- Email: [email protected]
Electrophysiological changes accompanying DSP-4 lesions of rat locus coeruleus neurons
.
317
Brain Research, 628 (1993) 317-320 Elsevier Science Publishers B.V.
. BRES 25889
Electrophysiological
changes accompanying DSP-4 lesions of rat locus coeruleus neurons
David SK. Magnuson al*, William A. Staines b, Kenneth C. Marshall a aDepartment of Physiology and b Department of Anatomy and Neurobiology, University of Ottawa, Ottawa, Ont., KlH 8M5, Canada (Accepted 10 August 1993)
Key words: Neurotoxin; DSP-4; Locus coeruleus; Noradrenergic
neuron; Electrophysiology; Ca’+ spike
The electrophysiological characteristics of intracellularly recorded locus coeruleus (LC) neurons in brain stem slices from DSP-4 treated animals have been compared to those from untreated controls. LC neurons from DSP-4 treated animals had action potentials and Ca2+ spikes (elicited in the presence of,TTX) of significantly reduced duration compared to controls. These observations suggest that chemical axotomy with DSP-4 reduces Ca2’ conductance in neurons of the locus coeruleus.
Studies of degeneration and regeneration of central neurons have benefitted greatly from the use of transmitter-specific toxins, which enter neurons by way of re-uptake systems, thereby accumulating to toxic levels selectively in neurons with the specific uptake carrier. One of these, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)” appears to act selectively on noradrenergic neurons of the locus coeruleus (LCj6. The actions of DSP4 are rapid, with an almost complete loss of noradrenergic staining in the neocortex, hippocampal formation and parts of the cerebellum at 24 h post-injection; the distribution of affected fibers parallels that of LC efferent innervation’.3-5. Fritschy and Grzanna5 have demonstrated that the acute loss of locus coeruleus efferents is followed by recovery or regeneration of the noradrenergic fibers to levels approaching those of controls by 1 year post-injection. Although the morphology of locus coeruleus neuronal somata appears normal even up to several weeks postDSP4 injection, with doses of 50 mg/kg or more substantial decreases in locus coeruleus cell numbers have been reported3,4. While the morphological and behavioral consequences of catecholamine-specific neurotoxins have been quite well documented, the electrophysiological
manifestations of such chemical lesions at the single cell level have been explored relatively little. In the present study, therefore, we have tested the hypothesis that chemical axotomy caused by treatment with moderate doses of the neurotoxin DSP4 elicits electrophysiological changes similar to those observed in other central neurons following mechanical axotomy. Transverse pontine slices (400 pm) were prepared from DSP-4 injected or uninjected control male Sprague-Dawley rats using slice preparation techniques similar to those described by Williams et al.“. Slices were maintained in a submersion-style slice chamber continuously perfused at 1.3-1.7 ml/min. with ACSF bubbled with 95% 0,/5% CO, at 31-33°C and pH 7.3-7.4. The ACSF was composed of (mM): NaCl 118, KC1 3.0, NaH,PO, 1.0, MgSO, 0.81, CaCl, 2.5, glucose 10, and NaHCO, 24. Intracellular recordings were made using glass electrodes (2 M KAc) with DC tip resistances between 100 and 150 MO, and Axon Instruments amplifiers/ acquisition hardware and software. The toxin DSP4 was administered by i.p. injection as described by Fritschy and Grzanna4. Injected animals weighed a minimum of 70 g and whenever possible, age matched control animals were used in alternate experiments for each survival time.
* Corresponding author. Present address: Department of Physiology, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Man., R3E OW3, Canada. Fax: (1) (204) X6-0932. Email: [email protected].
318 TABLE
I
Electrophysiological
properties of LC neurons following
DSP-4
treat-
ment Neurons tested from 1-41 days following DSP-4 injections. Data given as mean+S.D. (n). Records from cells not included had unacceptably high error during curve fitting by the PClamp 5.5 ‘Fit’ program.
DSP-4 injected Input resistance (M0) Time constant (ms) Resting potential (mV1 Afterhyperpolarization (mV1 AP height (mV1 AP threshold potential (mV) * P < 0.05; unpaired
175.4*57
(17) (9) -53.4+15 (16) 27.0+ 2.6 (18) 54.7* 6 (15) - 46.5 + 3.2 (18) * 34.3+
8
5msec 5msec Fig. I. On the left are two LC neuron action potentials, one from a DSP-4 treated animal and the other from a control animal. Each action potential is averaged from 8 or more sweeps. The spikes are similar in magnitude apart from the size of the shoulder on the falling phase, which is reduced in the DSP-4 treated neuron. The single arrow at left indicates the spike threshold potential, used to determine the falling phase durations as indicated by the double arrows. On the right are two Ca2+ -spikes from the same neurons as at left, recorded in the presence of 500 nM TTX. Each is averaged from at least 8 sweeps. The Ca2+ -spike recorded from the DSP-4 treated neuron is reduced in both amplitude and duration compared to that of control.
Uninjected controls
172.9+57 * 27.25 IO -55.8+ 6 27.3+ 5 53.7+ 5 - 45.9 + 3.4
(17) (14) (13) (14) (11) (141
f-test.
The histological manifestations of DSP-4 treatment have been thoroughly documented by Fritschy, Grzanna and their colleagues 4.18. In the present study, immunoperoxidase localization of tyrosine hydroxylase or dopamine p-hydroxylase’6, provided histological evidence that our DSP-4 injections of 30, 40 and 50 mg/kg were causing the expected depletion of NA terminal fields. Light microscopic examination of immunostained 14 pm cryostat sections from treated and control brains revealed, at 2 and 4 weeks following 30 mg/ kg injections of DSP4, an absence of NA terminal fields in the neo-, piriform, and olfactory cortices and the hippocampal formation. Other brain areas which do not receive NA innervation from the LC remained unchanged when compared to controls. Our findings agree, therefore, with those of Fritschy and Grzanna’ and show that 30 mg/ kg DSP-4 is sufficient to dramatically reduce NA terminal fields supplied by the LC; we therefore chose the lower dose of 30 mg/ kg for the bulk of our study. The electrophysiology of LC neurons contained in transverse pontine slices has been well documented”. Our findings largely parallel the observations by Williams et al., and apparent differences may be accounted for by the differing composition of the super-
TABLE
fusates used. The electrophysiological properties measured included the input resistance (R,), the time constant (tau), the threshold potential for action potential generation, the action potential amplitude, and the amplitude of the afterhyperpolarization following each action potential. The threshold potential, action potential amplitude, and afterhyperpolarization amplitude were unaffected by DSP-4 (Table I). The duration of the action potential was, however, significantly reduced in neurons recorded in slices from DSP4 treated animals. The action potentials recorded from treated neurons often appeared to have a less substantial shoulder on the falling phase compared to controls (Fig. 1). This suggested that the underlying Ca2+ spike was reduced in size after DSP-4 treatment, which was supported by experiments in which 300-600 nM ITX was added to the superfusate to remove the fast-Na+portion of the action potential. The TTX-insensitive spikes recorded in treated cells were found to have significantly reduced durations compared to untreated controls (Table 11 and Fig. 1). In addition, a significant increase in tau was observed for LC neurons following DSP-4 treatment (Table II). Since both rising and falling
11
Data given as mean_+S.D.
(n).
A. LC action potential duration following DSP-4 treatment Control * 1.59+0.14
3h
1.52kO.13
(21)
B. LC Ca2+-spike
I - 12 days
(6)
* 1.38kO.13
duration in TTXfollowing
I-12days
+ 1.91+0.08(11)
+ 1.73f0.16
*‘P
< 0.05; unpaired
differences
t-tests; control using unpaired
50 - 200 days
I - 41 days
1.53+0.14
1.59kO.17
* 1.4650.15
(14)
(6)
I - 200 days
(25)
1.49+0.16
(31)
DSP-4 treatment
Control
significant
(11)
20 - 41 days
(6)
vs. 1-12 days, control t-tests.
20 - 53 days
I - 53 days
1.89+0.19
1.77f0.19
vs. 1-41
(4)
(10)
days and control vs. l-12
days (Ca2+- spike duration)
showed
statistically
319
phas’es of action potentials are actively propagated events, an increase in tau is unlikely to alter the action potential duration, and the observed decrease in duration is not likely to be related to the change in tau. An increase in tau without a change in R,, suggests that the area of membrane participating in the capacitative response to current injection has increased, and may be explained by sprouting of processes close to the cell body or by increases in electrical coupling of neurons, which has been indicated in LC neurons of young adult rats (J.T. Williams and P. Osborne, personal communication). A proportion of LC neurons recorded in slices from either control or DSP-4 injected animals displayed some level of spontaneous activity, often accompanied by an oscillatory resting potential. In slices from untreated rats more than 75% of neurons showed rates of spontaneous activity greater than 0.5 Hz compared to only 48% of neurons in slices taken from treated rats. This observation may also suggest a drop in Ca*+ conductance, particularly since the spontaneous activity of LC neurons is thought to be due to a Ca2+ conductance persistent at the RMP”. A sensitive measure of voltage-dependent membrane properties is the F/Z relationship (firing/ injected current). In an effort to assess the effects of DSP-4 on these properties, we measured the firing characteristics of treated and untreated LC neurons in two ways. We compared the intervals between the first and second, and second and third spikes in a train induced by relatively large, 200 ms duration depolarizing current pulses. LC neurons recorded from untreated slices had a spike-train ratio of 0.749 k 0.14 (n = 8; &SD.) while those from treated slices had a ratio of 0.730 + 0.07 (n = 11; &-SD.). In the second analysis, the intervals (as a ratio) between the first pair of spikes were compared for two successive current steps which differed by 0.05 nA. The paired-interval ratio from cells recorded in control slices was 0.71 + 0.09 (n = 19; &S.D.) while that from cells in treated slices was 0.64 f 0.12 (n = 18; +S.D.>. These ratio means are not significantly different using unpaired t-tests. A total of 5 neurons were recorded with DSP-4 applied directly in the superfusate for a variety of time periods from 20 to 60 min, and 3 of these recordings were made for at least 2 h following application of the toxin. No consistent electrophysiological changes were observed during these recordings and the supposition might be made that the toxin’s actions are slow in onset and/or are dependent on the presence of neuronal terminals which are removed by the slicing procedure.
The major findings of the present work are in two areas. First of all, we have observed that the noradrenergic specific toxin DSP-4 causes measurable changes in the electrophysiology of LC neurons which may be comparable to those caused by mechanical axotomy of other central neurons. Secondly, we have found that the electrophysiological manifestations of chemical axotomy with DSP-4 observed under these experimental conditions appear to include a reduction of the action potential and TTX-insensitive Ca2+ spike durations, and the proportion of LC neurons which exhibit spontaneous activity at rest. An additional observation of interest is the time course of the electrophysiological changes, which may relate to either a recovery from a lengthy, albeit acute stage of toxicity, or may relate to the reestablishment of contact with target tissue. Unlike the findings reported for cat motoneurons and frog sympathetic ganglion neurons following mechanical axotomies2,7.‘2*‘“, we did not observe any changes in afterhyperpolarization amplitude or action potential height which might indicate that LC neuron K+conductances were decreased post-DSP-4. We did not make any direct measurements of K+conductance, and therefore, cannot rule-out an involvement of Kfin the observed changes to action potential duration. Our observations suggest that chemical axotomy with DSP-4 results in a reduction of LC neuron Ca2+ current(s) without significant alteration of other electrophysiological parameters. For the majority of central neurons, it might be expected that a significant reduction in Ca2+ flux would have additional indirect effects, due to changes in Ca 2+-dependent currents, particularly those of K+. The substantial resting Ca2+ influx of LC’neurons, in addition to the voltage-dependent currents which result in the Ca2+ spike” might supra-maximally activate Cazf-dependent conductances, so that a reduction of Ca*+ flux would have to be larger than that observed to have any secondary electrophysiological consequences. Similar transient reductions in calcium conductance have been reported following mechanical axotomy of vagal motoneurons in the guinea pig14. They further demonstrated that both a Ca’+-dependent K+current and the A-current were reduced, the latter presumed to account for a dramatic attenuation of the accommodative responses of these neurons to depolarizing current injection. In our assessment of LC neuron responses to current injection, no such additional changes were observed. Fritschy and Grzanna have shown dramatic increases in NA terminal fields, compared to uninjected controls, in some brain areas at 6 months and 1 year post-DSP-4 treatment. Assuming the majority of LC
320 neurons innervate multiple target sites, at varying distances from pre-terminal axons which remain intact after DSP-4-?, it seems reasonable to suggest that recovery of LC electrophysiology may accompany reestablishment of target tissue contact, and that cell death after this time involves cells which were unsuccessful at competing for target tissue sites. Suggestions that target organ influences are responsible for recovery of electrophysiological manifestations of axotomy have been proposed for motoneurons by Gustafsson and Pinter”, for vagal motoneurons by Laiwand et a1.14, and for frog sympathetic ganglion cells by Kelly et aLI2 and Jassar and Smith”‘. The authors would like to acknowledge the excellent technical assistance given by Dr. Tomasz Woloszyn, Ms. Babben Tinner-Staines and Ms. Lucy Pickavance. Supported by the Canadian Network of Centres of Excellence in Neural Regeneration and Functional Recovery. Bickford, P.C., Mosimann, W.F., Hoffer, B.J. and Freedman, R., Effects of the selective noradrenergic neurotoxin DSP-4 on cerebellar purkinje neuron electrophysiology, Life Sci., 34 (1984) 731-741. Foehring, R.C., Sypert, G.W. and Munson, J.B., Properties of self-reinnervated motor units of medial gastrocnemius of CatII. Axotomized motoneurons and time course of recovery, J. Neurophysiol., 55 (1990) 947-965. 3 Fritschy, J.-M., Geffard, M. and Grzanna, R., The response of noradrenergic axons to systemically administered DSP-4 in the rat: an immunocytochemical study using antibodies to noradrenaline and dopamine-beta-hydroxylase, J. Chem. Neurounat., 3 (19901309-321. 4 Fritschy, J.-M. and Grzanna, R., Immunohistochemical analysis of the neurotoxic effects of DSP-4 identifies two populations of noradrenergic axon terminals, Neuroscience, 30 (1989) 181-197. 5 Fritschy, J.-M. and Grzanna, R., Experimentally induced neuron loss in the locus coeruleus of adult rats, Exp. Neural., 111 (1991) f23-7.
6 Grzanna, R., Berger, LJ., Fritschy, J.-M. and Geffard, M.;Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects, J. Histochem. Cytochem., 37 (1989) 1435-1442. 7 Gustafsson, B., Changes in motoneurone electrical properties following axotomy, J. Physiol., 293 (1979) 197-215. 8 Gustafsson, B. and Pinter, M.J., Effects of axotomy on the distribution of passive electrical properties of cat motoneurons, J. Physiol., 356 (1984) 433-442. 9 Jassar, B.S. and Smith, P.A., Effects of axotomy on sodium currents in bullfrog sympathetic ganglion neurons, Can. J. Physiol. Pharm., 70 (1992) Axii. 10 Jassar, B.S. and Smith, P.A., Role of target organ in the maintenance of calcium currents in bullfrog sympathetic neurons, Sot. Veursci. Abstr., 17 (1992) 67. 11 Jonsson, G., Hallman, H., Ponzio, F. and Ross, S., DSP-4 (N-(2chloroethyll-N-ethyl-2-bromobenzylamine) - a useful denervation tool for central and peripheral noradrenaline neurons, Eur. J. Pharmacol., 72 (1981) 173-188. 12 Kelly, M.E.M., Bisby, M.A. and Lukowiak, K., Regeneration restores some of the altered electrical properties of axotomized bullfrog B-cells, J. Neurobiol., 19 (1988) 357-372. 13 Kelly, M.E.M, Bisby, M.A. and Lukowiak, K., The effects of axotomy on electrophysiological properties of B cells of bullfrog sympathetic ganglia conditioned by a previous lesion, Can. J. Physiol. Pharmacol., 66 (1988) 942-945. 14 Laiwand, R., Werman, R. and Yarom, Y., Electrophysiology of degenerating neurons in vagal motor nucleus of the guinea-pig following axotomy, J. Physiol., 404 (1988) 749-766. 15 Nakai, K., Jonsson, G. and Kasamatsu, T., Norepinephrinergic reinnervation of cat occipital cortex following localized lesions with h-hydroxydopamine, Neurosci Res., 4 (1987) 433-453. 16 Staines, W.A., Yamamoto, T., Daddona, P.E. and Nagy, J.I., Neuronal colocalization of adenosine deaminase, monoamine oxidaes and 5-hydroxytryptophan uptake in the tuberomammillary nucleus of the rat, Bruin Res. Bull., 17 (1986) 351-3 17 Williams, J.T., North, R.A., Shefner, S.A., Nishi, S. and Egan, T.M., Membrane properties of rat locus coeruleus neurons, Neuroscience, 13 (1984) 137-156. 18 Zaczek, R., Fritschy, J.-M., Gulp, S., De Souza, E. and Grzanna, R., Differential effects of DSP-4 on noradrenaline axons in cerebral cortex and hypothalamus may reflect heterogeneity of noradrenaline uptake sites, Brain Res., 522 (1990) 308-314.
317
Brain Research, 628 (1993) 317-320 Elsevier Science Publishers B.V.
. BRES 25889
Electrophysiological
changes accompanying DSP-4 lesions of rat locus coeruleus neurons
David SK. Magnuson al*, William A. Staines b, Kenneth C. Marshall a aDepartment of Physiology and b Department of Anatomy and Neurobiology, University of Ottawa, Ottawa, Ont., KlH 8M5, Canada (Accepted 10 August 1993)
Key words: Neurotoxin; DSP-4; Locus coeruleus; Noradrenergic
neuron; Electrophysiology; Ca’+ spike
The electrophysiological characteristics of intracellularly recorded locus coeruleus (LC) neurons in brain stem slices from DSP-4 treated animals have been compared to those from untreated controls. LC neurons from DSP-4 treated animals had action potentials and Ca2+ spikes (elicited in the presence of,TTX) of significantly reduced duration compared to controls. These observations suggest that chemical axotomy with DSP-4 reduces Ca2’ conductance in neurons of the locus coeruleus.
Studies of degeneration and regeneration of central neurons have benefitted greatly from the use of transmitter-specific toxins, which enter neurons by way of re-uptake systems, thereby accumulating to toxic levels selectively in neurons with the specific uptake carrier. One of these, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)” appears to act selectively on noradrenergic neurons of the locus coeruleus (LCj6. The actions of DSP4 are rapid, with an almost complete loss of noradrenergic staining in the neocortex, hippocampal formation and parts of the cerebellum at 24 h post-injection; the distribution of affected fibers parallels that of LC efferent innervation’.3-5. Fritschy and Grzanna5 have demonstrated that the acute loss of locus coeruleus efferents is followed by recovery or regeneration of the noradrenergic fibers to levels approaching those of controls by 1 year post-injection. Although the morphology of locus coeruleus neuronal somata appears normal even up to several weeks postDSP4 injection, with doses of 50 mg/kg or more substantial decreases in locus coeruleus cell numbers have been reported3,4. While the morphological and behavioral consequences of catecholamine-specific neurotoxins have been quite well documented, the electrophysiological
manifestations of such chemical lesions at the single cell level have been explored relatively little. In the present study, therefore, we have tested the hypothesis that chemical axotomy caused by treatment with moderate doses of the neurotoxin DSP4 elicits electrophysiological changes similar to those observed in other central neurons following mechanical axotomy. Transverse pontine slices (400 pm) were prepared from DSP-4 injected or uninjected control male Sprague-Dawley rats using slice preparation techniques similar to those described by Williams et al.“. Slices were maintained in a submersion-style slice chamber continuously perfused at 1.3-1.7 ml/min. with ACSF bubbled with 95% 0,/5% CO, at 31-33°C and pH 7.3-7.4. The ACSF was composed of (mM): NaCl 118, KC1 3.0, NaH,PO, 1.0, MgSO, 0.81, CaCl, 2.5, glucose 10, and NaHCO, 24. Intracellular recordings were made using glass electrodes (2 M KAc) with DC tip resistances between 100 and 150 MO, and Axon Instruments amplifiers/ acquisition hardware and software. The toxin DSP4 was administered by i.p. injection as described by Fritschy and Grzanna4. Injected animals weighed a minimum of 70 g and whenever possible, age matched control animals were used in alternate experiments for each survival time.
* Corresponding author. Present address: Department of Physiology, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Man., R3E OW3, Canada. Fax: (1) (204) X6-0932. Email: [email protected].
318 TABLE
I
Electrophysiological
properties of LC neurons following
DSP-4
treat-
ment Neurons tested from 1-41 days following DSP-4 injections. Data given as mean+S.D. (n). Records from cells not included had unacceptably high error during curve fitting by the PClamp 5.5 ‘Fit’ program.
DSP-4 injected Input resistance (M0) Time constant (ms) Resting potential (mV1 Afterhyperpolarization (mV1 AP height (mV1 AP threshold potential (mV) * P < 0.05; unpaired
175.4*57
(17) (9) -53.4+15 (16) 27.0+ 2.6 (18) 54.7* 6 (15) - 46.5 + 3.2 (18) * 34.3+
8
5msec 5msec Fig. I. On the left are two LC neuron action potentials, one from a DSP-4 treated animal and the other from a control animal. Each action potential is averaged from 8 or more sweeps. The spikes are similar in magnitude apart from the size of the shoulder on the falling phase, which is reduced in the DSP-4 treated neuron. The single arrow at left indicates the spike threshold potential, used to determine the falling phase durations as indicated by the double arrows. On the right are two Ca2+ -spikes from the same neurons as at left, recorded in the presence of 500 nM TTX. Each is averaged from at least 8 sweeps. The Ca2+ -spike recorded from the DSP-4 treated neuron is reduced in both amplitude and duration compared to that of control.
Uninjected controls
172.9+57 * 27.25 IO -55.8+ 6 27.3+ 5 53.7+ 5 - 45.9 + 3.4
(17) (14) (13) (14) (11) (141
f-test.
The histological manifestations of DSP-4 treatment have been thoroughly documented by Fritschy, Grzanna and their colleagues 4.18. In the present study, immunoperoxidase localization of tyrosine hydroxylase or dopamine p-hydroxylase’6, provided histological evidence that our DSP-4 injections of 30, 40 and 50 mg/kg were causing the expected depletion of NA terminal fields. Light microscopic examination of immunostained 14 pm cryostat sections from treated and control brains revealed, at 2 and 4 weeks following 30 mg/ kg injections of DSP4, an absence of NA terminal fields in the neo-, piriform, and olfactory cortices and the hippocampal formation. Other brain areas which do not receive NA innervation from the LC remained unchanged when compared to controls. Our findings agree, therefore, with those of Fritschy and Grzanna’ and show that 30 mg/ kg DSP-4 is sufficient to dramatically reduce NA terminal fields supplied by the LC; we therefore chose the lower dose of 30 mg/ kg for the bulk of our study. The electrophysiology of LC neurons contained in transverse pontine slices has been well documented”. Our findings largely parallel the observations by Williams et al., and apparent differences may be accounted for by the differing composition of the super-
TABLE
fusates used. The electrophysiological properties measured included the input resistance (R,), the time constant (tau), the threshold potential for action potential generation, the action potential amplitude, and the amplitude of the afterhyperpolarization following each action potential. The threshold potential, action potential amplitude, and afterhyperpolarization amplitude were unaffected by DSP-4 (Table I). The duration of the action potential was, however, significantly reduced in neurons recorded in slices from DSP4 treated animals. The action potentials recorded from treated neurons often appeared to have a less substantial shoulder on the falling phase compared to controls (Fig. 1). This suggested that the underlying Ca2+ spike was reduced in size after DSP-4 treatment, which was supported by experiments in which 300-600 nM ITX was added to the superfusate to remove the fast-Na+portion of the action potential. The TTX-insensitive spikes recorded in treated cells were found to have significantly reduced durations compared to untreated controls (Table 11 and Fig. 1). In addition, a significant increase in tau was observed for LC neurons following DSP-4 treatment (Table II). Since both rising and falling
11
Data given as mean_+S.D.
(n).
A. LC action potential duration following DSP-4 treatment Control * 1.59+0.14
3h
1.52kO.13
(21)
B. LC Ca2+-spike
I - 12 days
(6)
* 1.38kO.13
duration in TTXfollowing
I-12days
+ 1.91+0.08(11)
+ 1.73f0.16
*‘P
< 0.05; unpaired
differences
t-tests; control using unpaired
50 - 200 days
I - 41 days
1.53+0.14
1.59kO.17
* 1.4650.15
(14)
(6)
I - 200 days
(25)
1.49+0.16
(31)
DSP-4 treatment
Control
significant
(11)
20 - 41 days
(6)
vs. 1-12 days, control t-tests.
20 - 53 days
I - 53 days
1.89+0.19
1.77f0.19
vs. 1-41
(4)
(10)
days and control vs. l-12
days (Ca2+- spike duration)
showed
statistically
319
phas’es of action potentials are actively propagated events, an increase in tau is unlikely to alter the action potential duration, and the observed decrease in duration is not likely to be related to the change in tau. An increase in tau without a change in R,, suggests that the area of membrane participating in the capacitative response to current injection has increased, and may be explained by sprouting of processes close to the cell body or by increases in electrical coupling of neurons, which has been indicated in LC neurons of young adult rats (J.T. Williams and P. Osborne, personal communication). A proportion of LC neurons recorded in slices from either control or DSP-4 injected animals displayed some level of spontaneous activity, often accompanied by an oscillatory resting potential. In slices from untreated rats more than 75% of neurons showed rates of spontaneous activity greater than 0.5 Hz compared to only 48% of neurons in slices taken from treated rats. This observation may also suggest a drop in Ca*+ conductance, particularly since the spontaneous activity of LC neurons is thought to be due to a Ca2+ conductance persistent at the RMP”. A sensitive measure of voltage-dependent membrane properties is the F/Z relationship (firing/ injected current). In an effort to assess the effects of DSP-4 on these properties, we measured the firing characteristics of treated and untreated LC neurons in two ways. We compared the intervals between the first and second, and second and third spikes in a train induced by relatively large, 200 ms duration depolarizing current pulses. LC neurons recorded from untreated slices had a spike-train ratio of 0.749 k 0.14 (n = 8; &SD.) while those from treated slices had a ratio of 0.730 + 0.07 (n = 11; &-SD.). In the second analysis, the intervals (as a ratio) between the first pair of spikes were compared for two successive current steps which differed by 0.05 nA. The paired-interval ratio from cells recorded in control slices was 0.71 + 0.09 (n = 19; &S.D.) while that from cells in treated slices was 0.64 f 0.12 (n = 18; +S.D.>. These ratio means are not significantly different using unpaired t-tests. A total of 5 neurons were recorded with DSP-4 applied directly in the superfusate for a variety of time periods from 20 to 60 min, and 3 of these recordings were made for at least 2 h following application of the toxin. No consistent electrophysiological changes were observed during these recordings and the supposition might be made that the toxin’s actions are slow in onset and/or are dependent on the presence of neuronal terminals which are removed by the slicing procedure.
The major findings of the present work are in two areas. First of all, we have observed that the noradrenergic specific toxin DSP-4 causes measurable changes in the electrophysiology of LC neurons which may be comparable to those caused by mechanical axotomy of other central neurons. Secondly, we have found that the electrophysiological manifestations of chemical axotomy with DSP-4 observed under these experimental conditions appear to include a reduction of the action potential and TTX-insensitive Ca2+ spike durations, and the proportion of LC neurons which exhibit spontaneous activity at rest. An additional observation of interest is the time course of the electrophysiological changes, which may relate to either a recovery from a lengthy, albeit acute stage of toxicity, or may relate to the reestablishment of contact with target tissue. Unlike the findings reported for cat motoneurons and frog sympathetic ganglion neurons following mechanical axotomies2,7.‘2*‘“, we did not observe any changes in afterhyperpolarization amplitude or action potential height which might indicate that LC neuron K+conductances were decreased post-DSP-4. We did not make any direct measurements of K+conductance, and therefore, cannot rule-out an involvement of Kfin the observed changes to action potential duration. Our observations suggest that chemical axotomy with DSP-4 results in a reduction of LC neuron Ca2+ current(s) without significant alteration of other electrophysiological parameters. For the majority of central neurons, it might be expected that a significant reduction in Ca2+ flux would have additional indirect effects, due to changes in Ca 2+-dependent currents, particularly those of K+. The substantial resting Ca2+ influx of LC’neurons, in addition to the voltage-dependent currents which result in the Ca2+ spike” might supra-maximally activate Cazf-dependent conductances, so that a reduction of Ca*+ flux would have to be larger than that observed to have any secondary electrophysiological consequences. Similar transient reductions in calcium conductance have been reported following mechanical axotomy of vagal motoneurons in the guinea pig14. They further demonstrated that both a Ca’+-dependent K+current and the A-current were reduced, the latter presumed to account for a dramatic attenuation of the accommodative responses of these neurons to depolarizing current injection. In our assessment of LC neuron responses to current injection, no such additional changes were observed. Fritschy and Grzanna have shown dramatic increases in NA terminal fields, compared to uninjected controls, in some brain areas at 6 months and 1 year post-DSP-4 treatment. Assuming the majority of LC
320 neurons innervate multiple target sites, at varying distances from pre-terminal axons which remain intact after DSP-4-?, it seems reasonable to suggest that recovery of LC electrophysiology may accompany reestablishment of target tissue contact, and that cell death after this time involves cells which were unsuccessful at competing for target tissue sites. Suggestions that target organ influences are responsible for recovery of electrophysiological manifestations of axotomy have been proposed for motoneurons by Gustafsson and Pinter”, for vagal motoneurons by Laiwand et a1.14, and for frog sympathetic ganglion cells by Kelly et aLI2 and Jassar and Smith”‘. The authors would like to acknowledge the excellent technical assistance given by Dr. Tomasz Woloszyn, Ms. Babben Tinner-Staines and Ms. Lucy Pickavance. Supported by the Canadian Network of Centres of Excellence in Neural Regeneration and Functional Recovery. Bickford, P.C., Mosimann, W.F., Hoffer, B.J. and Freedman, R., Effects of the selective noradrenergic neurotoxin DSP-4 on cerebellar purkinje neuron electrophysiology, Life Sci., 34 (1984) 731-741. Foehring, R.C., Sypert, G.W. and Munson, J.B., Properties of self-reinnervated motor units of medial gastrocnemius of CatII. Axotomized motoneurons and time course of recovery, J. Neurophysiol., 55 (1990) 947-965. 3 Fritschy, J.-M., Geffard, M. and Grzanna, R., The response of noradrenergic axons to systemically administered DSP-4 in the rat: an immunocytochemical study using antibodies to noradrenaline and dopamine-beta-hydroxylase, J. Chem. Neurounat., 3 (19901309-321. 4 Fritschy, J.-M. and Grzanna, R., Immunohistochemical analysis of the neurotoxic effects of DSP-4 identifies two populations of noradrenergic axon terminals, Neuroscience, 30 (1989) 181-197. 5 Fritschy, J.-M. and Grzanna, R., Experimentally induced neuron loss in the locus coeruleus of adult rats, Exp. Neural., 111 (1991) f23-7.
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