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Decreased activity of locus coeruleus neurons in the rat after DSP-4 treatment
Neuroscience Letters, 40 (1983) 81-84
81
Elsevier Scientific Pubfishers Ireland Ltd.
DECRLASED ACTIVITY OF LOCUS COERULEUS NEURONS IN THE RAT ~ D S P 4 TREATMENT
H.R. OLPE, J. LASZLO, D,J. DOOLEY*, J. H E I D a n d M.W. STEINMANN
Research Department, Pharmaceuticals Division, CIBA-Geigy Ltd., CH-4002 Basel ($w:'t~erland) (Received April 12th, 1983; Revised version received June 28th, 1983; Accepted June 30th, 1983)
Key words: DSP-4 - noradrenergic neurotoxin - locus coendeus - firing rate - cell morphology - rat
The activity of noradrenergic neurons o f the rat locus cocruleus was investigated at 10 and 50 days after the administration o f DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine), a selective noradrenergic neurotoxin. The mean neuronal firing rate in control animals was 2.4 Hz. In contrast, DSP-4 animals had lower rates o f 1.2 Hz at 10 days and 1.7 Hz at 50 days. Histological examinations revealed no morphological changes o f locus coeruleus cell bodies at either the 10- or 50.day time points. These results suggest that DSP-4 can impair neuronal activity o f the locus coeruleus without altering the. structural appearance o f locus coeruleus perikarya.
A peripheral injection of DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) depletes norepinephrine (NE) in the rodent central nervous system [1, 4, 5, 9]. This depletion is long-lasting and rather selective in that neocortical concentrations of dopamine, 5-hydroxytryptamine, acetylcholine and several amino acids are practically unchanged by the neurotoxin [1, 5]. The noradrenergic neurons of the locus coeruleus (LC) appear to be the main target of DSP-4. Neuronal degeneration presumably begins at the presynaptic terminals as characterized by decrements in a number of parameters: histochemical fluorescence, concentration of NE, uptake of [3HINE, and dopamine-O-hydroxylase activity [1, 5, 9]. Morphologically, the noradrenergic axons are characterized by marked accumulations of NE distal to their termination areas [5]. In the present study, we investigated whether or not DSP-4 produces an effect on LC cell bodies by using histological and electrophysiological techniques. Male rats (Tif: R A I f (SPF)), initially weighing 220-240 g, were maintained and prepared as previously described [1]. Briefly, the animals were pretreated with CGP 6085 A (4-(5,6)-dimethyl-2-benzofuranyl)piperidine hydrochloride) (10 pmol/kg
* Present address: Beecham Pharmaceuticals, Medicinal Research Centre, Coldharbour Road, The Pinnacles, Harlow, Essex, C M I 9 SAD, U.K. 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
82
i.p.), an inhibitor of 5-hydroxytryptamine uptake, 30 min before the injection of DSP-4 (200 #mol/kg i.p.) or the control solution (0.9~/0 saline). The control and DSP-4 rats were then used in experiments 10 or 50 days later. At 10 and 50 days after DSP-4 injection, results of previous neurochemical experiments [1] indicate substantial reductions of NE (<35~/0 of control NE remaining) in neocortex and hippocampal formation. Central NE depletion in the DSP-4 animals was checked according to the method of Mogilnicka et al. [7], and confirmed after experiments (if applicable) by absence of the post-decapitation reflex [1]. For the histological experiments, brains were used from 16 animals (8 control, 8 DSP-4). At the 10- and 50-day time points, 4 control and 4 DSP-4 rats were deeply ~nesthetized with ether. The thoracic cavity of each animal was rapidly opened. Saline was then intracardiaily perfused; this perfusion was followed by a second of a phosphate-buffered mixture of paraformaldehyde and glutaraldehyde [6]. The brains were carefully removed, trimmed, and postfixed in Miilonig buffer solution for 2 days. The tissue was subsequently dehydrated, embedded in paraffin, sectioned coronally at ~ 4 #m, and stained with toluidine blue and eosin. Selected sections from the area of the LC were examined with a Leitz microscope. For the electrophysiological experiments, the firing rate of LC neurons was studied in 24 animals (8 control, 16 DSP-4). At the 10- and 50-day time points, 4 control and 8 DSP-4 rats were anesthetized with chloral hydrate (400 m g / k g i.p.) and placed in a stereotaxic apparatus. Additional anesthetic was injected as required. The body temperature of each animal was maintained between 36.5 and 38°C by a heating cushion. The LC was a~proached stereotaxically from above (1.7 mm posterior, 1. i mm lateral, 6.9-7.5 mm below the cortical surface). The incisor bar was set 5 m m above the interaural line. Single cell recording was performed via glass electrodes filled with 4 M NaC! or with 2 M NaCi saturated with Fast Green which was used to label the recording site. Noradrenergic neurons were characterized by a slow firing rate (0.5-6 Hz) with long-lasting action potentials ( ~ 2 ms) [2, 3]. All neurons responded to tail-pinching with a short phase of cell excitation followed by a prolonged phase of complete firing arrest. In those instances in which the recording site was labeled, the dye deposit was found to be located in the area of the LC (n = 3). The activity of each neuron was recorded for 3 min on a rate meter, and the mean activity of 8-10 neurons was determined for each rat. Statistical significance of changes in frequency distribution was assessed using the Kolmogoroff-Smirnoff test. The light microscopic examination revealed a normal appearance of the neuropil and perikarya in the area of the LC at the 10- and 50-day time points (Fig. 1). This finding is in contrast to a previous report [10] of perikaryal swelling after two injections of DSP-4. Such swelling, however, may have reflected the cumulative toxicity from repeated DSP-4 administration. ElectrophysiologicaUy, there was a clear reduction of the mean firing rate of LC neurons in DSP-4 rats. The mean rate in control animals was 2.4 + 1.1 Hz (x + v
83
Fig. 1. Micrograph of a coronal section through the locus coeruleus of a DSP-4 rat .s0 days after treatment. Note the normal structural organization of this nuclear region. Sections for 10 days after treatment (not shown) were identical to those for the 50-day time point. Abbreviations: Lc, locus coeruleus; Nmt, nucleus tractus mesencephalici nervi trigemini; V, ventriculus quartus, x 180.
S.E., n = 80), whereas that in DSP-4 animals was 1.4 _+ 0.9 H z (n = 70) and i.7 _+ 0.5 Hz (n = 70) at the 10- and 50-day time points, respectively. The frequencydistribution patterns o f L C firing in control and DSP-4 rats are depicted in Fig. 2. The distribution patterns for DSP-4 rats differed significantly f r o m the one for control rats ( P < 0 . 0 5 ) . The present study demonstrates that retrograde degeneration o f LC neurons, as a consequence o f D S P - 4 administration, does not extend to the cell bodies. Still, the decreased firing rate o f these noradrenergic neurons, c o u p l e d with the m a r k e d depletion o f NE in termination areas, must impede L C function. In this regard, it is o f interest that the DSP-4-induced shift in frequency-distribution pattern s h o w s a strong resemblance to the age-related shift for old rats [8]. The authors gratefully acknowledge the cooperation o f Dr. H . Bittiger.
84
number of neurons
5O 4O 30 20 10 0
~ 0-1
1-2
2-3
3-4
4--5
m 5--6
r-HZ
Fig. 2. Frequency distribution of activity of locus coeruleus neurons in control and DSP-4 rats 10 and 50 days after treatment. (The distribution for control is based on pooled values across the I0- and 50-day time points). Control rats, vertical lined columns; DSP-4 rats, 10 days, dotted columns; DSP-4 rats, 50 days, cross-hatched columns.
1 Dooley, D.J., Bittiger, H., Hauser, K.L., Bischoff, S.F. and Waldmeier, P.C., Alteration of central alpha2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin, Neuroscience, 9 (1983) 889-898. 2 Engberg, G. and Svensson, T.H., Pharmacological analysis of a cholinergic receptor mediated regulation of brain norepinephrine neurons, J. Neural Transm., 49 (1980) 137-150. 3 Faiers, A.A. and Mogenson, G.J., Electrophysiological identification of neurons in locus coeruleus, Exp. Neurol., 53 (1976) 254-266. 4 Jaim-Etcheverry, G. and Zieher, L.H., DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats, Brain Res., 188 (1980) 513-523. 5 Jonsson, G., Hallman, H., Ponzio, F. and Ross, S., DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) - a useful denervation tool for central and peripheral noradrenaline neurons, Europ. J. Pharmacol., 72 (1981) 173-188. 6 Karnovsky, M.J., A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy, J. Cell Biol., 27 (1965) 137-138. 7 Mogilnicka, E., Dooley, D.J., Boissard, C.G. and Delini-Stula, A., Altered hindlimb extension in the rat after DSP-4: a useful marker o f central noradrenergic depletion, Europ. J. Pharmacol., 87 (1983) 345-347. 8 0 l p e , H.R. and Steinmann, M.W., Age-related decline in the activity of noradrenergic neurons of tile rat locus coeruleus, Brain Res., 251 (1982) 174-176. 9 Ross, S.B., Long-term effects of N-2-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride on noradrenergic neurones in the rat brain and heart, Brit. J. Pharmacol., 58 (1976) 521-527. 10 Spyraki, C., Arbuthnott, G,W. and Fibiger, H.C., The effect of DSP-4 on some positively reinforced operant behaviors in the rat, Pharmacol. Biochem. Behav., 16 (1982) 197-202.
81
Elsevier Scientific Pubfishers Ireland Ltd.
DECRLASED ACTIVITY OF LOCUS COERULEUS NEURONS IN THE RAT ~ D S P 4 TREATMENT
H.R. OLPE, J. LASZLO, D,J. DOOLEY*, J. H E I D a n d M.W. STEINMANN
Research Department, Pharmaceuticals Division, CIBA-Geigy Ltd., CH-4002 Basel ($w:'t~erland) (Received April 12th, 1983; Revised version received June 28th, 1983; Accepted June 30th, 1983)
Key words: DSP-4 - noradrenergic neurotoxin - locus coendeus - firing rate - cell morphology - rat
The activity of noradrenergic neurons o f the rat locus cocruleus was investigated at 10 and 50 days after the administration o f DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine), a selective noradrenergic neurotoxin. The mean neuronal firing rate in control animals was 2.4 Hz. In contrast, DSP-4 animals had lower rates o f 1.2 Hz at 10 days and 1.7 Hz at 50 days. Histological examinations revealed no morphological changes o f locus coeruleus cell bodies at either the 10- or 50.day time points. These results suggest that DSP-4 can impair neuronal activity o f the locus coeruleus without altering the. structural appearance o f locus coeruleus perikarya.
A peripheral injection of DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) depletes norepinephrine (NE) in the rodent central nervous system [1, 4, 5, 9]. This depletion is long-lasting and rather selective in that neocortical concentrations of dopamine, 5-hydroxytryptamine, acetylcholine and several amino acids are practically unchanged by the neurotoxin [1, 5]. The noradrenergic neurons of the locus coeruleus (LC) appear to be the main target of DSP-4. Neuronal degeneration presumably begins at the presynaptic terminals as characterized by decrements in a number of parameters: histochemical fluorescence, concentration of NE, uptake of [3HINE, and dopamine-O-hydroxylase activity [1, 5, 9]. Morphologically, the noradrenergic axons are characterized by marked accumulations of NE distal to their termination areas [5]. In the present study, we investigated whether or not DSP-4 produces an effect on LC cell bodies by using histological and electrophysiological techniques. Male rats (Tif: R A I f (SPF)), initially weighing 220-240 g, were maintained and prepared as previously described [1]. Briefly, the animals were pretreated with CGP 6085 A (4-(5,6)-dimethyl-2-benzofuranyl)piperidine hydrochloride) (10 pmol/kg
* Present address: Beecham Pharmaceuticals, Medicinal Research Centre, Coldharbour Road, The Pinnacles, Harlow, Essex, C M I 9 SAD, U.K. 0304-3940/83/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
82
i.p.), an inhibitor of 5-hydroxytryptamine uptake, 30 min before the injection of DSP-4 (200 #mol/kg i.p.) or the control solution (0.9~/0 saline). The control and DSP-4 rats were then used in experiments 10 or 50 days later. At 10 and 50 days after DSP-4 injection, results of previous neurochemical experiments [1] indicate substantial reductions of NE (<35~/0 of control NE remaining) in neocortex and hippocampal formation. Central NE depletion in the DSP-4 animals was checked according to the method of Mogilnicka et al. [7], and confirmed after experiments (if applicable) by absence of the post-decapitation reflex [1]. For the histological experiments, brains were used from 16 animals (8 control, 8 DSP-4). At the 10- and 50-day time points, 4 control and 4 DSP-4 rats were deeply ~nesthetized with ether. The thoracic cavity of each animal was rapidly opened. Saline was then intracardiaily perfused; this perfusion was followed by a second of a phosphate-buffered mixture of paraformaldehyde and glutaraldehyde [6]. The brains were carefully removed, trimmed, and postfixed in Miilonig buffer solution for 2 days. The tissue was subsequently dehydrated, embedded in paraffin, sectioned coronally at ~ 4 #m, and stained with toluidine blue and eosin. Selected sections from the area of the LC were examined with a Leitz microscope. For the electrophysiological experiments, the firing rate of LC neurons was studied in 24 animals (8 control, 16 DSP-4). At the 10- and 50-day time points, 4 control and 8 DSP-4 rats were anesthetized with chloral hydrate (400 m g / k g i.p.) and placed in a stereotaxic apparatus. Additional anesthetic was injected as required. The body temperature of each animal was maintained between 36.5 and 38°C by a heating cushion. The LC was a~proached stereotaxically from above (1.7 mm posterior, 1. i mm lateral, 6.9-7.5 mm below the cortical surface). The incisor bar was set 5 m m above the interaural line. Single cell recording was performed via glass electrodes filled with 4 M NaC! or with 2 M NaCi saturated with Fast Green which was used to label the recording site. Noradrenergic neurons were characterized by a slow firing rate (0.5-6 Hz) with long-lasting action potentials ( ~ 2 ms) [2, 3]. All neurons responded to tail-pinching with a short phase of cell excitation followed by a prolonged phase of complete firing arrest. In those instances in which the recording site was labeled, the dye deposit was found to be located in the area of the LC (n = 3). The activity of each neuron was recorded for 3 min on a rate meter, and the mean activity of 8-10 neurons was determined for each rat. Statistical significance of changes in frequency distribution was assessed using the Kolmogoroff-Smirnoff test. The light microscopic examination revealed a normal appearance of the neuropil and perikarya in the area of the LC at the 10- and 50-day time points (Fig. 1). This finding is in contrast to a previous report [10] of perikaryal swelling after two injections of DSP-4. Such swelling, however, may have reflected the cumulative toxicity from repeated DSP-4 administration. ElectrophysiologicaUy, there was a clear reduction of the mean firing rate of LC neurons in DSP-4 rats. The mean rate in control animals was 2.4 + 1.1 Hz (x + v
83
Fig. 1. Micrograph of a coronal section through the locus coeruleus of a DSP-4 rat .s0 days after treatment. Note the normal structural organization of this nuclear region. Sections for 10 days after treatment (not shown) were identical to those for the 50-day time point. Abbreviations: Lc, locus coeruleus; Nmt, nucleus tractus mesencephalici nervi trigemini; V, ventriculus quartus, x 180.
S.E., n = 80), whereas that in DSP-4 animals was 1.4 _+ 0.9 H z (n = 70) and i.7 _+ 0.5 Hz (n = 70) at the 10- and 50-day time points, respectively. The frequencydistribution patterns o f L C firing in control and DSP-4 rats are depicted in Fig. 2. The distribution patterns for DSP-4 rats differed significantly f r o m the one for control rats ( P < 0 . 0 5 ) . The present study demonstrates that retrograde degeneration o f LC neurons, as a consequence o f D S P - 4 administration, does not extend to the cell bodies. Still, the decreased firing rate o f these noradrenergic neurons, c o u p l e d with the m a r k e d depletion o f NE in termination areas, must impede L C function. In this regard, it is o f interest that the DSP-4-induced shift in frequency-distribution pattern s h o w s a strong resemblance to the age-related shift for old rats [8]. The authors gratefully acknowledge the cooperation o f Dr. H . Bittiger.
84
number of neurons
5O 4O 30 20 10 0
~ 0-1
1-2
2-3
3-4
4--5
m 5--6
r-HZ
Fig. 2. Frequency distribution of activity of locus coeruleus neurons in control and DSP-4 rats 10 and 50 days after treatment. (The distribution for control is based on pooled values across the I0- and 50-day time points). Control rats, vertical lined columns; DSP-4 rats, 10 days, dotted columns; DSP-4 rats, 50 days, cross-hatched columns.
1 Dooley, D.J., Bittiger, H., Hauser, K.L., Bischoff, S.F. and Waldmeier, P.C., Alteration of central alpha2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin, Neuroscience, 9 (1983) 889-898. 2 Engberg, G. and Svensson, T.H., Pharmacological analysis of a cholinergic receptor mediated regulation of brain norepinephrine neurons, J. Neural Transm., 49 (1980) 137-150. 3 Faiers, A.A. and Mogenson, G.J., Electrophysiological identification of neurons in locus coeruleus, Exp. Neurol., 53 (1976) 254-266. 4 Jaim-Etcheverry, G. and Zieher, L.H., DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats, Brain Res., 188 (1980) 513-523. 5 Jonsson, G., Hallman, H., Ponzio, F. and Ross, S., DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) - a useful denervation tool for central and peripheral noradrenaline neurons, Europ. J. Pharmacol., 72 (1981) 173-188. 6 Karnovsky, M.J., A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy, J. Cell Biol., 27 (1965) 137-138. 7 Mogilnicka, E., Dooley, D.J., Boissard, C.G. and Delini-Stula, A., Altered hindlimb extension in the rat after DSP-4: a useful marker o f central noradrenergic depletion, Europ. J. Pharmacol., 87 (1983) 345-347. 8 0 l p e , H.R. and Steinmann, M.W., Age-related decline in the activity of noradrenergic neurons of tile rat locus coeruleus, Brain Res., 251 (1982) 174-176. 9 Ross, S.B., Long-term effects of N-2-chloroethyl-N-ethyl-2-bromobenzylamine hydrochloride on noradrenergic neurones in the rat brain and heart, Brit. J. Pharmacol., 58 (1976) 521-527. 10 Spyraki, C., Arbuthnott, G,W. and Fibiger, H.C., The effect of DSP-4 on some positively reinforced operant behaviors in the rat, Pharmacol. Biochem. Behav., 16 (1982) 197-202.