570
Brain Research, 136 (1977) 570- 577 t') Elsevier/North-Holland Biomedical Press
Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons
G. K. AGHAJANIAN, J. M. CEDARBAUM, R. Y. WANG Yale University School of Medicine and Connecticut Mental Health Center, New Haven, Conn. 06508 (u.s.A.)
(Accepted July 7th, 1977)
The locus coerulei (LC) in rats are composed of two well defined clusters of norepinephrine (NE)-containing neurons 3. These neurons typically respond to noxious stimuli with a brief burst of firing followed by a prolonged quiescent interval2,1L One mechanism which could account for such a poststimulus period of suppressed activity is that of collateral or mutual inhibition. There are both anatomical and physiological data which are consistent with this possibility. By electron histochemical and autoradiographic methods, presumed catecholaminergic nerve terminals have been found m the LC4A 1. Golgi studies show that LC neurons possess what appear to be collateral branchings of their axons26, ~9. Fluorescence histochemical studies suggest the possibility that dendro-dendritic junctions may also occur in the LC 28. Some catecholaminergic terminals in the LC may be derived from immunocytochemically identified epinephrine-containing neurons in the medulla oblongata (groups C1 and C~ of H6kfelt et al.12). Thus, catecholaminergic transmission could be mediated in the LC by NE collaterals and/or by epinephrine afferents. Physiological data are compatible with both possibilities since LC units are inhibited equally well by NE and epinephrine; moreover, the inhibitions produced by each are antagonized to the same degree by the a-adrenergic blocker piperoxane (PIP)L If there are axon collaterals (or dendro-dendritic junctions) within the LC, then a period of poststimulus suppression should occur after antidromic activation. Antidromic activation of LC neurons has been reported following stimulation of the hippocampus and cerebral and cerebellar cortices 'zl, the medial forebrain bundle and medial septal nucleus 7, and the dorsal noradrenergic bundle and cingulum 5. in none of these studies was there any mention of a period of poststimulus suppression in LC neuronal firing. In the present study, we found that antidromic activation of LC neurons via the dorsal NE bundle or cingulum was invariably followed by periods of poststimulation suppression. On this basis, further experiments were performed to determine if the suppression depends on antidromic invasion of N E fibers and can be reversed by PIP, an antagonist of NE in the LC". For the electrophysiological studies 30 male albino rats (Charles River) weighing 225-275 g were used. Twenty-three of the rats were stimulated in the dorsal NE bundle
571 (the main ascending LC pathway) and 7 in the cingulum (which carries LC fibers projecting to the cerebral cortex)25,27,31,33. Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic instrument. Burr holes were drilled 0.7-1.0 mm lateral to midline, 3 or 9 mm anterior to lambda (frontal planes A2180 and A8920, respectively of K6nig and Klippe116). Bipolar stimulating electrodes (NE100 or SNE-100, David Kopf) were then placed in the dorsal NE bundle (3.0 mm anterior to lambda, 0.7 mm lateral to midline, 6 mm below dural surface) and the cingulum (9.0 mm anterior to lambda, 1.0 mm lateral to midline, 2.75 mm below dural surface). Single-cells were recorded either by a dual micropipette assembly (50 #m tip separation) or by a 6-barrel electrode consisting of a 5-barrel electrode with a single barrel affixed to it (50 ~m tip separation) to permit combined differential recording and microiontophoresis, as previously described aS. For LC recordings, a burr hole was made 1.1 mm posterior to lambda and 1.1 mm lateral to the midline. As described previously2,8,17, 22, practical aids in finding the LC include: (1) depth of 5.5-6.0 mm below dural surface; (2) a zone or relative electrical silence just dorsal to LC corresponding to IVth ventricle; (3) the presence just lateral to LC of cells of the mesencephalic nucleus of Vth nerve, which are activated by proprioceptive stimulation in facial region; and (4) in the LC itself, a closely packed population of slowly firing neurons (1-5 spikes/sec) all responding to noxious stimulation (e.g. of contralateral paw) by a burst of firing followed by a quiescent period. In addition, these cells can be readily activated antidromically by stimulation of the dorsal NE bundle or, even more specifically, the cingulum 5. Ultimately, the recording electrode position was marked at the end of each experiment by passing --20 #A through the recording barrel for 10 min to deposit Fast Green 3° and located later by routine histology for confirmation of LC recording sites; stimulating electrode positions were also confirmed histologically. Procedures for differential recording and microiontophoresis were as described previously 9,~5. Solutions for microiontophoresis were as follows: NE bitartrate, 0.1 M, pH 4 (Regis); PIP HCI, 0.1 M, pH 4 (Rh6ne-Poulenc); and sotolol HC1 (Mead Johnson). A retaining current of --10 nA was applied to all drug barrels between periods of ejection. For histofluorescence studies, brains from 6 rats were prepared according to the method of Falck et al. 6. Rats were injected with 4/ag of 6-hydroxydopamine free base (2/~g//zl in a 1/~g/#l solution of ascorbic acid) 0.5 mm above the dorsal NE bundle. Brains were taken at 2-24 h after injection to examine for possible damage to the dorsal NE bundle. We found in all LC cells ipsilateral to stimulating electrodes in the dorsal NE bundle (n = 80 cells) or cingulum (n = 43 cells) that periods of suppressed firing could be demonstrated following single shocks at 1 Hz; the duration of these periods was proportional to current intensity (Fig. 1A). A detailed analysis of 10 LC cells after a graded series dorsal bundle stimulations (50 sweeps, 1 Hz) showed mean durations for periods of total poststimulus suppression as follows: (1) 0.25 mA, 102 msec; (2) 0.5 mA, 311 msec; (3) 1.0 mA, 525 msec. In this range of stimulating currents, the duration of suppressed firing was linearly related (r = 0.79) to current intensity. At
572
A i
J1
1.0 mA
J
.5 mA .....
3
.25mA 200 mS
Fig. 1. A: a series of poststimulus time histograms showing suppression in the firing of an LC neuron following stimulation of the dorsal NE bundle. The duration of the suppression was proportional to current strength; all stimuli were 0.5 msec in duration. At 0.25 mA no antidromic responses were evoked in this cell; at 1.0 mA there was 100 ~ antidromic activation (latency, 7 msec). The histograms were generated with a Nicolet 1072 averager (50 sweeps at 1 Hz). Bin width, 1 msec. B: storage-scope tracings showing collision between a spontaneous spike and an antidromically activated spike. In the upper tracing, when the dorsal bundle stimulus (arrow-head) follows the spontaneous spike (s) by 7.0 msec no collision occurred and the antidromic spike (a) is seen (6 msec latency). In the lower tracing, with .a 6 msec delay, collision occurred as indicated by a failure of the antidromic spike to appear. Dorsal bundle stimulation was at 1.5 mA (50~ above threshold for antidromic activation giving 100 ~ antidromic responses). The electrical stimulus was triggered by the spontaneous spikes at predetermined delays. Scale: 2 msec, 0.5 mV.
low currents, poststimulus suppression occurred in some cells in the absence o f antidromic spikes; however, at such low currents antidromic responses could be elicited in at least a portion o f the L C neuronal population. All 10 o f the above cells exhibited 1 0 0 ~ antidromic activation (mean latency, 7.2 msec) at the 1.0 m A current level; antidromic responses were recognized by their invariant latency, the occurrence of collision with spontaneous spikes (Fig. 1 B), and ability to follow at high frequencies (100 Hz) for at least the initial segment of the spike. In none of the cells tested in the contralateral L C (n ~ 15 cells) could antidromic responses be elicited after dorsal bundle stimulation; contralateral LC cells also did not show poststimulus periods of suppression except at currents high enough to produce o r t h o d r o m i c activation. L C cells could be activated orthodromically from m a n y sites in the ventral tegmentum and central gray; the nature of the pathways involved was not determined. Results obtained after stimulation o f the cingulum were similar to those seen after dorsal bundle stimulation except that only 70 ~ o f ipsilateral cells (n ~ 30) could be antidromically activated (mean latency 42 msec) even at currents up to 5 mA ; in the same animals, only 1 out of 20 contralateral cells tested showed antidromic responses and none of the latter showed a substantial (i.e. ~ 100 msec)poststimulus suppression of firing. The above latencies for antidromic activation of L C cells f r o m dorsal bundle and cingulum stimulations are almost identical to those reported by Faiers and MogensonS; thus, L C axon conduction velocities o f 0.59 m/sec (dorsal bundle) and 0.45 m/sec (cingulum)
573
CJ.
A.L
I
control
]
control
6-OHDA
1 [
PIP20nA
Bj.
u~U~
]
DJ.
control
I
control
t/~ Fig. 2. A: a prolonged poststimulus suppression in the firing of an LC cell evoked by dorsal NE bundle stimulation was seen before but not 1 h after 6-hydroxydopamine (6-OHDA) for both of these cells the current strength was 2.0 mA with a duration of 0.5 msec. In general, a sample of control cells was tested prior to 6-OHDA with a fixed stimulating electrode placement in the dorsal bundle; 6O H D A (4/~g free base in 2/~1 o f a 1 mg/ml solution of ascorbic acid) was then injected 0.5 m m above dorsal bundle, 2.5 m m anterior to lambda; a sample of LC cells was then tested at various times afterward (see text). B: under conditions similar to those in 'A', 5,7-dihydroxytryptamine (5,7-DHT; 4 / t g free base) failed to prevent either antidromic responses or poststimulus suppression produced by dorsal bundle stimulation (stimulation parameters: 1 Hz, 1.0 mA, 0.5 msec). C: PIP (20 nA, 5 min) reversed a prolonged poststimulus period of suppression in an LC cell (stimulation parameters: 1 Hz, 0.7 mA, 0.5 msec). D: under conditions as in 'C', SOT (20 nA, 5 min) failed to reverse poststimulus suppression (stimulation parameters: 1 Hz, 0.5 mA, 0.5 msec).
were calculated. The observation that antidromic responses occurred in an LC neuron contralateral to stimulation in the cingulum is consistent with anatomical 27 and physiological 7 evidence for some crossing of LC fibers. The fact that poststimulus suppression of individual cells did not depend on antidromic invasion and was linearly related to current intensity, suggests the occurrence of synaptic summation rather than a prolonged refractory period of the soma. Although this pattern is compatible with a collateral inhibitory mechanism, extrinsic afferents (e.g. from hypothalamus 2°) could also have been activated by stimulation in the vicinity of the dorsal bundle. Of course, the latter possibility would be less likely in the case of cingulum stimulation. Nevertheless, to distinguish between these possibilities, 6-hydroxydopamine, which in small amounts injected intracerebrally is a selective neurotoxin for catecholamine cells and fibers 13-15,1s,19,a~ was injected 0.5 mm above the dorsal NE bundle (n ---- 5 animals). LC cell responses to dorsal bundle stimulation were tested before the injections, and up to 24 h later. Prior to 6-hydroxydopamine, LC cells showed antidromic responses and poststimulus suppressions of firing as described above; however, within 1 h after 6-hydroxydopamine, antidromic responses could no longer be obtained and no poststimulus suppression occurred (Fig. 2A)
Fig. 3. A: fluorescence micrograph showing normal appearance of dorsal NE bundle contralateral to side of 6-OHDA injection (4/~g free base in 2/tl). Brain was taken for histofluorescence 6 h after placement of the 6-OHDA. B : on side of 6-OHDA injection (4/~g free base) swelling of proximal ends of LC axons can be seen throughout the dorsal bundle. There is no evidence of non-specific damage from the 6-OHDA in the bundle itself. A small (0.25 diameter) necrotic zone was seen in the immediate area of the injection site (0.5 mm above the bundle).
575 except at currents high enough to elicit orthodromic activation. This rapid interruption of impulse flow induced by 6-hydroxydopamine in the dorsal bundle is consistent with similarly rapid changes, measured indirectly, in central dopaminergic pathways a4. On the other hand, 5,7-dihydroxytryptamine (a neurotoxin with selectivity toward serotonergic neurons 1) did not disrupt the effects of dorsal bundle stimulation on LC neurons (Fig. 2B; n ---- 4 animals). The specificity of these changes is indicated by the fact that 5,7-dihydroxytryptamine but not 6-hydroxydopamine has been found to block transmission in serotonergic axons 35. Fluorescence histochemical examination of brains from animals injected with 6-hydroxydopamine showed extensive unilateral destruction of axons in the dorsal NE bundle (Fig. 3). The above results indicate that both antidromic responses and poststimulus suppression of LC cell activity are dependent on the integrity of NE axons emanating from the LC. This finding favors the view that the poststimulus suppression of LC cell activity is mediated via antidromic invasion of inhibitory NE collaterals within the LC and not via an extrinsic afferent pathway. If this were the case, then the a-antagonist PIP, which blocks NE-induced inhibition of LC cells z, should attenuate the effects of stimulation of the dorsal bundle or cingulum. Fourteen LC cells were tested before and during the microiontophoretic application of PIP (Fig. 2C). Before PIP, dorsal bundle stimulation produced a mean period of poststimulus suppression in these cells lasting 656 msec (mean current, 0.63 mA). After PIP (applied for an average of 5 min at 20 nA) the poststimulus suppression was reduced to a mean of 260 msec (P < 0.001, paired t-test); the microiontophoretic PIP caused little or no increase in baseline rate under these conditions. Systemically administered PIP (0.5-1.0 mg/kg, i.v.) produced an even more dramatic reversal of the poststimulus inhibition (n = 5 animals); however, under these conditions there was also a marked increase in baseline LC firing rate (cf. ref. 2). In contrast, the fl-adrenergic antagonist sotolol, which has activity at poststynaptic LC-innervated sites10,23 but which does not block the effects of NE on LC cells2, produced no attenuation of poststimulus suppressions of firing after dorsal bundle stimulation (Fig. 2D; n ---- 5 cells). Similar results were obtained after stimulation of the cingulum: PIP (20 nA, 5 min) reduced poststimulus suppression of LC cell firing (n = 5) from a mean of 370 msec to 154 msec (P < 0.025, paired t-test); sotolol again had no effect. Taken together, the above results provide strong indirect support for the hypothesis that the suppression of LC cell activity induced by stimulation of the dorsal bundle or cingulum is mediated via a direct NE-collateral inhibitory system. This conclusion is based primarily on the fact that the effect is dependent on antidromic invasion of NE fibers which emanate from the LC. Since the inhibition is attenuated by the direct application of PIP, which blocks the inhibition of LC cells by NE, it would appear unlikely that the inhibition is mediated through an axon-collateral activation of non-NE interneurons which might exert an inhibitory influence on LC cells. The existence of a collateral inhibitory mechanism could account for the poststimulus suppression of LC cell activity seen after orthodromic activation (e.g. by noxious stimulation2,17); studies are in progress to investigate this possibility. Another implication of a direct NE-mediated collateral inhibition in the LC is that drugs such as ampheta-
576 mine s a n d d e s i p r a m i n e ee which depress LC cell activity could do so, at least in part, by p o t e n t i a t i n g the action o f N E at collateral junctions. In p r e l i m i n a r y results, we find t h a t i o n t o p h o r e t i c a l l y a p p l i e d desipramine, which prevents the inactivation o f N E by blocking synaptic reuptake, causes a d r a m a t i c p r o l o n g a t i o n o f suppression in firing induced by d o r s a l N E bundle stimulation (n = 7 cells). Similarly, p a r t o f the mechanism t h r o u g h which the a - a n t a g o n i s t d r u g PIP activates LC neuronal firing 2 could be by b l o c k i n g tonic N E - m e d i a t e d collateral inhibition. In conclusion, the results o f this study suggest the presence o f a powerful, direct collateral i n h i b i t o r y system in the LC. A l p h a - a d r e n e r g i c a u t o r e c e p t o r s '~, r a t h e r t h a n fi-adrenergic receptors, which are f o u n d in certain LC p o s t s y n a p t i c areas10, 23, a p p e a r to m e d i a t e the i n h i b i t o r y responses. Collateral inhibition may account for certain physiological p r o p e r t i e s o f LC neurons such as the gating of o r t h o d r o m i c activations (e.g. to noxious stimulation). In a d d i t i o n , interactions with collateral inhibitory mechanisms m a y explain, in part, the effects on LC activity of drugs which can modify N E - t r a n s m i s s i o n (e.g. a m p h e t a m i n e , d e s i p r a m i n e a n d PIP). W e t h a n k N. M a r g i o t t a and A. Lorette for their excellent technical assistance. S u p p o r t e d by U S P H S G r a n t s MH-17871 ; MH-14459 a n d the State o f Connecticut.
1 Baumgarten, H. G. and Lachenmayer, L., 5,7-Dihydroxytryptamine: improvement in chemical lesioning of indoleamine neurons in the mammalian brain, Z. Zellforsch., 135 (1972) 399-414. 2 Cedarbaum, J. M. and Aghajanian, G. K., Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and activation by the a-antagonist piperoxane, Brain Research, 112 (1976) 413419. 3 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, suppl. 232 (1965) 1-55. 4 Descarries, L. et Droz, B., Incorporation de noradrenaline-aH (NA-ZH) dans le systeme nerveux central du rat adulte. Etude radioautographique en microscopie electronique, C. R. Acad. Soc. (Paris), 266 (1968) 2480-2482. 5 Faiers, A. A. and Mogenson, G. J., Electrophysiological identification of neurons in locus coeruleus, Exp. Neurol., 53 (1976) 254-266. 6 Falck, B., Hillarp, N. A., Thieme, G. and Torp, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde, J. Histochem. Cytochern., I0 (1962) 348-354. 7 German, D. C. and Fetz, E., Responses of primate locus coeruleus and subcoeruleus neurons to stimulation at reinforcing brain sites and to natural reinforcers, Brain Research, 109 (1976) 497515. 8 Graham, A. W. and Aghajanian, G. K., Effects of amphetamine on single cell activity in a catecholamine nucleus, the locus coeruleus, Nature (Lond.), 234 (1971) 100-102. 9 Haigler, H. J. and Aghajanian, G. K., Lysergic acid diethylamide and serotonin : a comparison ot effects on serotonergic neurons and neurons containing a serotonergic input, J. Pharmacol. exp. Ther., 188 (1974) 688-699. 10 Hoffer, B. J., Siggins, G. R. and Bloom, F. E., Studies on norepinephrine containing afferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis, Brain Research, 25 (1971) 523-534. 11 HSkfelt, T., On the ultrastructural localization of noradrenaline in the central nervous system of the rat, Z. Zellforsch., 79 (1967) 110-117. 12 H6kfelt, T., Fuxe, K., Goldstein, M. and Johnsson, O., lmmunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Research, 66 (1974) 235-251.
577 13 H6kfelt, T. and Ungerstedt, U., Specificity of 6-hydroxydopamine induced degeneration of central monoamine neurons: an electron and fluorescence microscopic study with special reference to intracerebral injection on the nigrostriatal dopamine system, Brain Research, 60 (1973) 269-297. 14 Javoy, F., Sotelo, C., Herbet, A. and Agid, Y., Specificity of dopaminergic neuronal degeneration induced by intracerebral injection of 6-hydroxydopamine in the nigrostriatal dopamine system, Brain Research, 102 (1976) 201-215. 15 Kelly, P. H., Joyce, E. M., Minneman, K. P. and Phillipson, O. T., Specificity of 6-hydroxydopamine-induced destruction of mesolimbic or nigrostriatal dopamine-containing terminals, Brain Research, 122 (1977) 382-387. 16 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Krieger, New York, 162 pp. 17 Korf, J., Bunney, B. S. and Aghajanian, G. K., Noradrenergic neurons: morphine inhibition of spontaneous activity, Europ. J. PharmacoL, 25 (1974) 165-169. 18 Lidbrink, P. and Jonsson, G., Noradrenaline nerve terminals in the cerebral cortex: effects on noradrenaline uptake and storage followingaxonal lesions with 6-hydroxydopamine, J. Neurochem., 22 (1974) 617-626. 19 Maler, L., Fibiger, H. C. and McGeer, P. L., Demonstration of the nigrostriatal projection by silver staining after nigral injections of 6-hydroxydopamine, Exp. Neurol., 40 (1973) 505-515. 20 Mizuno, N. and Nakamura, Y., Direct hypothalamic projections to the locus coeruleus, Brain Research, 19 (1970) 160-163. 21 Nakamura, S. and Iwama, K., Antidromic activation of rat locus coeruleus neurons from hippocampus, cerebral and cerebellar cortices, Brain Research, 99 (1975) 372-376. 22 Nyb~ck, H., Walters, J. R., Aghajanian, G. K. and Roth, R. H., Tricyclic antidepressants: effects on the firing rate of brain noradrenergic neurons, Europ. J. Pharmacol., 32 (1975) 302-312. 23 Segal, M. and Bloom, F. E., The action of norepinephrlne in the rat hippocampus. I. Iontophoretic studies, Brain Research, 72 (1974) 79-97. 24 Segal, M. and Bloom, F. E., The action of norepinephrine in the rat hippocampus. II. Activation of the input pathway, Brain Research, 72 (1974) 99-114. 25 Segal, M., Pickel, V. and Bloom, F., The projections of the nucleus locus coeruleus: an autoradiographic study, Life Sci., 13 (1973) 817-821. 26 Shimizu, N. and Imamoto, K., Fine structure of the locus coeruleus in the rat, Arch. histoL jap., 31 (1970) 229-246. 27 Shimizu, N., Ohnishi, S., Tohyama, M. and Maeda, T., Demonstration by degeneration silver method of the ascending projection from the locus coeruleus, Exp. Brain Res., 20 (1974) 181-192. 28 Sladek, J. R., Jr. and Parnavelas, J. G., Catecholamine-containing dendrites in primate brain, Brain Research, 100 (1975) 657-662. 29 Swanson, L. W., The locus coeruleus: a cytoarchitectonic, Golgi and immunocytochemical study in the albino rat, Brain Research, 110 (1976) 39-56. 30 Thomas, R. C. and Wilson, V. J., Precise localization of Renshaw cells with a new marking technique, Nature (Lond.), 206 (1965) 211-213. 31 Tohyama, M., Maeda, T. and Shimizu, N., Detailed noradrenaline pathways of locus coeruleus to the cerebral cortex with use of 6-hydroxydopa, Brain Research, 79 (1974) 139-144. 32 VonVoightlander, P. F. and Moore, K. E., Turning behavior of mice with unilateral 6-hydroxydopamine lesions in striatum: effects of apomorphine, L-DOPA, amantadine, amphetamine and other psychomotor stimulants, Neuropharmacology, 12 (1973) 451-462. 33 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., suppl. 367 (1971) 1--48. 34 Ungerstedt, U., Histochemical studies on the effect of intracerebral and intraventricular injections of 6-hydroxydopamine on monoamine neurons in the rat brain. In T. Malmfors and H. Thoenen (Eds.), 6-Hydroxydopamine and Catecholamine Neurons, North-Holland, New York, 1971, pp. 101-129. 35 Wang, R. Y. and Aghajanian, G. K., Inhibition of neurons in the amygdala by dorsal raphe stimulation: mediation through a direct serotonergic pathway, Brain Research, 120 (1977) 85-102. 36 Wang, R. Y. and Aghajanian, G. K., Antidromically identified serotonergic neurons in the rat midbrain: evidence for collateral inhibition, Brain Research (1977) in press.
Brain Research, 136 (1977) 570- 577 t') Elsevier/North-Holland Biomedical Press
Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons
G. K. AGHAJANIAN, J. M. CEDARBAUM, R. Y. WANG Yale University School of Medicine and Connecticut Mental Health Center, New Haven, Conn. 06508 (u.s.A.)
(Accepted July 7th, 1977)
The locus coerulei (LC) in rats are composed of two well defined clusters of norepinephrine (NE)-containing neurons 3. These neurons typically respond to noxious stimuli with a brief burst of firing followed by a prolonged quiescent interval2,1L One mechanism which could account for such a poststimulus period of suppressed activity is that of collateral or mutual inhibition. There are both anatomical and physiological data which are consistent with this possibility. By electron histochemical and autoradiographic methods, presumed catecholaminergic nerve terminals have been found m the LC4A 1. Golgi studies show that LC neurons possess what appear to be collateral branchings of their axons26, ~9. Fluorescence histochemical studies suggest the possibility that dendro-dendritic junctions may also occur in the LC 28. Some catecholaminergic terminals in the LC may be derived from immunocytochemically identified epinephrine-containing neurons in the medulla oblongata (groups C1 and C~ of H6kfelt et al.12). Thus, catecholaminergic transmission could be mediated in the LC by NE collaterals and/or by epinephrine afferents. Physiological data are compatible with both possibilities since LC units are inhibited equally well by NE and epinephrine; moreover, the inhibitions produced by each are antagonized to the same degree by the a-adrenergic blocker piperoxane (PIP)L If there are axon collaterals (or dendro-dendritic junctions) within the LC, then a period of poststimulus suppression should occur after antidromic activation. Antidromic activation of LC neurons has been reported following stimulation of the hippocampus and cerebral and cerebellar cortices 'zl, the medial forebrain bundle and medial septal nucleus 7, and the dorsal noradrenergic bundle and cingulum 5. in none of these studies was there any mention of a period of poststimulus suppression in LC neuronal firing. In the present study, we found that antidromic activation of LC neurons via the dorsal NE bundle or cingulum was invariably followed by periods of poststimulation suppression. On this basis, further experiments were performed to determine if the suppression depends on antidromic invasion of N E fibers and can be reversed by PIP, an antagonist of NE in the LC". For the electrophysiological studies 30 male albino rats (Charles River) weighing 225-275 g were used. Twenty-three of the rats were stimulated in the dorsal NE bundle
571 (the main ascending LC pathway) and 7 in the cingulum (which carries LC fibers projecting to the cerebral cortex)25,27,31,33. Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic instrument. Burr holes were drilled 0.7-1.0 mm lateral to midline, 3 or 9 mm anterior to lambda (frontal planes A2180 and A8920, respectively of K6nig and Klippe116). Bipolar stimulating electrodes (NE100 or SNE-100, David Kopf) were then placed in the dorsal NE bundle (3.0 mm anterior to lambda, 0.7 mm lateral to midline, 6 mm below dural surface) and the cingulum (9.0 mm anterior to lambda, 1.0 mm lateral to midline, 2.75 mm below dural surface). Single-cells were recorded either by a dual micropipette assembly (50 #m tip separation) or by a 6-barrel electrode consisting of a 5-barrel electrode with a single barrel affixed to it (50 ~m tip separation) to permit combined differential recording and microiontophoresis, as previously described aS. For LC recordings, a burr hole was made 1.1 mm posterior to lambda and 1.1 mm lateral to the midline. As described previously2,8,17, 22, practical aids in finding the LC include: (1) depth of 5.5-6.0 mm below dural surface; (2) a zone or relative electrical silence just dorsal to LC corresponding to IVth ventricle; (3) the presence just lateral to LC of cells of the mesencephalic nucleus of Vth nerve, which are activated by proprioceptive stimulation in facial region; and (4) in the LC itself, a closely packed population of slowly firing neurons (1-5 spikes/sec) all responding to noxious stimulation (e.g. of contralateral paw) by a burst of firing followed by a quiescent period. In addition, these cells can be readily activated antidromically by stimulation of the dorsal NE bundle or, even more specifically, the cingulum 5. Ultimately, the recording electrode position was marked at the end of each experiment by passing --20 #A through the recording barrel for 10 min to deposit Fast Green 3° and located later by routine histology for confirmation of LC recording sites; stimulating electrode positions were also confirmed histologically. Procedures for differential recording and microiontophoresis were as described previously 9,~5. Solutions for microiontophoresis were as follows: NE bitartrate, 0.1 M, pH 4 (Regis); PIP HCI, 0.1 M, pH 4 (Rh6ne-Poulenc); and sotolol HC1 (Mead Johnson). A retaining current of --10 nA was applied to all drug barrels between periods of ejection. For histofluorescence studies, brains from 6 rats were prepared according to the method of Falck et al. 6. Rats were injected with 4/ag of 6-hydroxydopamine free base (2/~g//zl in a 1/~g/#l solution of ascorbic acid) 0.5 mm above the dorsal NE bundle. Brains were taken at 2-24 h after injection to examine for possible damage to the dorsal NE bundle. We found in all LC cells ipsilateral to stimulating electrodes in the dorsal NE bundle (n = 80 cells) or cingulum (n = 43 cells) that periods of suppressed firing could be demonstrated following single shocks at 1 Hz; the duration of these periods was proportional to current intensity (Fig. 1A). A detailed analysis of 10 LC cells after a graded series dorsal bundle stimulations (50 sweeps, 1 Hz) showed mean durations for periods of total poststimulus suppression as follows: (1) 0.25 mA, 102 msec; (2) 0.5 mA, 311 msec; (3) 1.0 mA, 525 msec. In this range of stimulating currents, the duration of suppressed firing was linearly related (r = 0.79) to current intensity. At
572
A i
J1
1.0 mA
J
.5 mA .....
3
.25mA 200 mS
Fig. 1. A: a series of poststimulus time histograms showing suppression in the firing of an LC neuron following stimulation of the dorsal NE bundle. The duration of the suppression was proportional to current strength; all stimuli were 0.5 msec in duration. At 0.25 mA no antidromic responses were evoked in this cell; at 1.0 mA there was 100 ~ antidromic activation (latency, 7 msec). The histograms were generated with a Nicolet 1072 averager (50 sweeps at 1 Hz). Bin width, 1 msec. B: storage-scope tracings showing collision between a spontaneous spike and an antidromically activated spike. In the upper tracing, when the dorsal bundle stimulus (arrow-head) follows the spontaneous spike (s) by 7.0 msec no collision occurred and the antidromic spike (a) is seen (6 msec latency). In the lower tracing, with .a 6 msec delay, collision occurred as indicated by a failure of the antidromic spike to appear. Dorsal bundle stimulation was at 1.5 mA (50~ above threshold for antidromic activation giving 100 ~ antidromic responses). The electrical stimulus was triggered by the spontaneous spikes at predetermined delays. Scale: 2 msec, 0.5 mV.
low currents, poststimulus suppression occurred in some cells in the absence o f antidromic spikes; however, at such low currents antidromic responses could be elicited in at least a portion o f the L C neuronal population. All 10 o f the above cells exhibited 1 0 0 ~ antidromic activation (mean latency, 7.2 msec) at the 1.0 m A current level; antidromic responses were recognized by their invariant latency, the occurrence of collision with spontaneous spikes (Fig. 1 B), and ability to follow at high frequencies (100 Hz) for at least the initial segment of the spike. In none of the cells tested in the contralateral L C (n ~ 15 cells) could antidromic responses be elicited after dorsal bundle stimulation; contralateral LC cells also did not show poststimulus periods of suppression except at currents high enough to produce o r t h o d r o m i c activation. L C cells could be activated orthodromically from m a n y sites in the ventral tegmentum and central gray; the nature of the pathways involved was not determined. Results obtained after stimulation o f the cingulum were similar to those seen after dorsal bundle stimulation except that only 70 ~ o f ipsilateral cells (n ~ 30) could be antidromically activated (mean latency 42 msec) even at currents up to 5 mA ; in the same animals, only 1 out of 20 contralateral cells tested showed antidromic responses and none of the latter showed a substantial (i.e. ~ 100 msec)poststimulus suppression of firing. The above latencies for antidromic activation of L C cells f r o m dorsal bundle and cingulum stimulations are almost identical to those reported by Faiers and MogensonS; thus, L C axon conduction velocities o f 0.59 m/sec (dorsal bundle) and 0.45 m/sec (cingulum)
573
CJ.
A.L
I
control
]
control
6-OHDA
1 [
PIP20nA
Bj.
u~U~
]
DJ.
control
I
control
t/~ Fig. 2. A: a prolonged poststimulus suppression in the firing of an LC cell evoked by dorsal NE bundle stimulation was seen before but not 1 h after 6-hydroxydopamine (6-OHDA) for both of these cells the current strength was 2.0 mA with a duration of 0.5 msec. In general, a sample of control cells was tested prior to 6-OHDA with a fixed stimulating electrode placement in the dorsal bundle; 6O H D A (4/~g free base in 2/~1 o f a 1 mg/ml solution of ascorbic acid) was then injected 0.5 m m above dorsal bundle, 2.5 m m anterior to lambda; a sample of LC cells was then tested at various times afterward (see text). B: under conditions similar to those in 'A', 5,7-dihydroxytryptamine (5,7-DHT; 4 / t g free base) failed to prevent either antidromic responses or poststimulus suppression produced by dorsal bundle stimulation (stimulation parameters: 1 Hz, 1.0 mA, 0.5 msec). C: PIP (20 nA, 5 min) reversed a prolonged poststimulus period of suppression in an LC cell (stimulation parameters: 1 Hz, 0.7 mA, 0.5 msec). D: under conditions as in 'C', SOT (20 nA, 5 min) failed to reverse poststimulus suppression (stimulation parameters: 1 Hz, 0.5 mA, 0.5 msec).
were calculated. The observation that antidromic responses occurred in an LC neuron contralateral to stimulation in the cingulum is consistent with anatomical 27 and physiological 7 evidence for some crossing of LC fibers. The fact that poststimulus suppression of individual cells did not depend on antidromic invasion and was linearly related to current intensity, suggests the occurrence of synaptic summation rather than a prolonged refractory period of the soma. Although this pattern is compatible with a collateral inhibitory mechanism, extrinsic afferents (e.g. from hypothalamus 2°) could also have been activated by stimulation in the vicinity of the dorsal bundle. Of course, the latter possibility would be less likely in the case of cingulum stimulation. Nevertheless, to distinguish between these possibilities, 6-hydroxydopamine, which in small amounts injected intracerebrally is a selective neurotoxin for catecholamine cells and fibers 13-15,1s,19,a~ was injected 0.5 mm above the dorsal NE bundle (n ---- 5 animals). LC cell responses to dorsal bundle stimulation were tested before the injections, and up to 24 h later. Prior to 6-hydroxydopamine, LC cells showed antidromic responses and poststimulus suppressions of firing as described above; however, within 1 h after 6-hydroxydopamine, antidromic responses could no longer be obtained and no poststimulus suppression occurred (Fig. 2A)
Fig. 3. A: fluorescence micrograph showing normal appearance of dorsal NE bundle contralateral to side of 6-OHDA injection (4/~g free base in 2/tl). Brain was taken for histofluorescence 6 h after placement of the 6-OHDA. B : on side of 6-OHDA injection (4/~g free base) swelling of proximal ends of LC axons can be seen throughout the dorsal bundle. There is no evidence of non-specific damage from the 6-OHDA in the bundle itself. A small (0.25 diameter) necrotic zone was seen in the immediate area of the injection site (0.5 mm above the bundle).
575 except at currents high enough to elicit orthodromic activation. This rapid interruption of impulse flow induced by 6-hydroxydopamine in the dorsal bundle is consistent with similarly rapid changes, measured indirectly, in central dopaminergic pathways a4. On the other hand, 5,7-dihydroxytryptamine (a neurotoxin with selectivity toward serotonergic neurons 1) did not disrupt the effects of dorsal bundle stimulation on LC neurons (Fig. 2B; n ---- 4 animals). The specificity of these changes is indicated by the fact that 5,7-dihydroxytryptamine but not 6-hydroxydopamine has been found to block transmission in serotonergic axons 35. Fluorescence histochemical examination of brains from animals injected with 6-hydroxydopamine showed extensive unilateral destruction of axons in the dorsal NE bundle (Fig. 3). The above results indicate that both antidromic responses and poststimulus suppression of LC cell activity are dependent on the integrity of NE axons emanating from the LC. This finding favors the view that the poststimulus suppression of LC cell activity is mediated via antidromic invasion of inhibitory NE collaterals within the LC and not via an extrinsic afferent pathway. If this were the case, then the a-antagonist PIP, which blocks NE-induced inhibition of LC cells z, should attenuate the effects of stimulation of the dorsal bundle or cingulum. Fourteen LC cells were tested before and during the microiontophoretic application of PIP (Fig. 2C). Before PIP, dorsal bundle stimulation produced a mean period of poststimulus suppression in these cells lasting 656 msec (mean current, 0.63 mA). After PIP (applied for an average of 5 min at 20 nA) the poststimulus suppression was reduced to a mean of 260 msec (P < 0.001, paired t-test); the microiontophoretic PIP caused little or no increase in baseline rate under these conditions. Systemically administered PIP (0.5-1.0 mg/kg, i.v.) produced an even more dramatic reversal of the poststimulus inhibition (n = 5 animals); however, under these conditions there was also a marked increase in baseline LC firing rate (cf. ref. 2). In contrast, the fl-adrenergic antagonist sotolol, which has activity at poststynaptic LC-innervated sites10,23 but which does not block the effects of NE on LC cells2, produced no attenuation of poststimulus suppressions of firing after dorsal bundle stimulation (Fig. 2D; n ---- 5 cells). Similar results were obtained after stimulation of the cingulum: PIP (20 nA, 5 min) reduced poststimulus suppression of LC cell firing (n = 5) from a mean of 370 msec to 154 msec (P < 0.025, paired t-test); sotolol again had no effect. Taken together, the above results provide strong indirect support for the hypothesis that the suppression of LC cell activity induced by stimulation of the dorsal bundle or cingulum is mediated via a direct NE-collateral inhibitory system. This conclusion is based primarily on the fact that the effect is dependent on antidromic invasion of NE fibers which emanate from the LC. Since the inhibition is attenuated by the direct application of PIP, which blocks the inhibition of LC cells by NE, it would appear unlikely that the inhibition is mediated through an axon-collateral activation of non-NE interneurons which might exert an inhibitory influence on LC cells. The existence of a collateral inhibitory mechanism could account for the poststimulus suppression of LC cell activity seen after orthodromic activation (e.g. by noxious stimulation2,17); studies are in progress to investigate this possibility. Another implication of a direct NE-mediated collateral inhibition in the LC is that drugs such as ampheta-
576 mine s a n d d e s i p r a m i n e ee which depress LC cell activity could do so, at least in part, by p o t e n t i a t i n g the action o f N E at collateral junctions. In p r e l i m i n a r y results, we find t h a t i o n t o p h o r e t i c a l l y a p p l i e d desipramine, which prevents the inactivation o f N E by blocking synaptic reuptake, causes a d r a m a t i c p r o l o n g a t i o n o f suppression in firing induced by d o r s a l N E bundle stimulation (n = 7 cells). Similarly, p a r t o f the mechanism t h r o u g h which the a - a n t a g o n i s t d r u g PIP activates LC neuronal firing 2 could be by b l o c k i n g tonic N E - m e d i a t e d collateral inhibition. In conclusion, the results o f this study suggest the presence o f a powerful, direct collateral i n h i b i t o r y system in the LC. A l p h a - a d r e n e r g i c a u t o r e c e p t o r s '~, r a t h e r t h a n fi-adrenergic receptors, which are f o u n d in certain LC p o s t s y n a p t i c areas10, 23, a p p e a r to m e d i a t e the i n h i b i t o r y responses. Collateral inhibition may account for certain physiological p r o p e r t i e s o f LC neurons such as the gating of o r t h o d r o m i c activations (e.g. to noxious stimulation). In a d d i t i o n , interactions with collateral inhibitory mechanisms m a y explain, in part, the effects on LC activity of drugs which can modify N E - t r a n s m i s s i o n (e.g. a m p h e t a m i n e , d e s i p r a m i n e a n d PIP). W e t h a n k N. M a r g i o t t a and A. Lorette for their excellent technical assistance. S u p p o r t e d by U S P H S G r a n t s MH-17871 ; MH-14459 a n d the State o f Connecticut.
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