating electrodes stereotactically in the 2 medial lemnisci (co-ordinates A5, L4.5, V1.8). The dorsal column nuclei were exposed by removing the atlanto-occipital membrane and a portion of the occipital bone. Extra* To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © Elsevier BiomedicalPress
178 cellular spike potentials were recorded through double-barrelled electrodes. The barrel used for recording was one of 2 types: 3 M NaCl-filled glass or platinum-coated tungsten microelectrodes 7. The drug was applied in three ways. Electrophoresis from the second microelectrode barrel filled with a 0.025-0.5 g/litre 4-AP solution in distilled water using currents of 100-300 nA for 10-30 s (WPI 160E Electrophoresis Programmer). Pressure ejection of 10-30 nl quantities of the same strength 4-AP in saline with a WPI 1400 Nanoliter pump. In 3 experiments (12 cells) a microdrop of 4AP (0.025-0.25 g/litre) was applied on the surface of the dorsal column nuclei close to the recording glass micropipette as previous described1,10. The responses to natural stimuli were examined in 60 cells in the gracile and cuneate nuclei. Forty-two of the 60 responded antidromically with short latency and high frequency following to contralateral medial lemniscus stimulation. The cells responded to hair movement (n - : 21), to touch (n ~-- 22), to pressure (n -~ 6), and to hair and touch (n -- 1 i). These cRFs remained stable if repeatedly examined for periods up to 1 h. Once the c R F had been established and found to be stable, the drug was applied and effects were seen within 1-2 rain (2-3 rain after surface microdrop application). These effects persisted for 10-20 min. On occasions a second dose was applied at 5 min with a further increase of effect but subsequent applications produced Touch c! n
4-AP 0.025 mg.lml. A
50 nI.115sec. ._..~
C
I ~ , . ~ ~....~lt J~..~lk.
k
L[..
iiitdiI 1 tll I lqlIIll'11111 i i.ii I 0.2mV. t
20 msec.
L Fig. 1. Expansion of the cRF of a cuneate neurone on the underside of the forelimb, induced by baroejection of t2.5 nl of 0.025 g/litre solution of 4-AP. The upper trace shows the evoked single unit response to natural stimulation (brushing) of the area marked below. Dark areas represent the control cRF, speckled areas the increase in cRF 3 min after 4-AP application. A" control extracellular record of the response evoked by brushing the cRF before 4-AP application. B: response evoked by brushing the control cRF region after 4-AP application. C: response evoked by brushing the new area included in the cRF after 4-AP application. Prior to 4-AP, brushing this (speckled) area failed to evoke any discharge in this neurone.
179 TABLE I Alterations in RFs of cuneate and gracile neurones induced by 4-AP application Modality
Number Method of 4-AP application Surface Baroejection
Hair
1ontophoresis
Field changes Expansion Shrinkage
No change
21
4
13
4
16
2
3
Touch 22 Hair + touch 11 Pressure 6 Total 60
6 1 1 12
5 8 4 30
11 2 1 18
19 9 5 49
1 0 1 4
2 2 0 7
Relay cells
16 13 9 4 42
no additional increase. F o r 49 of the 60 units, the cRF expanded after 4-AP, in 4 units it contracted and 7 were unaffected. A typical example of expanded cRF is shown in Fig. 1. An example of a smaller expansion was a cell responding to touch stimuli on 2 toes before and to 3 toes after 4-AP. Much larger expansions were seen, particularly of proximal cRFs, including 3 examples of extension to the contralateral body surface. Table I shows that cells responding to all 4 categories of stimulus showed a similar reaction. The mode of application did not effect the results other than the latency of onset. The cells with axons projecting to the thalamus responded in the same manner as the cells which could not be driven from the medial lemniscus. Some cells increased their ongoing activity with 4-AP but this was not a necessary correlate of cRF expansion since expansion was observed without an increase of spontaneous activity. Where inhibitory fields were observed, they persisted and increased, except for one cell where the inhibitory zone disappeared. F o r 10 cells, the response to electrical stimulation of the skin was tested. Before the drug, threshold stimuli in the cRF produced a typical short latency burst response 2, while outside the cRF the same stimulus produced either no response or delayed variable responses. After the 4-AP, the response to stimulation within the original cRF had a faster latency (0.2-0.3 ms) and with less latency variation. The stimuli in the area outside the original cRF but within the expanded RF now produced brisk responses with the same latency as those evoked after the 4-AP from the original cRF. These results support the suggestion that there exists a subliminal fringe around the normal cutaneous R F and that some of these inputs can become effective in exciting cells with natural and electrical stimuli when 4-AP is applied. Furthermore, the results of electrical stimulation suggest that some of these novel inputs are in monosynaptic contact with the cells. The appearance of these newly effective afferents cannot be due to the disappearance of normal inhibitions since, as in the spinal cordS, 6, 4-AP increases the strength of inhibition as well as of excitation. The work was supported by two grants from the Lebanese National Research Council. We wish to thank Mr. M. Shakarji for technical assistance.
180 1 Chanelet, J. and Lonchampt, P., Etude microregionale de Faction du flaxedil sur la moelle fombaire, C.R. Soc. Biol., 161 (1967) 1934-1938. 2 Dostrovsky, J. O., Jabbur, S. and Millar, J., Neurones in cat gracile nucleus with both local and widefield inputs, J. Physiol. (Lond.), 278 (1978) 365-375. 3 Dostrovsky, J. O., Millar, J. and Wall, P. D., The immediate shift of afferent drive of dorsal column cells following deafferentation, Exp. Neurol., 52 (1976) 480~195. 4 Glowinski, A. J., Kennedy, H., Martin, K. A. C. and Whitteridge, D., Rapid changes in ocular dominance produced by 4-aminopyridine in monocularly deprived lambs, J. Physiol. (Lond.), 296 (1979) 64-65P. 5 Lemeignan, M., Analysis of the action of 4-aminopyridineon the cat lumbar spinal cord. I. Modification of the afferent volley, the monosynaptic discharge and the polysynaptic evoked responses, Neuropharmacology, 11 (1972) 551-558. 6 Lemeignan, M., Analysis of the effects of 4-aminopyridineon the lumbar spinal cord of the cat. II. Modification of certain spinal inhibitory phenomena, post-tetanic potentiation and dorsal root potentials, Neuropharmaeology, 12 (1973)641-651. 7 Merrill, E. G. and Ainsworth, A., Glass coated platinum plated tungsten microelectrodes, Med. Biol. Engng, 10 (1972) 662-672. 8 Millar, J. and Basbaum, A. I., Topography of the projection of the body surface of the cat to the gracile and cuneate nuclei, Exp. Neurol., 50 (1976) 658-672. 9 Millar, J., Basbaum, A. I. and Walt, P. D., Restructuring of the somatotopic map and the appearance of abnormal neuronal activity in the gracile nucleus after partial deafferentation, Exp. Neurol., 50 (1976) 658-672. 10 Saad6, N. E., Chanelet, J. and Lonchampt, P., Action facilitatrice de microinjections de 4-aminopyridine sur les activit6s m6dullaire r6flexes de la GrenouiUe, C.R. Soc. Biol., 165 (1971) 20692077. 11 Sherratt, R. M., Bostock, H. and Sears, T. A., Effects of 4-aminopyridine on normal and demyelinated mammalian nerve fibres, Nature (Lond.), 283 (1980) 570-572. 12 Thesleff, S., Aminopyridines and synaptic transmission, Neuroscienee, 5 (1980) 1413-1419. 13 Wall, P. D., The presence of ineffective synapses and the circumstances which unmask them, Phil. Trans. B, 278 (1977) 361-372.