EXPERIMENTAL
53, 254266
NEUROLOGY
Electrophysiological
(1976)
Identification Locus Coeruleus’
of Neurons
in
A. A. FAIERS AND G. J. MOGENSON Departments
of Physiology London,
and Psychology, The University Ontario, Canada N6A 5Cl Received
March
of Western
Ontario,
29,1976
In 17 urethane-anesthetized rats 35 neurons, histologically verified as being situated in the loxrs coeruleus, were driven antidromically (latency, 44 msec) by electrical stimulation of the supracallosal bundle. Neurons of the locus coeruleus were also activated antidromically by stimulation of sites along the dorsal noradrenergic bundle in the midbrain (8-msec latency) and the hypothalamus (12-msec latency), and by stimulation of sites in the olfactory bulb (latency, 39 msec). Conduction velocity from these sites to the locus coeruleus was estimated to be 0.4 to 0.6 m/set. Refractory periods of fibers in the dorsal noradrenergic bundle were determined at twice threshold and in the supracallosal bundle at intensities just above threshold; refractory periods ranging from 4 to 20 msec were observed. Because neurons both in and near the locus coeruleus were antidromically activated by stimulation of the dorsal noradrenergic bundle whereas stimulation of the supracallosal bundle antidromically activated only neurons in the locus coeruleus, stimulation of the dorsal noradrenergic bundle could not be used to identify locus coeruleus neurons. It is concluded that a subpopulation of neurons in the locus coeruleus can be identified by their slow, steady firing rate (2.6 per second) and long-latency antidromic response to stimulation of the supracallosal bundle. The electrophysiological properties of locus coeruleus neurons are considered in relation to neuroanatomical and functional studies of the locus coeruleus.
INTRODUCTION The projections of the nucleus locus coeruleus have been mapped using a variety of techniques including histofluorescence (2, 8, 14, 17, 20, 30) radioautography (21)) silver staining (27)) and retrograde transport of 1 This work was supported by grants from the Medical Research Council of Canada and the National Research Council of Canada. The authors wish to express their appreciation to Dr. J. D. Cooke, D. Carter, and Miss B. Box for their criticism of the manuscript. 2.54 Copyright All rights
1976 by Academic Press, Inc. 8 reproduction CI in any form reserved.
IDENTIFICATION
OF
LOCUS
COERULEUS
NEURONS
2 55
horseradish peroxidase (13. 35). These studies have shown the locus coeruleus to consist mainly of medium size noraclrenergic cell bodies whose fine axons project to many areas of the brain including hippocampus, cerebral and cerebellar cortices, amygdala. and some thalamic and hypothalnmic nuclei. The major rostra1 projection of the locus coeruleus is through the dorsal noradrenergic bundle which ascendsin the midbrain beside lhe dorsal longitudinal fasciculus of Shultz and then passesventrally into the lateral hypothalamus. The locus coeruleus also projects rostrally through the central tegmental tract and the periventricular pathway (17). The hippocampus is innervated by a rostra1 extension of dorsal noradrenergic bundle which projects through the cingulate cortex forming the supracallosal bundle. The functional significance of this structure has been investigated. The effects of stimulation of the locus coeruleus on single unit activity in severa1 regions of the brain have been found to be inhibitory (11, 24). The locus coeruleus has been postulated to play a role in sleep-wake cycles (4, 15, lG), regulation of water intake (19), and learning (3). Self-stimulation is a behavior for which the noradrenergic fiber system originating in the locus coeruleus may be important; the supporting evidence has been summarized recently by German and Bowden (8). However, some reports have questioned the role of the locus coeruleus in this behavior (1). The refractory periods of neurons in regions through which the dorsal noradrenergic bundle travels, and which may be involved in self-stimulation elicited from these regions, have been determined (7, 9, 22) and the refractory period attributed to locus coeruleus neurons (S) . In spite of the many anatomical and functional studies concerning the locus coeruleus the electrophysiological properties of these neurons remain virtually unknown. Meaningful electrophysiological experiments demand positive identification of the cells from which recordings are made. The small size of the locus coeruleus (extending approximately 1 mm rostrocaudally, 0.3 mm mediolaterally, and 0.6 mm dorsoventrally) and the presence of nonnoradrenergic neurons in the nucleus (26) makes it imperative that criteria in addition to the position of the recording electrode tip be used for this identification. The experiments reported here were designed to provide a means of positively identifying locus coeruleus units during the course of an electrophysiological experiment and to gather data on the electrophysiological properties of these neurons. Three sites were selected for antidromically stimulating locus coeruleus neurons : supracallosal bundle, dorsal noradrenergic bundle in the region of the midbrain, and dorsal noradrenergic bundle in the region of the lateral hypothalamus. A fourth site, the olfactory bulb, from which locus coeruleus units could be driven antidromically,
256
FAIERS
AND
MOGENSON
was found while studying the responses neurons to various orthodromic inputs.
of identified
locus
coeruleus
METHODS Seventeen male Wistar rats (Woodlyn Farms, Guelph, Ontario) weighing between 440 and 480 g and anesthetized with urethane (120 mg/lOO g, ip ) were used. A plastic t-tube was inserted in the trachea. Rectal temperature was monitored and maintained between 36 and 38 C. Rats were held in a Kopf stereotaxic frame so that the head of the rat was horizontal; a strip of bone several millimeters wide and extending from the olfactory bulb to the posterior part of cerebellum was removed with a dental drill. Two strips of bone containing bregma and lambda were left intact. The clura covering cerebellum was deflected and the exposed cortices were covered with mineral oil. Stainless-steel microelectrodes (10) were used for recording. The locus coeruleus was approached at 15” to a plane passing vertically through the ear bars to avoid piercing the transverse sinus ; coordinates used were 4.5 to 5.0 nm posterior to lambda, 0.9 to 1.3 mm lateral to lambda, and 5.5 to 6.2 rnnl ventral to cerebellar cortex. Standard electrophysiological recording techniques were used. The antidromic conduction of an action potential was verified in each case by the collision technique. Coaxial bipolar electrodes (SNE-100, Rhodes Instruments, Los Angeles, California) were visually guided to the olfactory bundle and supracallosal bundle and stereotaxically guided to the midbrain region of the dorsal noradrenergic bundle (6.5 mm posterior and 1.0 mm lateral to bregma and 5.6 mm ventral to cortex) and the hypothalamic region of the dorsal noradrenergic bundle (4.0 mm posterior and 1.5 mm lateral to bregma and 7.0 mn ventral to cortex). All stimulation sites were ipsilateral to the recording site. Monophasic square pulses of 1 to 2 msec duration were delivered to stimulating electrodes from a Grass S44 stimulator; stimulation voltage was 5 to 15 V for dorsal noradrenergic bundle sites, 10 to 20 V for olfactory bulb and 10 to 30 V for supracallosal bundle. Stimulation and recording sites were marked by depositing iron from the electrode tip, and standard histological procedures were used (5). The locations of the locus coeruleus, dorsal noradrenergic bundle, and supracallosal bundle were identified from the brain maps of Ungerstedt (30) and Jacobowitz and Palkovits (14, 20). Unit activity was accepted as being recorded from a neuron in the locus coeruleus if it was antidromitally activated by stimulation of the supracallosal bundle and the recording electrode tip was histologically verified to have been in the locus coeruleus. To estimate the length of the conduction pathway from the locus coeruleus to the various stimulation sites, sagittal sections were made of the
IDEiYTJFICATJON
OF
LOCUS
COERULEGS
257
NEURONS
brains of three rats in the same weight range as those used for the electrophysiological experiments. Iron deposits, two in the dorsoventral and two in the rostrocaudal plane in the brain of each rat, were made at a known separation so that shrinkage could be calculated. Drawings were made of a sagittal section through the locus coeruleus. and the noradrenergic pathon this drawing. way. as shown by Ungerstedt (30 j, was superimposed The length of the pathway was measured and corrected for shrinkage. No attempt was made to account for the lateral deviations of the pathway. RESULTS
.Jxtidroluic
R~rs~o~~~es to Stimulation
of the Supracallosal Bundle.
Thirty-five neurons, histologically verified as being located in the locus coeruleus (Fig. 1). were antidromically driven at a latency of 44 r+ 2 msec (range 23 to 83 msec) by stimulation of sites along the anterior portion of supracallosal bundle (Fig. 2A ). The antidromic nature of the response was established for each unit by collision of the antidromic action potential with a spontaneous action potential (Figs. 3B and C. 4B and C j. Threshold for most of these responses was approximately 20 V. The refractory
FIG. cording
1. Photomicrographs of iron deposits (arrows) marking electrodes in the 10x1s coeruleus. Calibration mark 1 mm.
the
position
of re-
258
FAIERS
AND
MOGENSON
c
FIG. 2. Photomicrographs of iron deposits (arrows) made to mark sites of stimulation. A-supracallosal bundle. B-dorsal noradrenergic bundle in region of lateral hypothalamus, C-olfactory bundle, and D-dorsal noradrenergic bundle in region of midbrain. Calibration marks 1 mm.
period was determined at 30 V for six of the 35 units and was found to be 15 * 2 msec (range 8 to 20 msec ; Figs. 3C and D). The length of the pathway from the locus coeruleus to supracallosal stimulation sites was estimated to be 18 mm from which an average conduction velocity of 0.4 m/set was calculated. No units histologically located outside the locus coeruleus were antidromically driven by stimulation of the supracallosal bundle although two units, whose location could not be histologically verified, were driven antidromically at a latency of 15 msec. Spontaneous Activity. The spontaneous firing rate was determined for 26 of the 35 neurons and found to be 2.6 -C 1.2/set (range 0.9 to 5.l/sec ; Figs. 3A, 4A). The average firing rate of these neurons remained constant while the electrophysiological experiments were being performed (a few were held for more than 3 hr). The action potentials had a mean amplitude of 250 PV (range 80 to 1000 pV> and a spike duration of approximately 2 msec. Antidromic Responses to Stivvmlation of the Dorsal Noradrenergic Bundle. Sites in the midbrain portion of the dorsal noradrenergic bundle (Fig. 2D) were stimulated while recording from 22 of the 35 neurons antidromically activated from the supracallosal bundle ; 14 of the 22 were antidromically activated from the dorsal noradrenergic bundle at a latency
IDENTIFICATION
OF
LOCUS
COERULEUS
XEURONS
2.50
(range G to IO msec; Figs. 3G and H, 4F and G). A short-latency (approximately 5 msec) orthodromic response was elicited from the dorsal noradrenergic bundle after stimulation of six of the eight neurons which did not respond antidromically and this orthodromic activity may have cancelled the antidromic response. Refractory period was determined at twice threshold for four of the antidromically activated neurons and was found to be 6.0 + 1 msec (range 4 to 8 msec; Figs. 31, 4H). The distance from these stimulation sites to the locus coeruleus was estimated to be 4 to 5 mm giving an average conduction velocity of 0.5 to 0.6 m/set. of S k 0.4 msec
FIG. 3. Single unit activity of a locus coeruleus neuron. A-Spontaneous activity. B-Antidromic response (starred in this and subsequent traces) to stimulation of supracallosal bundle at 30 V and Z-msec pulse duration; five sweeps. C-Antidromic responses to twin pulse stimulation of supracallosal bundle, 30 msec separation, same parameters as in B; three sweeps. Note that one of the three responses to the first three stimuli fails because of spontaneously occurring action potential (downward pointing arrow). D-Twin pulse stimulation of supracallosal bundle at 20 msec separation, stimulation parameters as in B; three sweeps. Note that two of three antidromic responses to the second stimulus fail to occur. E-Antidromic response to stimulation of olfactory bulb at 15 V and Z-msec pulse duration; five sweeps. FStimulation of olfactory bulb at same parameters as in E; one sweep. Note that antidromic response fails to be recorded because of collision with spontaneously occurring action potential. G-Antidromic response to stimulation of dorsal noradrenergic bundle in region of midbrain at 8 V and 2-msec pulse duration; 5 sweeps. H-Stimulation of dorsal noradrenergic bundle in region of midbrain at same parameters as in G; one sweep. Note collision. I-Antidromic responses to twin pulse stimulation of dorsal noradrenergic bundle in region of midbrain at 8-msec separation, 16 V and Z-msec pulse duration; six sweeps. Note the variable latency of the response to the second stimulus. Onset of stimulus artifact is marked by upward pointing arrows. Antidromic responses are marked by stars. Time calibration (horizontal bar at lower right) is 2 set in A, 10 msec in B through F and 5 msec in G through I. All recordings from same neuron. Positivity downward.
260
FAIERS
AND
MOGENSON
FIG. 4. Single unit activity recorded from a neuron located in locus coeruleus. ASpontaneous activity of unit. B-Antidromic response (starred in this and subsequent traces) to stimulation of supracallosal bundle at 30 V and 2-msec pulse duration; four sweeps. C-Stimulation of supracallosal bundle at same parameters as in B; one sweep. Note collision. D-Antidromic response to stimulation of olfactory bulb at 12 V and 2-msec pulse duration; four sweeps. E-Stimulation of olfactory bulb at same parameters as in D; one sweep. Note collision. F-Antidromic response to stimulation of dorsal noradrenergic bundle in region of midbrain, at 8 V and 1.5msec pulse duration; four sweeps. G-Stimulation of dorsal noradrenergic bundle in region of midbrain at same parameters as in F; one sweep. Note collision. H-Twin pulse stimulation of dorsal noradrenergic bundle in region of midbrain at 14 msec separation, 15 V and 1.5msec pulse duration; five sweeps. Note failure of two antidromic responses and variable latency of three responses to second stimulus. I-Antidromic response to stimulation of dorsal noradrenergic bundle in region of lateral hypothalamus at 8 V and 1.5-msec pulse duration; five sweeps. J-Stimulation of dorsal noradrenergic bundle in region of lateral hypothalamus at same parameters as in I; one sweep. Note collision. Onset of stimulus artifact is marked by upward pointing arrows. Antidromic responses are marked by stars. Time calibration is 5 set in A, 10 msec in A through E and 5 msec in F through J. All recordings were made from the same neuron. Positivity downward.
Twenty of the 35 neurons antidromically activated from the supracallosal bundle were recorded from while stimulating the dorsal noradrenergic bundle in the region of the lateral hypothalamus (Fig. 2B) ; 15 of these 20 were antidromically activated at a latency of 12 -t 0.3 msec (range 10 to 15 msec; Figs. 41, J). In four of the five neurons for which antidromic activation could not be demonstrated, the antidromic response may have been cancelled by a short-latency (4 to 10 msec) orthodromic response elicited from the lateral hypothalamic region of the dorsal noradrenergic bundle. The refractory period was determined at twice threshold
IDENTIFICATION
OF
LOCUS
COERULEUS
NEURONS
261
for four of the antidromically activated neurons (10 * 2 msec; range 5 to 14 msec). It was estimated from sagittal brain sections that the dorsal noradrenergic bundle stimulation sites in the lateral hypothalamus lay 6 to 7 mm from the locus coeruleus giving a conduction velocity of 0.5 to 0.6 m/set. Neurons advenergic
Responding Antidromically Bundle but not to Stimulation
to Stimulation of the Dorsal of the Supracallosal Bundle.
Nor-
The location of 21 neurons which did not respond antidromically to stimulation of supracallosal bundle but did respond antidromically to stimulation of dorsal noradrenergic bundle sites was histologically verified. Eleven neurons were driven antidromically by stimulation of both lateral hypothalamus and midbrain regions of the supracallosal bundle, six neurons were driven antidromically only by stimulation of the midbrain region, and four others only by stimulation of lateral hypothalamic region of the dorsal noradrenergic bundle. Nine of these 21 neurons were situated in locus coeruleus and the antidromic response of these neurons was similar in latency (8 + 0.7 msec for midbrain; 14.5 +- 0.8 msec for lateral hypothalamus) to that of the locus coeruleus neurons responding to stimulation of the supracallosal bundle. However, 12 neurons located outside of, but close to the locus coeruleus in the dorsal tegmental nucleus of Gudden, reticular formation, and mesencephalic nucleus of trigeminal nerve, also responded antidromically to stimulation of the dorsal noradrenergic bundle at latencies in the samerange as the latencies of the identified locus coeruleus neurons. Antidvowtic Responses to Stimulation of the Olfactory Bulb. The olfactory bulb (Fig. 2C) was stimulated while recording from 21 of the 35 identified neurons ; seven neurons responded antidromically (as verified by collision) at a latency of 39 2 4.5 msec (range 26 to 60 msec; Figs. 3E and F, 4D and E). Twelve neurons were orthodromically activated by stimulation of the olfactory bulb at a latency of 35 f 5 msec (range 20 to 60 msec). Stimulation sites in the olfactory bulb were approximately the same distance from the locus coeruleus as those in the supracallosal bundle and the average conduction velocity was estimated as similar to that along the supracallosal bundle (0.4 m/set). Refractory period was not tested at any of these sites. DISCUSSION Electrical stimulation of the supracallosal bundle was shown to activate antidromically neurons of the locus coeruleus at a characteristically long latency thus providing a procedure for identifying a subpopulation of locus coeruleus neurons while an electrophysiological experiment is in progress. Additional characteristics of the antidromically activated locus coeruleus
262
FAIERS
AND
MOGENSON
neurons were their slow, steady firing rate, slow conduction velocity, and the long refractory periods of their axons. The iron deposits made at the sites from which antidromically-driven units were recorded were observed to be in the locus coeruleus. It was not necessary to use histofluorescence to identify this cell group because the locus coeruleus is easily recognized in stained sections of the rat brain. The mean discharge rate of locus coeruleus neurons was lower than that reported by Chu and Bloom (4) in the unanesthetized cat. They reported a firing frequency of 4.2 + 1.1 set during slow wave sleep for neurons identified by the proximity of the microelectrode tip to histofluorescent cell bodies of the locus coeruleus and a correlation between the firing rate of these neurons and the attentiveness of the animal was observed. The conduction velocity calculated for the axons of locus coeruleus neurons was 0.4 to 0.6 m/set. Similar conduction velocities have been recorded for peripheral autonomic fibers of comparable diameter (23). Lateral deviations of the pathway from the locus coeruleus to the supracallosal bundle were not taken into consideration, and the path length used to calculate conduction velocity was probably underestimated. In addition, fibers from the locus coeruleus can reach neocortex through either the internal or external capsule (29) and fibers of this pathway might have been stimulated. If either or both of these factors were operative the actual conduction velocity would be slower than the conduction velocity reported here. Because the conduction velocity for the path from supracallosal bundle to the locus coeruleus was slower than that from dorsal noradrenergic bundle sites to the locus coeruleus it may be inferred that these fibers are tapered. A recent study (18) has reported similar conduction velocities based on the latency of the antidromic response to stimulation of the dorsal noradrenergic bundle in the midbrain. Latencies of the antidromic response to stimulation of cerebral cortex (20 to 70 msec) were similar to the latency of the antidromic response to stimulation of the supracallosal bundle and olfactory bundle reported here. The refractory periods of the axons in dorsal noradrenergic bundle were determined at twice stimulus threshold and found to be 6 and 10 msec, respectively, for the midbrain and hypothalamic sites. Because the number of neurons tested in each case was small it is felt that the difference in refractory periods obtained from these two sites may not be significant; it is unlikely that the fibers of the locus coeruleus neurons are of appreciably smaller diameter in the hypothalamic region of the ‘dorsal noradrenergic bundle. Refractory periods exceeding 3 msec have been observed for unmyelinated fibers 1 pm in diameter and a plot of diameter against absolute refractory period shows that the refractory period increases markedly at diameter less than 1 pm (12). The refractory period of the axons at supracallosal bundle sites was determined to be 15 msec at 30 V; although this
IDENTIFICATION
OF
LOCUS
COERULEUS
IGEURONS
263
is only 10 V higher than threshold for the antidromic response, it is likely that the absolute refractory period of axons in the supracallosal bundle is also long. The high voltage and long pulse durations necessary to antidromically drive locus coeruleus neurons from the supracallosal bundle may be due to the small diameter of the fibers in this part of the pathway. It has recently been demonstrated, using the retrograde transport of horseradish peroxidase, that the locus coeruleus projects to the olfactory bulb (13). This projection was verified in the present study by antidromitally driving locus coeruleus neurons by stimulation of the olfactory bulb. Because locus coeruleus neurons could be antidromically activated by stimulation of either the olfactory bulb or supracallosal bundle, some locus coeruleus neurons must send collaterals to both of these structures. Only seven neurons were antidromically activated by stimulation of the olfactory bulb; however, in many cases a shorter-latency orthodromic response to stimulation of the olfactory bulb may have obscured the antidromic response. The latency and conduction velocity of antidromic responses to stimulation of the olfactory bulb were similar to those elicited from the supracallosal bundle. Because no projections to the olfactory bulb have been found to originate near the locus coeruleus (13)) an antidromic response to stimulation of the olfactory bulb can be used to identify locus coeruleus neurons. An antidromic response to stimulation of the stria terminalis also might be used to identify locus coeruleus neurons since they project through this pathway (21 j ; a different subpopulation of locus coeruleus neurons might be identified this way. Some of the locus coeruleus neurons antidromically activated by stimulation of the supracallosal bundle could not be driven antidromically by stimulation of the dorsal noradrenergic bundle. It is possible that some of the fibers stimulated in the supracallosal bundle passed caudally in the midbrain through the central tegmental tract situated ventrolateral to the dorsal noradrenergic bundle so that they were not close enough to the stimulating electrode in this pathway to be driven. In some cases a short-latency orthodromic response observed after stimulation of the dorsal noradrenergic bundle may have obscured the antidromic response to stimulation of these sites. A direct projection from the bed nucleus of the stria terminalis to the locus coeruleus through the medial forebrain bundle and central tegmental field has been demonstrated anatomically (38) and perhaps these fibers were stimulated. Because it was shown that cells in the area surrounding the locus coeruleus, as well as locus coeruleus cells, could be antidromically activated by stimulation of the two dorsal noradrenergic bundle sites (hypothalamic and midbrain), these stimulation sites could not be used to provide an unambiguous identification of locus coeruleus neurons. The study reported here has some implications for speculations concern-
264
FAIERS
AND
MOGENSON
ing the functional role of locus coeruleus neurons. It has been postulated that the noradrenergic system originating in the locus coeruleus is involved in self-stimulation at sites in the medial forebrain bundle (near the dorsal noradrenergic bundle) and the refractory period of the neurons subserving this behavior has been determined, using behavioral techniques, to be 0.5 to 1.1 msec (7, 9). Similar refractory periods have been determined electrophysiologically for neurons in medial forebrain bundle implicated in selfstimulation (22). Because these refractory periods are much shorter than those reported in this study for fibers of the dorsal noradrenergic bundle it would appear that the refractory periods measured in previous studies were not those of the noradrenergic fibers originating in the locus coeruleus as has been suggested (8). Although this finding does not in any way prove that the locus coeruleus is not involved in self-stimulation behavior, the long refractory period of locus coeruleus neurons will have to be accounted for in future speculations concerning the involvement of this system in self-stimulation. This study provides a means of identifying locus coeruleus neurons during the course of an electrophysiological experiment by their long-latency antidromic response to stimulation of supracallosal bundle or olfactory bulb and their characteristic slow rate of firing. The antidromic response to stimulation of dorsal noradrenergic bundle sites cannot be used to identify locus coeruleus neurons because neurons near the locus coeruleus are also antidromically activated. Considering the small size of the locus coeruleus and the presence of nonnoradrenergic neurons which do not project from the locus coeruleus, this method of identifying locus coeruleus neurons should prove useful in future experiments on the electrophysiological properties of locus coeruleus neurons. The technique may also be used to suggest or verify the existence of anatomical projections of the locus coeruleus. REFERENCES 1. AMARAL, D. G., and A. ROUTTENBERG. 1975. Locus coeruleus and intracranial self-stimulation: A cautionary note. Behav. Biol. 13 : 331-338. 2. ANDBN, N. E., A. DAHLSTRBM, K. FUXE, K. LARSSON, L. OLSON, and U. UNGERS-T. 1966. Ascending monoamine neurons to the telencephalon and diencephalon. Acta Physiol. Stand. 67: 313-326. 3. ANLEZARK, G. M., T. J. CROW, and A. P. GREENWAY. 1973. Impaired learning and decreased cortical norepinephrine after bilateral locus coeruleus lesions. Science 181: 682-684. 4. CHU, N. D., and F. E. BLOOM. 1973. Norepinephrine-containing neurons: Changes in spontaneous discharge patterns during sleeping and waking. Science 179: 90%910. 5. FAIERS, A.
A., F. R. CALARESU, and G. J. MOGENSON. 1975. Pathway mediating hypotension elicited by stimulation of the amygdala in the rat. -&rev. J. Physiol. 228 : 1358-1366.
IDENTIFICATION
OF
LOCUS
COERULEUS
265
NEURONS
6. FUXE, K. 1965. Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine terminal in the central nervous system. Acta Physiol. Stand. 64 (Suppl 247) : 37-85. 7. GALLISTEL, C. R., E. ROLLS, and D. GREENE. 1969. Neuron function inferred from behavioral and electrophysiological estimates of refractory period. Science 166: 1028-1030. 8. GERMAX, D. C., and D. M. Bownax. 1974. Catecholamine systems as the neural substrate for intracranial self-stimulation: A hypothesis. Bra& Rcs. 73: 381419. 9. GERMAN, D. C., and F. A. HOLLOWAY. 1972. Behaviorally determined neurophysiological properties of MFB self-stimulation fibers. Physiol. Behazr. 9: 823-829. 10. GREEN, J. D. 1958. A simple microelectrode for recording from the central nervous system. Natwe (LOIKICIII) 182: 962. 11. HOFFER, B. J., G. R. SICCIKS, A. P. OLIVER, and F. E. BLOOM. 1973. Activation of the pathway from locus coeruleus to rat cerebellar purkinje neurons: Pharmacological evidence of noradrenergic central inhibition. J. Pltarntacol. Erp. They. 184 : 553-569. 12. HURSH, J. B. 1939. The properties of growing fibers. ‘4111~~. J. PhysioZ. 127: 140153. 13. JACOBOWITZ, D. M., and R. D. BROADWELT.. 1975. Origin of afferent fibers to the olfactory bulb of the rat. Nerrrosci. Abstr. 1 : 677. 14. JACOBOWITZ, D. M., and M. PALKOVITS. 1974. Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. I. Forebrain (telencephalon, diencephalon). J. Conzp. Ncnrol. 157 : 13-28. 1.5. JONES, B. E., P. BOBILIXR, C. PIN, and M. JOUYET. 1973. The effect of lesions of catecholamine containing neurons upon monoamine content of the brain and EEB and behavioral waking in the cat. BraLz Rcs. 58: 157-177. 16. LIDBRINK, P. 1974. The effect of lesions of ascending noradrenaline pathways on sleep and waking in the rat. Brain Rcs. 74: 19-20. 17. LINDVALL, O., and A. BJ~RKLUXD. 1974. The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method. Acta Physiol. Scalzd. Suppl. 412: l-48. 18. NAKAMCR.4, S., and K. IWAMA. 1975. Antidromic activation of the rat locus coeruleus neurons from hippocampus, cerebral and cerebellar cortices. Brain
Res. 99: 372-376, 19. OSUMI, Y., R. OISHI, H. FUJIWARA, and S. TAKAORI. 1973. Hyperdipsia induced by bilateral destruction of the locus coeruleus in rats. Bvaix Res. 86: 419-427. 20. PALKO~ITS, M., and D. M. JACOBOWITZ. 1975. Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephalon). /. Contp. Ncu~ol. 157 : 29-42. 21. PICKLE, V. R;I., M. SEGAL, and F. E. BLOOM. 1974. A radioautographic study of the efferent pathways of the nucleus locus coeruleus. J. Camp. Neural. 155: 15112. 22. ROLLS, E. T. 1971. Absolute refractory period of neurons involved in MFB selfstimulation. Pizysiol. Beizav. 7 : 311-315. 23. RUCH, T. C., and H. D. PATTON. 1965. “Pllysiology and Biophysics," p. Z-31. W. B. Saunders, Philadelphia. ‘24. SEGAL, M., and F. E. BLOOM. 1974. The action of norepinephrine on the rat hippocampus. II. Activation of the input pathway. Brain Res. 72: 99-114. 25. SEGAL,
M.,
and
S. LANDIS.
1974. Afferents
to the hippocampus
of the
rat
studied
266
26. 27. 28. 29. 30.
FAIERS
AND
MOGENSON
with the method of retrograde transport of horseradish peroxidase. Brab Res. 7’8: I-15. SHIMIZU, N., and K. IMAMOTO. 1970. Fine structure of the locus coeruleus in the rat. Arch. Histol. Jap. 31: 229-246. SHIMIZU, N., S. OHNISHI, M. TOHYAMA, and T. MAEDA. 1974. Demonstration by degeneration silver method of the ascending projection from the locus coeruleus. Exp. Bra& Research. 20: 181-192. SWANSON, L. W., and C. B. SAPER. 1975. Direct neural inputs to locus coeruleus from basal forebrain. Nezlrosci. Abstr. 1: 683. TOHYAMA, M., T. MAEDA, and H. SHIMIZU. 1974. Detailed noradrenaline pathways of locus coeruleus neurons to the cerebral cortex with use of 6-hydroxydopa. Brain lies. 79: 139-144. UNGERSTEDT, U. 1971. Stereotaxic mapping of monoamine pathways in the rat brain. Acta Physiol. Stand. 82(Suppl. 367) : l-48.
53, 254266
NEUROLOGY
Electrophysiological
(1976)
Identification Locus Coeruleus’
of Neurons
in
A. A. FAIERS AND G. J. MOGENSON Departments
of Physiology London,
and Psychology, The University Ontario, Canada N6A 5Cl Received
March
of Western
Ontario,
29,1976
In 17 urethane-anesthetized rats 35 neurons, histologically verified as being situated in the loxrs coeruleus, were driven antidromically (latency, 44 msec) by electrical stimulation of the supracallosal bundle. Neurons of the locus coeruleus were also activated antidromically by stimulation of sites along the dorsal noradrenergic bundle in the midbrain (8-msec latency) and the hypothalamus (12-msec latency), and by stimulation of sites in the olfactory bulb (latency, 39 msec). Conduction velocity from these sites to the locus coeruleus was estimated to be 0.4 to 0.6 m/set. Refractory periods of fibers in the dorsal noradrenergic bundle were determined at twice threshold and in the supracallosal bundle at intensities just above threshold; refractory periods ranging from 4 to 20 msec were observed. Because neurons both in and near the locus coeruleus were antidromically activated by stimulation of the dorsal noradrenergic bundle whereas stimulation of the supracallosal bundle antidromically activated only neurons in the locus coeruleus, stimulation of the dorsal noradrenergic bundle could not be used to identify locus coeruleus neurons. It is concluded that a subpopulation of neurons in the locus coeruleus can be identified by their slow, steady firing rate (2.6 per second) and long-latency antidromic response to stimulation of the supracallosal bundle. The electrophysiological properties of locus coeruleus neurons are considered in relation to neuroanatomical and functional studies of the locus coeruleus.
INTRODUCTION The projections of the nucleus locus coeruleus have been mapped using a variety of techniques including histofluorescence (2, 8, 14, 17, 20, 30) radioautography (21)) silver staining (27)) and retrograde transport of 1 This work was supported by grants from the Medical Research Council of Canada and the National Research Council of Canada. The authors wish to express their appreciation to Dr. J. D. Cooke, D. Carter, and Miss B. Box for their criticism of the manuscript. 2.54 Copyright All rights
1976 by Academic Press, Inc. 8 reproduction CI in any form reserved.
IDENTIFICATION
OF
LOCUS
COERULEUS
NEURONS
2 55
horseradish peroxidase (13. 35). These studies have shown the locus coeruleus to consist mainly of medium size noraclrenergic cell bodies whose fine axons project to many areas of the brain including hippocampus, cerebral and cerebellar cortices, amygdala. and some thalamic and hypothalnmic nuclei. The major rostra1 projection of the locus coeruleus is through the dorsal noradrenergic bundle which ascendsin the midbrain beside lhe dorsal longitudinal fasciculus of Shultz and then passesventrally into the lateral hypothalamus. The locus coeruleus also projects rostrally through the central tegmental tract and the periventricular pathway (17). The hippocampus is innervated by a rostra1 extension of dorsal noradrenergic bundle which projects through the cingulate cortex forming the supracallosal bundle. The functional significance of this structure has been investigated. The effects of stimulation of the locus coeruleus on single unit activity in severa1 regions of the brain have been found to be inhibitory (11, 24). The locus coeruleus has been postulated to play a role in sleep-wake cycles (4, 15, lG), regulation of water intake (19), and learning (3). Self-stimulation is a behavior for which the noradrenergic fiber system originating in the locus coeruleus may be important; the supporting evidence has been summarized recently by German and Bowden (8). However, some reports have questioned the role of the locus coeruleus in this behavior (1). The refractory periods of neurons in regions through which the dorsal noradrenergic bundle travels, and which may be involved in self-stimulation elicited from these regions, have been determined (7, 9, 22) and the refractory period attributed to locus coeruleus neurons (S) . In spite of the many anatomical and functional studies concerning the locus coeruleus the electrophysiological properties of these neurons remain virtually unknown. Meaningful electrophysiological experiments demand positive identification of the cells from which recordings are made. The small size of the locus coeruleus (extending approximately 1 mm rostrocaudally, 0.3 mm mediolaterally, and 0.6 mm dorsoventrally) and the presence of nonnoradrenergic neurons in the nucleus (26) makes it imperative that criteria in addition to the position of the recording electrode tip be used for this identification. The experiments reported here were designed to provide a means of positively identifying locus coeruleus units during the course of an electrophysiological experiment and to gather data on the electrophysiological properties of these neurons. Three sites were selected for antidromically stimulating locus coeruleus neurons : supracallosal bundle, dorsal noradrenergic bundle in the region of the midbrain, and dorsal noradrenergic bundle in the region of the lateral hypothalamus. A fourth site, the olfactory bulb, from which locus coeruleus units could be driven antidromically,
256
FAIERS
AND
MOGENSON
was found while studying the responses neurons to various orthodromic inputs.
of identified
locus
coeruleus
METHODS Seventeen male Wistar rats (Woodlyn Farms, Guelph, Ontario) weighing between 440 and 480 g and anesthetized with urethane (120 mg/lOO g, ip ) were used. A plastic t-tube was inserted in the trachea. Rectal temperature was monitored and maintained between 36 and 38 C. Rats were held in a Kopf stereotaxic frame so that the head of the rat was horizontal; a strip of bone several millimeters wide and extending from the olfactory bulb to the posterior part of cerebellum was removed with a dental drill. Two strips of bone containing bregma and lambda were left intact. The clura covering cerebellum was deflected and the exposed cortices were covered with mineral oil. Stainless-steel microelectrodes (10) were used for recording. The locus coeruleus was approached at 15” to a plane passing vertically through the ear bars to avoid piercing the transverse sinus ; coordinates used were 4.5 to 5.0 nm posterior to lambda, 0.9 to 1.3 mm lateral to lambda, and 5.5 to 6.2 rnnl ventral to cerebellar cortex. Standard electrophysiological recording techniques were used. The antidromic conduction of an action potential was verified in each case by the collision technique. Coaxial bipolar electrodes (SNE-100, Rhodes Instruments, Los Angeles, California) were visually guided to the olfactory bundle and supracallosal bundle and stereotaxically guided to the midbrain region of the dorsal noradrenergic bundle (6.5 mm posterior and 1.0 mm lateral to bregma and 5.6 mm ventral to cortex) and the hypothalamic region of the dorsal noradrenergic bundle (4.0 mm posterior and 1.5 mm lateral to bregma and 7.0 mn ventral to cortex). All stimulation sites were ipsilateral to the recording site. Monophasic square pulses of 1 to 2 msec duration were delivered to stimulating electrodes from a Grass S44 stimulator; stimulation voltage was 5 to 15 V for dorsal noradrenergic bundle sites, 10 to 20 V for olfactory bulb and 10 to 30 V for supracallosal bundle. Stimulation and recording sites were marked by depositing iron from the electrode tip, and standard histological procedures were used (5). The locations of the locus coeruleus, dorsal noradrenergic bundle, and supracallosal bundle were identified from the brain maps of Ungerstedt (30) and Jacobowitz and Palkovits (14, 20). Unit activity was accepted as being recorded from a neuron in the locus coeruleus if it was antidromitally activated by stimulation of the supracallosal bundle and the recording electrode tip was histologically verified to have been in the locus coeruleus. To estimate the length of the conduction pathway from the locus coeruleus to the various stimulation sites, sagittal sections were made of the
IDEiYTJFICATJON
OF
LOCUS
COERULEGS
257
NEURONS
brains of three rats in the same weight range as those used for the electrophysiological experiments. Iron deposits, two in the dorsoventral and two in the rostrocaudal plane in the brain of each rat, were made at a known separation so that shrinkage could be calculated. Drawings were made of a sagittal section through the locus coeruleus. and the noradrenergic pathon this drawing. way. as shown by Ungerstedt (30 j, was superimposed The length of the pathway was measured and corrected for shrinkage. No attempt was made to account for the lateral deviations of the pathway. RESULTS
.Jxtidroluic
R~rs~o~~~es to Stimulation
of the Supracallosal Bundle.
Thirty-five neurons, histologically verified as being located in the locus coeruleus (Fig. 1). were antidromically driven at a latency of 44 r+ 2 msec (range 23 to 83 msec) by stimulation of sites along the anterior portion of supracallosal bundle (Fig. 2A ). The antidromic nature of the response was established for each unit by collision of the antidromic action potential with a spontaneous action potential (Figs. 3B and C. 4B and C j. Threshold for most of these responses was approximately 20 V. The refractory
FIG. cording
1. Photomicrographs of iron deposits (arrows) marking electrodes in the 10x1s coeruleus. Calibration mark 1 mm.
the
position
of re-
258
FAIERS
AND
MOGENSON
c
FIG. 2. Photomicrographs of iron deposits (arrows) made to mark sites of stimulation. A-supracallosal bundle. B-dorsal noradrenergic bundle in region of lateral hypothalamus, C-olfactory bundle, and D-dorsal noradrenergic bundle in region of midbrain. Calibration marks 1 mm.
period was determined at 30 V for six of the 35 units and was found to be 15 * 2 msec (range 8 to 20 msec ; Figs. 3C and D). The length of the pathway from the locus coeruleus to supracallosal stimulation sites was estimated to be 18 mm from which an average conduction velocity of 0.4 m/set was calculated. No units histologically located outside the locus coeruleus were antidromically driven by stimulation of the supracallosal bundle although two units, whose location could not be histologically verified, were driven antidromically at a latency of 15 msec. Spontaneous Activity. The spontaneous firing rate was determined for 26 of the 35 neurons and found to be 2.6 -C 1.2/set (range 0.9 to 5.l/sec ; Figs. 3A, 4A). The average firing rate of these neurons remained constant while the electrophysiological experiments were being performed (a few were held for more than 3 hr). The action potentials had a mean amplitude of 250 PV (range 80 to 1000 pV> and a spike duration of approximately 2 msec. Antidromic Responses to Stivvmlation of the Dorsal Noradrenergic Bundle. Sites in the midbrain portion of the dorsal noradrenergic bundle (Fig. 2D) were stimulated while recording from 22 of the 35 neurons antidromically activated from the supracallosal bundle ; 14 of the 22 were antidromically activated from the dorsal noradrenergic bundle at a latency
IDENTIFICATION
OF
LOCUS
COERULEUS
XEURONS
2.50
(range G to IO msec; Figs. 3G and H, 4F and G). A short-latency (approximately 5 msec) orthodromic response was elicited from the dorsal noradrenergic bundle after stimulation of six of the eight neurons which did not respond antidromically and this orthodromic activity may have cancelled the antidromic response. Refractory period was determined at twice threshold for four of the antidromically activated neurons and was found to be 6.0 + 1 msec (range 4 to 8 msec; Figs. 31, 4H). The distance from these stimulation sites to the locus coeruleus was estimated to be 4 to 5 mm giving an average conduction velocity of 0.5 to 0.6 m/set. of S k 0.4 msec
FIG. 3. Single unit activity of a locus coeruleus neuron. A-Spontaneous activity. B-Antidromic response (starred in this and subsequent traces) to stimulation of supracallosal bundle at 30 V and Z-msec pulse duration; five sweeps. C-Antidromic responses to twin pulse stimulation of supracallosal bundle, 30 msec separation, same parameters as in B; three sweeps. Note that one of the three responses to the first three stimuli fails because of spontaneously occurring action potential (downward pointing arrow). D-Twin pulse stimulation of supracallosal bundle at 20 msec separation, stimulation parameters as in B; three sweeps. Note that two of three antidromic responses to the second stimulus fail to occur. E-Antidromic response to stimulation of olfactory bulb at 15 V and Z-msec pulse duration; five sweeps. FStimulation of olfactory bulb at same parameters as in E; one sweep. Note that antidromic response fails to be recorded because of collision with spontaneously occurring action potential. G-Antidromic response to stimulation of dorsal noradrenergic bundle in region of midbrain at 8 V and 2-msec pulse duration; 5 sweeps. H-Stimulation of dorsal noradrenergic bundle in region of midbrain at same parameters as in G; one sweep. Note collision. I-Antidromic responses to twin pulse stimulation of dorsal noradrenergic bundle in region of midbrain at 8-msec separation, 16 V and Z-msec pulse duration; six sweeps. Note the variable latency of the response to the second stimulus. Onset of stimulus artifact is marked by upward pointing arrows. Antidromic responses are marked by stars. Time calibration (horizontal bar at lower right) is 2 set in A, 10 msec in B through F and 5 msec in G through I. All recordings from same neuron. Positivity downward.
260
FAIERS
AND
MOGENSON
FIG. 4. Single unit activity recorded from a neuron located in locus coeruleus. ASpontaneous activity of unit. B-Antidromic response (starred in this and subsequent traces) to stimulation of supracallosal bundle at 30 V and 2-msec pulse duration; four sweeps. C-Stimulation of supracallosal bundle at same parameters as in B; one sweep. Note collision. D-Antidromic response to stimulation of olfactory bulb at 12 V and 2-msec pulse duration; four sweeps. E-Stimulation of olfactory bulb at same parameters as in D; one sweep. Note collision. F-Antidromic response to stimulation of dorsal noradrenergic bundle in region of midbrain, at 8 V and 1.5msec pulse duration; four sweeps. G-Stimulation of dorsal noradrenergic bundle in region of midbrain at same parameters as in F; one sweep. Note collision. H-Twin pulse stimulation of dorsal noradrenergic bundle in region of midbrain at 14 msec separation, 15 V and 1.5msec pulse duration; five sweeps. Note failure of two antidromic responses and variable latency of three responses to second stimulus. I-Antidromic response to stimulation of dorsal noradrenergic bundle in region of lateral hypothalamus at 8 V and 1.5-msec pulse duration; five sweeps. J-Stimulation of dorsal noradrenergic bundle in region of lateral hypothalamus at same parameters as in I; one sweep. Note collision. Onset of stimulus artifact is marked by upward pointing arrows. Antidromic responses are marked by stars. Time calibration is 5 set in A, 10 msec in A through E and 5 msec in F through J. All recordings were made from the same neuron. Positivity downward.
Twenty of the 35 neurons antidromically activated from the supracallosal bundle were recorded from while stimulating the dorsal noradrenergic bundle in the region of the lateral hypothalamus (Fig. 2B) ; 15 of these 20 were antidromically activated at a latency of 12 -t 0.3 msec (range 10 to 15 msec; Figs. 41, J). In four of the five neurons for which antidromic activation could not be demonstrated, the antidromic response may have been cancelled by a short-latency (4 to 10 msec) orthodromic response elicited from the lateral hypothalamic region of the dorsal noradrenergic bundle. The refractory period was determined at twice threshold
IDENTIFICATION
OF
LOCUS
COERULEUS
NEURONS
261
for four of the antidromically activated neurons (10 * 2 msec; range 5 to 14 msec). It was estimated from sagittal brain sections that the dorsal noradrenergic bundle stimulation sites in the lateral hypothalamus lay 6 to 7 mm from the locus coeruleus giving a conduction velocity of 0.5 to 0.6 m/set. Neurons advenergic
Responding Antidromically Bundle but not to Stimulation
to Stimulation of the Dorsal of the Supracallosal Bundle.
Nor-
The location of 21 neurons which did not respond antidromically to stimulation of supracallosal bundle but did respond antidromically to stimulation of dorsal noradrenergic bundle sites was histologically verified. Eleven neurons were driven antidromically by stimulation of both lateral hypothalamus and midbrain regions of the supracallosal bundle, six neurons were driven antidromically only by stimulation of the midbrain region, and four others only by stimulation of lateral hypothalamic region of the dorsal noradrenergic bundle. Nine of these 21 neurons were situated in locus coeruleus and the antidromic response of these neurons was similar in latency (8 + 0.7 msec for midbrain; 14.5 +- 0.8 msec for lateral hypothalamus) to that of the locus coeruleus neurons responding to stimulation of the supracallosal bundle. However, 12 neurons located outside of, but close to the locus coeruleus in the dorsal tegmental nucleus of Gudden, reticular formation, and mesencephalic nucleus of trigeminal nerve, also responded antidromically to stimulation of the dorsal noradrenergic bundle at latencies in the samerange as the latencies of the identified locus coeruleus neurons. Antidvowtic Responses to Stimulation of the Olfactory Bulb. The olfactory bulb (Fig. 2C) was stimulated while recording from 21 of the 35 identified neurons ; seven neurons responded antidromically (as verified by collision) at a latency of 39 2 4.5 msec (range 26 to 60 msec; Figs. 3E and F, 4D and E). Twelve neurons were orthodromically activated by stimulation of the olfactory bulb at a latency of 35 f 5 msec (range 20 to 60 msec). Stimulation sites in the olfactory bulb were approximately the same distance from the locus coeruleus as those in the supracallosal bundle and the average conduction velocity was estimated as similar to that along the supracallosal bundle (0.4 m/set). Refractory period was not tested at any of these sites. DISCUSSION Electrical stimulation of the supracallosal bundle was shown to activate antidromically neurons of the locus coeruleus at a characteristically long latency thus providing a procedure for identifying a subpopulation of locus coeruleus neurons while an electrophysiological experiment is in progress. Additional characteristics of the antidromically activated locus coeruleus
262
FAIERS
AND
MOGENSON
neurons were their slow, steady firing rate, slow conduction velocity, and the long refractory periods of their axons. The iron deposits made at the sites from which antidromically-driven units were recorded were observed to be in the locus coeruleus. It was not necessary to use histofluorescence to identify this cell group because the locus coeruleus is easily recognized in stained sections of the rat brain. The mean discharge rate of locus coeruleus neurons was lower than that reported by Chu and Bloom (4) in the unanesthetized cat. They reported a firing frequency of 4.2 + 1.1 set during slow wave sleep for neurons identified by the proximity of the microelectrode tip to histofluorescent cell bodies of the locus coeruleus and a correlation between the firing rate of these neurons and the attentiveness of the animal was observed. The conduction velocity calculated for the axons of locus coeruleus neurons was 0.4 to 0.6 m/set. Similar conduction velocities have been recorded for peripheral autonomic fibers of comparable diameter (23). Lateral deviations of the pathway from the locus coeruleus to the supracallosal bundle were not taken into consideration, and the path length used to calculate conduction velocity was probably underestimated. In addition, fibers from the locus coeruleus can reach neocortex through either the internal or external capsule (29) and fibers of this pathway might have been stimulated. If either or both of these factors were operative the actual conduction velocity would be slower than the conduction velocity reported here. Because the conduction velocity for the path from supracallosal bundle to the locus coeruleus was slower than that from dorsal noradrenergic bundle sites to the locus coeruleus it may be inferred that these fibers are tapered. A recent study (18) has reported similar conduction velocities based on the latency of the antidromic response to stimulation of the dorsal noradrenergic bundle in the midbrain. Latencies of the antidromic response to stimulation of cerebral cortex (20 to 70 msec) were similar to the latency of the antidromic response to stimulation of the supracallosal bundle and olfactory bundle reported here. The refractory periods of the axons in dorsal noradrenergic bundle were determined at twice stimulus threshold and found to be 6 and 10 msec, respectively, for the midbrain and hypothalamic sites. Because the number of neurons tested in each case was small it is felt that the difference in refractory periods obtained from these two sites may not be significant; it is unlikely that the fibers of the locus coeruleus neurons are of appreciably smaller diameter in the hypothalamic region of the ‘dorsal noradrenergic bundle. Refractory periods exceeding 3 msec have been observed for unmyelinated fibers 1 pm in diameter and a plot of diameter against absolute refractory period shows that the refractory period increases markedly at diameter less than 1 pm (12). The refractory period of the axons at supracallosal bundle sites was determined to be 15 msec at 30 V; although this
IDENTIFICATION
OF
LOCUS
COERULEUS
IGEURONS
263
is only 10 V higher than threshold for the antidromic response, it is likely that the absolute refractory period of axons in the supracallosal bundle is also long. The high voltage and long pulse durations necessary to antidromically drive locus coeruleus neurons from the supracallosal bundle may be due to the small diameter of the fibers in this part of the pathway. It has recently been demonstrated, using the retrograde transport of horseradish peroxidase, that the locus coeruleus projects to the olfactory bulb (13). This projection was verified in the present study by antidromitally driving locus coeruleus neurons by stimulation of the olfactory bulb. Because locus coeruleus neurons could be antidromically activated by stimulation of either the olfactory bulb or supracallosal bundle, some locus coeruleus neurons must send collaterals to both of these structures. Only seven neurons were antidromically activated by stimulation of the olfactory bulb; however, in many cases a shorter-latency orthodromic response to stimulation of the olfactory bulb may have obscured the antidromic response. The latency and conduction velocity of antidromic responses to stimulation of the olfactory bulb were similar to those elicited from the supracallosal bundle. Because no projections to the olfactory bulb have been found to originate near the locus coeruleus (13)) an antidromic response to stimulation of the olfactory bulb can be used to identify locus coeruleus neurons. An antidromic response to stimulation of the stria terminalis also might be used to identify locus coeruleus neurons since they project through this pathway (21 j ; a different subpopulation of locus coeruleus neurons might be identified this way. Some of the locus coeruleus neurons antidromically activated by stimulation of the supracallosal bundle could not be driven antidromically by stimulation of the dorsal noradrenergic bundle. It is possible that some of the fibers stimulated in the supracallosal bundle passed caudally in the midbrain through the central tegmental tract situated ventrolateral to the dorsal noradrenergic bundle so that they were not close enough to the stimulating electrode in this pathway to be driven. In some cases a short-latency orthodromic response observed after stimulation of the dorsal noradrenergic bundle may have obscured the antidromic response to stimulation of these sites. A direct projection from the bed nucleus of the stria terminalis to the locus coeruleus through the medial forebrain bundle and central tegmental field has been demonstrated anatomically (38) and perhaps these fibers were stimulated. Because it was shown that cells in the area surrounding the locus coeruleus, as well as locus coeruleus cells, could be antidromically activated by stimulation of the two dorsal noradrenergic bundle sites (hypothalamic and midbrain), these stimulation sites could not be used to provide an unambiguous identification of locus coeruleus neurons. The study reported here has some implications for speculations concern-
264
FAIERS
AND
MOGENSON
ing the functional role of locus coeruleus neurons. It has been postulated that the noradrenergic system originating in the locus coeruleus is involved in self-stimulation at sites in the medial forebrain bundle (near the dorsal noradrenergic bundle) and the refractory period of the neurons subserving this behavior has been determined, using behavioral techniques, to be 0.5 to 1.1 msec (7, 9). Similar refractory periods have been determined electrophysiologically for neurons in medial forebrain bundle implicated in selfstimulation (22). Because these refractory periods are much shorter than those reported in this study for fibers of the dorsal noradrenergic bundle it would appear that the refractory periods measured in previous studies were not those of the noradrenergic fibers originating in the locus coeruleus as has been suggested (8). Although this finding does not in any way prove that the locus coeruleus is not involved in self-stimulation behavior, the long refractory period of locus coeruleus neurons will have to be accounted for in future speculations concerning the involvement of this system in self-stimulation. This study provides a means of identifying locus coeruleus neurons during the course of an electrophysiological experiment by their long-latency antidromic response to stimulation of supracallosal bundle or olfactory bulb and their characteristic slow rate of firing. The antidromic response to stimulation of dorsal noradrenergic bundle sites cannot be used to identify locus coeruleus neurons because neurons near the locus coeruleus are also antidromically activated. Considering the small size of the locus coeruleus and the presence of nonnoradrenergic neurons which do not project from the locus coeruleus, this method of identifying locus coeruleus neurons should prove useful in future experiments on the electrophysiological properties of locus coeruleus neurons. The technique may also be used to suggest or verify the existence of anatomical projections of the locus coeruleus. REFERENCES 1. AMARAL, D. G., and A. ROUTTENBERG. 1975. Locus coeruleus and intracranial self-stimulation: A cautionary note. Behav. Biol. 13 : 331-338. 2. ANDBN, N. E., A. DAHLSTRBM, K. FUXE, K. LARSSON, L. OLSON, and U. UNGERS-T. 1966. Ascending monoamine neurons to the telencephalon and diencephalon. Acta Physiol. Stand. 67: 313-326. 3. ANLEZARK, G. M., T. J. CROW, and A. P. GREENWAY. 1973. Impaired learning and decreased cortical norepinephrine after bilateral locus coeruleus lesions. Science 181: 682-684. 4. CHU, N. D., and F. E. BLOOM. 1973. Norepinephrine-containing neurons: Changes in spontaneous discharge patterns during sleeping and waking. Science 179: 90%910. 5. FAIERS, A.
A., F. R. CALARESU, and G. J. MOGENSON. 1975. Pathway mediating hypotension elicited by stimulation of the amygdala in the rat. -&rev. J. Physiol. 228 : 1358-1366.
IDENTIFICATION
OF
LOCUS
COERULEUS
265
NEURONS
6. FUXE, K. 1965. Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine terminal in the central nervous system. Acta Physiol. Stand. 64 (Suppl 247) : 37-85. 7. GALLISTEL, C. R., E. ROLLS, and D. GREENE. 1969. Neuron function inferred from behavioral and electrophysiological estimates of refractory period. Science 166: 1028-1030. 8. GERMAX, D. C., and D. M. Bownax. 1974. Catecholamine systems as the neural substrate for intracranial self-stimulation: A hypothesis. Bra& Rcs. 73: 381419. 9. GERMAN, D. C., and F. A. HOLLOWAY. 1972. Behaviorally determined neurophysiological properties of MFB self-stimulation fibers. Physiol. Behazr. 9: 823-829. 10. GREEN, J. D. 1958. A simple microelectrode for recording from the central nervous system. Natwe (LOIKICIII) 182: 962. 11. HOFFER, B. J., G. R. SICCIKS, A. P. OLIVER, and F. E. BLOOM. 1973. Activation of the pathway from locus coeruleus to rat cerebellar purkinje neurons: Pharmacological evidence of noradrenergic central inhibition. J. Pltarntacol. Erp. They. 184 : 553-569. 12. HURSH, J. B. 1939. The properties of growing fibers. ‘4111~~. J. PhysioZ. 127: 140153. 13. JACOBOWITZ, D. M., and R. D. BROADWELT.. 1975. Origin of afferent fibers to the olfactory bulb of the rat. Nerrrosci. Abstr. 1 : 677. 14. JACOBOWITZ, D. M., and M. PALKOVITS. 1974. Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. I. Forebrain (telencephalon, diencephalon). J. Conzp. Ncnrol. 157 : 13-28. 1.5. JONES, B. E., P. BOBILIXR, C. PIN, and M. JOUYET. 1973. The effect of lesions of catecholamine containing neurons upon monoamine content of the brain and EEB and behavioral waking in the cat. BraLz Rcs. 58: 157-177. 16. LIDBRINK, P. 1974. The effect of lesions of ascending noradrenaline pathways on sleep and waking in the rat. Brain Rcs. 74: 19-20. 17. LINDVALL, O., and A. BJ~RKLUXD. 1974. The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method. Acta Physiol. Scalzd. Suppl. 412: l-48. 18. NAKAMCR.4, S., and K. IWAMA. 1975. Antidromic activation of the rat locus coeruleus neurons from hippocampus, cerebral and cerebellar cortices. Brain
Res. 99: 372-376, 19. OSUMI, Y., R. OISHI, H. FUJIWARA, and S. TAKAORI. 1973. Hyperdipsia induced by bilateral destruction of the locus coeruleus in rats. Bvaix Res. 86: 419-427. 20. PALKO~ITS, M., and D. M. JACOBOWITZ. 1975. Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephalon). /. Contp. Ncu~ol. 157 : 29-42. 21. PICKLE, V. R;I., M. SEGAL, and F. E. BLOOM. 1974. A radioautographic study of the efferent pathways of the nucleus locus coeruleus. J. Camp. Neural. 155: 15112. 22. ROLLS, E. T. 1971. Absolute refractory period of neurons involved in MFB selfstimulation. Pizysiol. Beizav. 7 : 311-315. 23. RUCH, T. C., and H. D. PATTON. 1965. “Pllysiology and Biophysics," p. Z-31. W. B. Saunders, Philadelphia. ‘24. SEGAL, M., and F. E. BLOOM. 1974. The action of norepinephrine on the rat hippocampus. II. Activation of the input pathway. Brain Res. 72: 99-114. 25. SEGAL,
M.,
and
S. LANDIS.
1974. Afferents
to the hippocampus
of the
rat
studied
266
26. 27. 28. 29. 30.
FAIERS
AND
MOGENSON
with the method of retrograde transport of horseradish peroxidase. Brab Res. 7’8: I-15. SHIMIZU, N., and K. IMAMOTO. 1970. Fine structure of the locus coeruleus in the rat. Arch. Histol. Jap. 31: 229-246. SHIMIZU, N., S. OHNISHI, M. TOHYAMA, and T. MAEDA. 1974. Demonstration by degeneration silver method of the ascending projection from the locus coeruleus. Exp. Bra& Research. 20: 181-192. SWANSON, L. W., and C. B. SAPER. 1975. Direct neural inputs to locus coeruleus from basal forebrain. Nezlrosci. Abstr. 1: 683. TOHYAMA, M., T. MAEDA, and H. SHIMIZU. 1974. Detailed noradrenaline pathways of locus coeruleus neurons to the cerebral cortex with use of 6-hydroxydopa. Brain lies. 79: 139-144. UNGERSTEDT, U. 1971. Stereotaxic mapping of monoamine pathways in the rat brain. Acta Physiol. Stand. 82(Suppl. 367) : l-48.
