214
Bruin Research,
4; ElsevieriNorth-Holland
Selective
degeneration
177
( 1979)2 14. ilY Biomedical Press
of neurons induced by cobalt ions
D. DAGAN and Y. SARNE Behavioral Biology Unit, Techniort Faculty of Medicine, Haifi, and Dept. of Physiology and Pharmarology, Sackler School of Medicine, Tel Aviv University, Tel Aviv Clsrael)
(Accepted July 19th, 1979)
in studies on neuronal plasticity, specificity of neural connections, and mutual effeCtSof neurons, it is often useful to degenerate specific nerve cellsW1~. Degeneration is also used to trace pathways in the CPW. The most widely used method for degeneration is severance ofaxons from their somata, this usually involves the destruction of non-neural elements as well, e.g. blood vessels, glia, connective tissue, etc. Furthermore, it is often inapplicable in heterogeneous systems when degeneration of specific nerve cells is desired. Recently, intracellular injection of proteolytic enzymes was used to cause degeneration of single neurons l-l’, however, this method is not applicable to systems where a large population of small neurons is to be degenerated. We propose an alternative approach for degeneration, namely, topical application of substances which are taken up by the neurons, transported or diffused along the cell’s processes, and cause destruction of the cell. Cobalt chloride is often used as a neuronal stain by intracellular injection. backfilling techniques or extracellular microelectrode application13. Cobalt ions are known to compete with calcium which is essential for many neuronal functions14. Thus, externally applied cobalt chloride may cause degeneration of specific neuronal pathways. To develop the cobalt chloride degeneration method, we used thecercal sensory neurons of crickets (Gryllus bimaculatus). The cerci are two caudal sensory appendages bearing some 2000 sensilla and sensory neurons with somata located peripherally, beneath the external cuticles. The axonal processes of these sensory neurons enter the terminal ganglion and synapse on small and giant interneurons which send ascending axons through the abdominal connectives 5. Glass capillaries, 2 mm in diameter filled with 0.5 M CoCIZ were inserted over one cercus while the contralateral cercus served as a control. Following l-8 h of such treatment the capillary was removed. Cereal nerves were tested for electrophysiological, biochemical and morphological parameters of integrity l-4 days following the application of CoClr. Electrical stimulation of the treated cereal nerve failed to evoke activity in postsynaptic cells (Fig. IB) recorded from the abdominal connectives, while normal activity was recorded following stimulation of the control cereal nerve (Fig. 1 A). The application of CoCl2 does not affect postsynaptic elements, as can be seen in Fig. 1C
215 B
A
W++++++-
D
Fig. 1. Postsynaptic activity evoked by cereal nerve stimulation. A and B : extracellular recording from connectives. A: stimulation of control cereal nerve evokes both giant and small fiber activity within the connectives. B: no activity is evoked following stimulation of CoClz-treated cereal nerve 48 h after treatment. C and D: intracellular recording from a medial giant neuron* following stimulation of control (C) and treated(D) cereal nerves. This giant interneuron normally responds to stimulation of both cereal nerves. Giant axon activity in C indicates that absence of activity in D is not due to destruction of the postsynaptic giant interneuron. Calibration: 20 msec; 2 mV (A and B) 20 mV (C and D).
and 1D. Here the electrical intracellularly.
Electrical
activity
stimulation
of a medial giant interneuron
(MGI)
is recorded
of the treated cereal nerve did not evoke any res-
ponses (Fig. ID) in the giant interneuron, although normally it can be activated by electrical stimulation of either cereal nerve. The integrity of this giant interneuron is demonstrated in Fig. 1C where synaptic and spiking activity were generated by stimulation of the contralateral untreated cereal nerve. Thus the external application of CoCla affected the cereal nerve itself and not postsynaptic elements. In order to test the extent of the effect on the cereal nerve a biochemical assay was used. The putative neurotransmitter of the cereal system in insects is acetylcholine (ACh)ls which is synthesized by the enzyme choline acetyltransferase (CAT). Following degeneration by external application of CoCls, CAT activity in the cereal nerve was markedly reduced. Choline acetyltransferase activity was measured by scintillation counting of labeled ACh synthesized from [3H]AcCoA incubated with homogenates of the cereal nervesa. Crickets kept at 30 “C for 3 days following cobalt chloride treatment showed a reduction of 87 x, (S.D. = 8, n = 11) in CAT activity in the treated cereal nerves as compared to the control contralateral cereal nerves. Reduction in CAT activity reaches values of 94% (S.D. = 4, n = 5) after 8 days. The decline in CAT activity is not due to the presence of residual cobalt chloride or degeneration products in the enzyme assay. This was ascertained by using mixtures of control and treated CN homogenates which gave CAT activity values equal to the sum of the two homogenates assayed separately. The decrease in CAT activity following application of CoCl2 was found to be temperature-dependent: at 20 “C, 3 days following treatment, only a slight (< 1%) reduction in activity was observed as compared to the 87 “/, reduction at 30 “C. A similar time course and temperature dependence of CAT activity reduction was found in cereal nerves of 28 crickets following mechanical amputation of the cercus. Amputation removes the somata and initial axonal regions situated in the cercus itselfs. * See ref. 8 for identification
criteria.
216 The morphological appearance of neurons is often used as the maiu criterion for degeneration. Electron micrographs of cereal nerve cross-sections, 4 clays after CoCItreatment,
show marked axonal degeneration
(Fig. 2). Only a few axons remain intact.
Fig. 2. Electron micrographs of cereal nerve cross- sections. CNs were dissected and fixed iu 1“/; gluteraldehyde-100 mM cacodylate buffer and osmium post-fixed. A and B: normal, untreated cereal nerve. C and D: CN 4 days after CoClz treatment. Note clear axonal profiles in untreated nerve surrounded by glia wrappings (A and B). In the degenerating nerve (C and D) only few axons remain intact, others are characterized by swollen mitochondria and aggregations of electron dense axopiasm. CaIibration: 5 !Lrn A and B, 1.5 /drn C and D.
217
ofnormal
the majority lack the clear delineations of glia membrane wrappings Other visible signs of degeneration include membrane invaginations, axoplasm
aggregations
with swollen
mitochondria,
lysosomes
Similar results are obtained after cereal amputation. Progressive covering of cereal hairs with petroleum
electron
and myelin
evident
by proper
selection
of stimulus
MGI activity can be evoked after covering these conditions,
the distal two-thirds
abdominal cord. Utilizing applicability of the CoCls destroy the proximal input, tion of cobalt chloride may affecting the distal sensilla.
parameters.
the proximal
evokes activity
bodies.
jelly from the tip to the base
of a cercus shows that the MGI has a stronger input from the proximal especially
axons. dense
regionlO. This is
At 900 Hz, 8.5 dB no
one-third
of a cercus. Under
in small fibers ascending
in the
this proximo - distal gradient one can demonstrate the method for selective degeneration. In order to selectively amputation cannot be used. Nevertheless, local applicacause degeneration of these proximal sensory cells without This was achieved by inserting an empty glass capillary
over one cercus. The capillary was then filled with the aid of a thin J-shaped micropipet from the end close to the base of the cercus. ln this manner only a small proximal region of the cercus was covered with the cobalt chloride solution. Following 1 h of CoCla application the solution was withdrawn and the capillary removed. The animals were then tested 8-10 days post-treatment electrophysiologically. The results of these experiments are summarized in Fig. 3. Acoustic stimulation evoked giant interneuron activity in the connective on the control (non-treated) side, while no giant fiber activity could be recorded from the ipsilateral connective (on the treated side). This treatment did not affect the distal sensilla on the cercus which could be activated by stronger high frequency stimulation, whereupon they activated small fibers in the connective but no giant fiber activity was evoked (Fig. 3C). These results show that the application of CoClz abolished activity originating in the proximal, treated region of the cercus while the distal, untreated area, remained effective in evoking electrical activity in response to acoustic stimulation. The degeneration is not a result of either osmotic or mechanical effects of the treatment. This was shown in control experiments where a 0.5 M CaClz solution was used and had no effect on the integrity
of the cereal system.
possible mode of action of CoCla come from the similarity
Indications
as to the
in time course and temper-
ature dependence of degeneration neuron is filled relatively rapidly
induced either by Co& or amputation. Although a with cobalt ions t2, the degeneration proceeds at the same slow time course of that induced by mechanical axotomy. This suggests that the degenerating effect of COCIZ commences in the somata of treated neurons and mimics the effect of separating the soma from its axon rather than having a direct effect on the axon. It has been shown for other systems that calcium ions are necessary for loading of material that is to be transported from the soma centrifugally”. Cobalt ions interfere
with this process7 and prevent the transport tional axotomy.
of essential substances,
thus causing func-
The method for degeneration of specific inputs described here is applicable where other methods are not suitable. Surgical intervention, the common method for inducing neuronal degeneration causes damage to non-neural elements as well, while the
_-s_--Fig. 3. Electrical activity recorded with hook electrodes from the connectives in response to acoustic stimulation. A-D upper trace: recording from connective on control side; lower trace: connective on treated side. The proximal one-third of the treated cercus was covered with 0.5 M COG% for 1 h 10 days prior to recordings. A : stimulation at 325 Hz, 70 dB evokes giant intemeuron activity on the control side and only small fiber activity on the treated side. Giant fiber activity is identified by the large spikes. The small fiber activity in treated side originates in the contralateral, control cercus and is abolished by covering the proximal one-third of this cercus with petroleum jelly (B). C: stronger stimulation (900 Hz, 85 dB chosen due to relative lower sensitivity of distal region)-evokes smali Bber activity on the treated side. The spikes in the control connective originate from contralateral activation since the control cercus was completely covered with petroleum jelly. This activity is abolished when the distal two-thirds of the treated cercus are covered with petroleum jelly. Areas of the cercus covered with petroleum jelly are indicated on the right. Shaded areas of the cercus indicates the region treated with cobalt chloride. Stimulus duration: 40 msec(A and B), 400 msec (C and D).
CoClz permits degeneration of neurons, leaving the sensory organ itself intact. Some suggested applications of the method include degeneration of proximal elements where amputation cannot be applied (vide infra) and degeneration of centrally located cell bodies within the central nervous system by using the backfilling techniquels in vivo. We are now using the CoCla degeneration method in our studies of neuronal development and interaction between different sensory inputs impinging on a common target neuron. When CoCla is applied to cerci of cricket nymphs, with regenerative capacity, new functional sensory cells develop within 10 days following the degeneration of existing cells?. This indicates that epidermal cells, located at the baseof the cercus, are not destroyed by application of CoCls and retain their potential for regeneration of a cercus.
219 This National
research
was supported
Science Foundation
Israel Center
Psychobiology,
We are grateful technical reading
assistance
by grants
(62.5) Jerusalem, Charles
to A. Dorman
from
Smith Family for electron
and to Prof. 1. Parnas
the
United
States-Israel
Israel, and by Grant 221/79-l
Bi-
from the
Foundation. microscopy,
and Prof.
to Y. Margolin
R. Hammerschlag
for
for critical
of the manuscript.
l Bowling, D., Nicholls, J. and Parnas, I., Destruction of a single cell in the C.N.S. of the leech as a means of analysing its connexions and functional role, J. Physiol. (Land.), 282 (I 978) 1699180. 2 Dagan, D. and Sarne, Y., Evidence for cholinergic nature of cockroach giant fibers, J. conrp. Pkysiol., 126 (1978) 157-160. 3 Dagan, D., Lecker, S., Margolin, Y. and Sarne, Y., Delayed differentiation of an insect sensory system: development of biochemical and electrophysiological properties prior to sensory activation, in preparation. 4 Dravid, A. R. and Hammerschlag, R., Axoplasmic transport of proteins in vitro in primary afferent neurons of frog spinal cord: effect of Ca ++-free incubation conditions, J. Neurochem., 24 (1975) 71 l-718. 5 Edwards, J. S. and Palka, J., The cerci and abdominal giant fibres of the house cricket Achern domesticus, Proc. roy. Sot. B, I85 (1974) 83-103. 6 Hess, A., Experimental anatomical studies of pathways in the severed central nerve cord of the cockroach, J. Morphol., 103 (1958) 479-499. 7 Lavoie, P. A., Hammerschlag, R. and Tjan, A., Cobalt ions inhibit fast axonal transport of [sH]glycoproteins but not glycosylation, Bruin Reseurch, 149 (1978) 535-540. 8 Murphey, R. K., Palka, J. and Hustert, R., The cercus-to-giant interneurons, J. romp. Physiol.,
I I9 (1977) 2855300. 9 Palka, J. and Edwards, J. S., The cerci and abdominal giant fibres of the house cricket, Acheta domesticus. II. Regeneration and effects of chronic deprivation, Proc. Roy. Sot. B, 185 (1974) 105-121. IO Palka, J. and Oldberg, R., The cercus-to-giant interneuron system of crickets. III. Receptive field organization, J. camp. Physiol., 119 (1977) 301-317. I I Parnas, J. and Bowling, D., Killing of single neurones by intracellular injection of proteolytic enzymes, Nature (Land.), 270 (1977) 626-628. I2 Pitman, R. M., Tweedle, C. D. and Cohen, M. J., The form of nerve cells determination by cobalt impregnation. In S. B. Kater and C. Nicholson (Eds.), Intracellular Staining in Neurobiology, Springer-Verlag, Berlin, 1973, pp. 83-97. I3 Rehbein, H. G., Kalmring, K. and Romer, H., Structure and function of acoustic neurons in the thoracic ventral nerve cord ofLocusta migratoria (Acrididae), J. camp. Physiol., 95 (1974) 263-280. 14 Rubin, R. P., Calcium and the Secretory Process, Plenum Press, New York, 1974. 15 Sanes, J. R., Hildebrand, J. G. and Prescott, D. J., Differentiation of insect sensory neurons in absence of their normal synaptic targets, Develop. Biol., 52 (1976) 121-127. 16 Shankland, D. L., Rose, J. A. and Donniger, C., The cholinergic nature of the cereal nerve-giant fibre synapse in the sixth abdominal ganglion of the American cockroach, Periplaneta americana (L) J. Neurobiol., 2 (1971) 247-262. 17 Tsukahara, N., Hultborm, H. and Murakami, F., Sprouting of corticorubral synapses in red nucleus neurones after destruction of the nucleus interpositus of cerebellum, Experientia (Base/), 30 (1974) 57-58.
Bruin Research,
4; ElsevieriNorth-Holland
Selective
degeneration
177
( 1979)2 14. ilY Biomedical Press
of neurons induced by cobalt ions
D. DAGAN and Y. SARNE Behavioral Biology Unit, Techniort Faculty of Medicine, Haifi, and Dept. of Physiology and Pharmarology, Sackler School of Medicine, Tel Aviv University, Tel Aviv Clsrael)
(Accepted July 19th, 1979)
in studies on neuronal plasticity, specificity of neural connections, and mutual effeCtSof neurons, it is often useful to degenerate specific nerve cellsW1~. Degeneration is also used to trace pathways in the CPW. The most widely used method for degeneration is severance ofaxons from their somata, this usually involves the destruction of non-neural elements as well, e.g. blood vessels, glia, connective tissue, etc. Furthermore, it is often inapplicable in heterogeneous systems when degeneration of specific nerve cells is desired. Recently, intracellular injection of proteolytic enzymes was used to cause degeneration of single neurons l-l’, however, this method is not applicable to systems where a large population of small neurons is to be degenerated. We propose an alternative approach for degeneration, namely, topical application of substances which are taken up by the neurons, transported or diffused along the cell’s processes, and cause destruction of the cell. Cobalt chloride is often used as a neuronal stain by intracellular injection. backfilling techniques or extracellular microelectrode application13. Cobalt ions are known to compete with calcium which is essential for many neuronal functions14. Thus, externally applied cobalt chloride may cause degeneration of specific neuronal pathways. To develop the cobalt chloride degeneration method, we used thecercal sensory neurons of crickets (Gryllus bimaculatus). The cerci are two caudal sensory appendages bearing some 2000 sensilla and sensory neurons with somata located peripherally, beneath the external cuticles. The axonal processes of these sensory neurons enter the terminal ganglion and synapse on small and giant interneurons which send ascending axons through the abdominal connectives 5. Glass capillaries, 2 mm in diameter filled with 0.5 M CoCIZ were inserted over one cercus while the contralateral cercus served as a control. Following l-8 h of such treatment the capillary was removed. Cereal nerves were tested for electrophysiological, biochemical and morphological parameters of integrity l-4 days following the application of CoClr. Electrical stimulation of the treated cereal nerve failed to evoke activity in postsynaptic cells (Fig. IB) recorded from the abdominal connectives, while normal activity was recorded following stimulation of the control cereal nerve (Fig. 1 A). The application of CoCl2 does not affect postsynaptic elements, as can be seen in Fig. 1C
215 B
A
W++++++-
D
Fig. 1. Postsynaptic activity evoked by cereal nerve stimulation. A and B : extracellular recording from connectives. A: stimulation of control cereal nerve evokes both giant and small fiber activity within the connectives. B: no activity is evoked following stimulation of CoClz-treated cereal nerve 48 h after treatment. C and D: intracellular recording from a medial giant neuron* following stimulation of control (C) and treated(D) cereal nerves. This giant interneuron normally responds to stimulation of both cereal nerves. Giant axon activity in C indicates that absence of activity in D is not due to destruction of the postsynaptic giant interneuron. Calibration: 20 msec; 2 mV (A and B) 20 mV (C and D).
and 1D. Here the electrical intracellularly.
Electrical
activity
stimulation
of a medial giant interneuron
(MGI)
is recorded
of the treated cereal nerve did not evoke any res-
ponses (Fig. ID) in the giant interneuron, although normally it can be activated by electrical stimulation of either cereal nerve. The integrity of this giant interneuron is demonstrated in Fig. 1C where synaptic and spiking activity were generated by stimulation of the contralateral untreated cereal nerve. Thus the external application of CoCla affected the cereal nerve itself and not postsynaptic elements. In order to test the extent of the effect on the cereal nerve a biochemical assay was used. The putative neurotransmitter of the cereal system in insects is acetylcholine (ACh)ls which is synthesized by the enzyme choline acetyltransferase (CAT). Following degeneration by external application of CoCls, CAT activity in the cereal nerve was markedly reduced. Choline acetyltransferase activity was measured by scintillation counting of labeled ACh synthesized from [3H]AcCoA incubated with homogenates of the cereal nervesa. Crickets kept at 30 “C for 3 days following cobalt chloride treatment showed a reduction of 87 x, (S.D. = 8, n = 11) in CAT activity in the treated cereal nerves as compared to the control contralateral cereal nerves. Reduction in CAT activity reaches values of 94% (S.D. = 4, n = 5) after 8 days. The decline in CAT activity is not due to the presence of residual cobalt chloride or degeneration products in the enzyme assay. This was ascertained by using mixtures of control and treated CN homogenates which gave CAT activity values equal to the sum of the two homogenates assayed separately. The decrease in CAT activity following application of CoCl2 was found to be temperature-dependent: at 20 “C, 3 days following treatment, only a slight (< 1%) reduction in activity was observed as compared to the 87 “/, reduction at 30 “C. A similar time course and temperature dependence of CAT activity reduction was found in cereal nerves of 28 crickets following mechanical amputation of the cercus. Amputation removes the somata and initial axonal regions situated in the cercus itselfs. * See ref. 8 for identification
criteria.
216 The morphological appearance of neurons is often used as the maiu criterion for degeneration. Electron micrographs of cereal nerve cross-sections, 4 clays after CoCItreatment,
show marked axonal degeneration
(Fig. 2). Only a few axons remain intact.
Fig. 2. Electron micrographs of cereal nerve cross- sections. CNs were dissected and fixed iu 1“/; gluteraldehyde-100 mM cacodylate buffer and osmium post-fixed. A and B: normal, untreated cereal nerve. C and D: CN 4 days after CoClz treatment. Note clear axonal profiles in untreated nerve surrounded by glia wrappings (A and B). In the degenerating nerve (C and D) only few axons remain intact, others are characterized by swollen mitochondria and aggregations of electron dense axopiasm. CaIibration: 5 !Lrn A and B, 1.5 /drn C and D.
217
ofnormal
the majority lack the clear delineations of glia membrane wrappings Other visible signs of degeneration include membrane invaginations, axoplasm
aggregations
with swollen
mitochondria,
lysosomes
Similar results are obtained after cereal amputation. Progressive covering of cereal hairs with petroleum
electron
and myelin
evident
by proper
selection
of stimulus
MGI activity can be evoked after covering these conditions,
the distal two-thirds
abdominal cord. Utilizing applicability of the CoCls destroy the proximal input, tion of cobalt chloride may affecting the distal sensilla.
parameters.
the proximal
evokes activity
bodies.
jelly from the tip to the base
of a cercus shows that the MGI has a stronger input from the proximal especially
axons. dense
regionlO. This is
At 900 Hz, 8.5 dB no
one-third
of a cercus. Under
in small fibers ascending
in the
this proximo - distal gradient one can demonstrate the method for selective degeneration. In order to selectively amputation cannot be used. Nevertheless, local applicacause degeneration of these proximal sensory cells without This was achieved by inserting an empty glass capillary
over one cercus. The capillary was then filled with the aid of a thin J-shaped micropipet from the end close to the base of the cercus. ln this manner only a small proximal region of the cercus was covered with the cobalt chloride solution. Following 1 h of CoCla application the solution was withdrawn and the capillary removed. The animals were then tested 8-10 days post-treatment electrophysiologically. The results of these experiments are summarized in Fig. 3. Acoustic stimulation evoked giant interneuron activity in the connective on the control (non-treated) side, while no giant fiber activity could be recorded from the ipsilateral connective (on the treated side). This treatment did not affect the distal sensilla on the cercus which could be activated by stronger high frequency stimulation, whereupon they activated small fibers in the connective but no giant fiber activity was evoked (Fig. 3C). These results show that the application of CoClz abolished activity originating in the proximal, treated region of the cercus while the distal, untreated area, remained effective in evoking electrical activity in response to acoustic stimulation. The degeneration is not a result of either osmotic or mechanical effects of the treatment. This was shown in control experiments where a 0.5 M CaClz solution was used and had no effect on the integrity
of the cereal system.
possible mode of action of CoCla come from the similarity
Indications
as to the
in time course and temper-
ature dependence of degeneration neuron is filled relatively rapidly
induced either by Co& or amputation. Although a with cobalt ions t2, the degeneration proceeds at the same slow time course of that induced by mechanical axotomy. This suggests that the degenerating effect of COCIZ commences in the somata of treated neurons and mimics the effect of separating the soma from its axon rather than having a direct effect on the axon. It has been shown for other systems that calcium ions are necessary for loading of material that is to be transported from the soma centrifugally”. Cobalt ions interfere
with this process7 and prevent the transport tional axotomy.
of essential substances,
thus causing func-
The method for degeneration of specific inputs described here is applicable where other methods are not suitable. Surgical intervention, the common method for inducing neuronal degeneration causes damage to non-neural elements as well, while the
_-s_--Fig. 3. Electrical activity recorded with hook electrodes from the connectives in response to acoustic stimulation. A-D upper trace: recording from connective on control side; lower trace: connective on treated side. The proximal one-third of the treated cercus was covered with 0.5 M COG% for 1 h 10 days prior to recordings. A : stimulation at 325 Hz, 70 dB evokes giant intemeuron activity on the control side and only small fiber activity on the treated side. Giant fiber activity is identified by the large spikes. The small fiber activity in treated side originates in the contralateral, control cercus and is abolished by covering the proximal one-third of this cercus with petroleum jelly (B). C: stronger stimulation (900 Hz, 85 dB chosen due to relative lower sensitivity of distal region)-evokes smali Bber activity on the treated side. The spikes in the control connective originate from contralateral activation since the control cercus was completely covered with petroleum jelly. This activity is abolished when the distal two-thirds of the treated cercus are covered with petroleum jelly. Areas of the cercus covered with petroleum jelly are indicated on the right. Shaded areas of the cercus indicates the region treated with cobalt chloride. Stimulus duration: 40 msec(A and B), 400 msec (C and D).
CoClz permits degeneration of neurons, leaving the sensory organ itself intact. Some suggested applications of the method include degeneration of proximal elements where amputation cannot be applied (vide infra) and degeneration of centrally located cell bodies within the central nervous system by using the backfilling techniquels in vivo. We are now using the CoCla degeneration method in our studies of neuronal development and interaction between different sensory inputs impinging on a common target neuron. When CoCla is applied to cerci of cricket nymphs, with regenerative capacity, new functional sensory cells develop within 10 days following the degeneration of existing cells?. This indicates that epidermal cells, located at the baseof the cercus, are not destroyed by application of CoCls and retain their potential for regeneration of a cercus.
219 This National
research
was supported
Science Foundation
Israel Center
Psychobiology,
We are grateful technical reading
assistance
by grants
(62.5) Jerusalem, Charles
to A. Dorman
from
Smith Family for electron
and to Prof. 1. Parnas
the
United
States-Israel
Israel, and by Grant 221/79-l
Bi-
from the
Foundation. microscopy,
and Prof.
to Y. Margolin
R. Hammerschlag
for
for critical
of the manuscript.
l Bowling, D., Nicholls, J. and Parnas, I., Destruction of a single cell in the C.N.S. of the leech as a means of analysing its connexions and functional role, J. Physiol. (Land.), 282 (I 978) 1699180. 2 Dagan, D. and Sarne, Y., Evidence for cholinergic nature of cockroach giant fibers, J. conrp. Pkysiol., 126 (1978) 157-160. 3 Dagan, D., Lecker, S., Margolin, Y. and Sarne, Y., Delayed differentiation of an insect sensory system: development of biochemical and electrophysiological properties prior to sensory activation, in preparation. 4 Dravid, A. R. and Hammerschlag, R., Axoplasmic transport of proteins in vitro in primary afferent neurons of frog spinal cord: effect of Ca ++-free incubation conditions, J. Neurochem., 24 (1975) 71 l-718. 5 Edwards, J. S. and Palka, J., The cerci and abdominal giant fibres of the house cricket Achern domesticus, Proc. roy. Sot. B, I85 (1974) 83-103. 6 Hess, A., Experimental anatomical studies of pathways in the severed central nerve cord of the cockroach, J. Morphol., 103 (1958) 479-499. 7 Lavoie, P. A., Hammerschlag, R. and Tjan, A., Cobalt ions inhibit fast axonal transport of [sH]glycoproteins but not glycosylation, Bruin Reseurch, 149 (1978) 535-540. 8 Murphey, R. K., Palka, J. and Hustert, R., The cercus-to-giant interneurons, J. romp. Physiol.,
I I9 (1977) 2855300. 9 Palka, J. and Edwards, J. S., The cerci and abdominal giant fibres of the house cricket, Acheta domesticus. II. Regeneration and effects of chronic deprivation, Proc. Roy. Sot. B, 185 (1974) 105-121. IO Palka, J. and Oldberg, R., The cercus-to-giant interneuron system of crickets. III. Receptive field organization, J. camp. Physiol., 119 (1977) 301-317. I I Parnas, J. and Bowling, D., Killing of single neurones by intracellular injection of proteolytic enzymes, Nature (Land.), 270 (1977) 626-628. I2 Pitman, R. M., Tweedle, C. D. and Cohen, M. J., The form of nerve cells determination by cobalt impregnation. In S. B. Kater and C. Nicholson (Eds.), Intracellular Staining in Neurobiology, Springer-Verlag, Berlin, 1973, pp. 83-97. I3 Rehbein, H. G., Kalmring, K. and Romer, H., Structure and function of acoustic neurons in the thoracic ventral nerve cord ofLocusta migratoria (Acrididae), J. camp. Physiol., 95 (1974) 263-280. 14 Rubin, R. P., Calcium and the Secretory Process, Plenum Press, New York, 1974. 15 Sanes, J. R., Hildebrand, J. G. and Prescott, D. J., Differentiation of insect sensory neurons in absence of their normal synaptic targets, Develop. Biol., 52 (1976) 121-127. 16 Shankland, D. L., Rose, J. A. and Donniger, C., The cholinergic nature of the cereal nerve-giant fibre synapse in the sixth abdominal ganglion of the American cockroach, Periplaneta americana (L) J. Neurobiol., 2 (1971) 247-262. 17 Tsukahara, N., Hultborm, H. and Murakami, F., Sprouting of corticorubral synapses in red nucleus neurones after destruction of the nucleus interpositus of cerebellum, Experientia (Base/), 30 (1974) 57-58.
