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Locus coeruleus-to-dorsal raphe input examined by electrophysiological and morphological methods
Brain Research Sulktin, All rights of reproduction
Vol. 2, pp. 209-221, 1977. Copyright 63 ANKHO in any form reserved. Printed in the U.S.A.
International
Inc.
Locus Coeruleus-to-Dorsal Raphe Input Examined by Electrophysiological and Morphological Methods’ C. D. ANDERSON,’
Worcester Foundation
D. A. PASQUIER,
for l?xperimental
W. B. FORBES AND P. J. MORGANE
SioIogy,
222 Maple Avenue,
Shrewsbury,
MA 01545
(Received 4 May 1977) ANDERSON, C. D., D. A. PASQUIER, W. B. FORBES AND P. J. MORGANE. f.ocus coerufeus-to-dorsonl raphe input by electrophysiologicaf and morphological methods. BRAIN RES. BULL. 2(3) 209-221, 1971. - In order to examine the hypothesis that the locus coeruleus (LC) projects directly to the nucleus raphe dorsalis (DR), electrical stimulation was applied to the LC of rats while recording from single neurons in the region of the DR. Slow firing units of the DR were not influenced by the stimulation, although faster firing units in the nearby substantia grisea centralis (SGC) were. These latter cells become oscillatory in their firing rates during LC stimulation. In parallel studies a retrograde transport technique was employed to obtain morphological evidence regarding projections to DR. Placements of horseradish peroxidase precisely in the DR resulted in very sparse labeling in the LC, although positive transport occurred to other areas. The results indicate that the LC does not project directly to slow firing DR neurons, but does influence faster firing cells in the region of the XC, probably by complex routes. Suggestions are made for the integration of these findings with earlier fluorescence studies.
examined
Dorsal raphe
Single units
Locus coeruleus
Horseradish peroxidase
‘l‘hc objective of the present investigation was to examine the locus coeruleus-dorsal raphe relationship by studies of neurophysiological influences on single neurons in the nucleus raphe dorsalis and inimediately surrounding areas from electrical stimulation of the locus coeruleus, and by morphological studies of retrograde cell labeling in the locus coeruleus following transport of horseradish peroxidase injected into the nucleus raphe dorsalis.
AS ONE suggestion to help account for the many sources of evidence linking serotonergic and noradrenergic actions in the regulation of sleep states, Jouvet has proposed that projections from the locus coeruleus may reach the nucleus raphe dorsalis and exert an inhibitory control there [6], Neuronal cell groups more remote from the locus coeruleus than the nucleus raphe dorsalis are known to receive connections from the locus coeruleus. Direct projections from the locus coeruleus have been reported to produce noradrener~c inhibition in cerebellar Purkinje cells [ 5,201 and in hippocampal pyramidal cells [ 161. Retrograde transport of horseradish peroxidase has provided additional evidence of locus coeruleus projections to the hippocampus oftherat [17] andthecat [13]. Morphological evidence for projections from the locus coeruleus to the nucleus raphe dorsalis has been provided by Loizou through fluorescence studies in the rat [7]. By noting locations with reduced fluorescence in material from animals with lesions of the locus coeruleus (relative to material from control animals), Loizou inferred that the locus coeruleus projects to the nucleus raphe dorsalis. Chu and Bloom [3] observed enhancement of green fluorescence in the ventrolateral portion of the nucleus raphe dorsalis following more rostra1 unilateral hemisections of the dorsal mesencephalon in the cat. Thus, considerable support for the hypothesis of direct locus coeruleus-todorsal raphe projections issues from these fluorescence studies. ’ Supported by NSF Grant BNS 74-02620
Brainstem relations
METHOD
Electrophysiologicu2
Procedui-e
A series of 60 adult male Long-Evans rats were used for the electrophysiological experiments. Chloral hydrate has been the anesthesia of choice in studies of the nucleus raphe dorsalis because it is known to minimally affect firing rates there [2,8]. However, it is possible that circuits leading from the locus coeruleus to the nucleus raphe dorsalis could be affected by the anesthesia. Therefore, in order to make our experiments more general, 35 rats were anesthetized under chloral hydrate (350 mg/kg) and 25 under urethane (I 200 mg/kg). Glass micropipettes of 5 to 12 fin resistance (tip diameter less than 1 urn) were filled with 2 M NaCl solution for use in recording single neurons. The saline filler was saturated with a Procion Red dye for iontophoretic delivery to mark tip locations. Potentials were fed into a conventional high impedance amplifying system with the
and Supplemental Support under Small College Grant Program.
2 Also at Providence College, Providence, RI.
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210 output monitored via a speaker and an oscilloscope and recorded on magnetic tape for subsequent analysis. Stimulating electrodes were bipolar stainless steel with approximately 0.5 mm tip separation. Stimulation was delivered from constant-current isolation units. Stimulus pulses were biphasic, lasting 0.2 msec for each pulse pair, and delivered singly or in trains of 1 to 11 sec. Frequencies of trains were from 4 to 50 Hz, but were usually 10 HZ as used in earlier analyses of locus coeruleus stimulation effects on other areas [ 5,201. Stimulation currents were generally in the 90- 150 PA range to avoid current spread associated with the proximity of stimulating and recording sites. A further objective in the use of these current parameters was to avoid activation of nuclei outside the locus coeruleus [ 5,201 The surgical approach was made through the cerebellum in order to avoid the venous sinuses. The stimulation electrode was located by standard stereotaxic procedures (coordinates: P 1.8, L 1.1, V 6.5 from lambda [4] ). Then the recording microelectrode was moved to the vicinity of the nucleus raphe dorsalis (coordinates: A 0.8, L 0.0, V 7.0 from lambda [4] ), and a motor-driven hydraulic microdrive was used to search for units with raphe-like characteristics. The rectal temperature was maintained near 37.S’C by a circulating-water heating pad. Following each experiment the microelectrode placement was marked by iontophoretically driving a small quantity of dye from the tip by a cathodal current increased slowly from IO to 40 PA over a 30 min period while the current was monitored to avoid blocking. In some animals the stimulating electrode location was marked by passing a small anodal current (300psec pulses, 200 PA, 200 Hz) for one min. In all experiments histological identification of both stimulating and recording sites were routinely obtained using Nissl methods. For the analysis of the unit data stored on magnetic tapes, firing rates before and following stimulation of the locus coeruleus were evaluated by using ink recorder write-outs of Schmidt trigger outputs, photographs of storage oscilloscope displays, and poststimulus time histograms. The latter were produced on an Ortec system which collects up to 128 counts in each of 255 bins. Comparisons between pre- and poststimulation firing rates were made by counts of the number of discharges during two four-second periods: the first, immediately prior to stimulation, and the second, immediately following it. Repeated samples of firing data were made on each unit; the samples varied in number from 8 to 40 depending upon experimental conditions. These data were analyzed by determining the number of pre- minus poststimulation differences that were positive (or negative) and applying a sign test to evaluate for significance [ 191 A two-tailed test and 0.05 confidence interval were adopted as the criteria for significance. Retrograde
ET AL.
description of the material and examples showing representative injection sites have been reported previously [lo]. The material was studied under bright- and dark-field illumination. Control injections were made in the regions around the nucleus raphe dorsalis and in the cerebellum. RESULTS
The requirement of histological confirmation in both electrode sites limited the data analysis to 35 units recorded in 14 animals. Of these 35 units, only 13 met the neurophysiological criteria for dorsal raphe units: firing rates not exceeding 3 per set and absence of bursting [ 2,8]. Using post-stimulus time histograms, together with online monitoring, visual study of storage oscilloscope displays, and statistical study of ink recorder writeouts of Schmidt trigger outputs, we did not see any consistent effects of locus coeruleus stimulation upon the activity of 13 units recorded from verified sites in the nucleus raphe dorsalis. None of the data analyses reached the 0.05 confidence level when the sign test was applied as described in the method section. Figure 1 is an illustration of a recording from a unit in the nucleus raphe dorsalis of Rat 28. During this recording electrical stimulation was applied to the locus coeruleus as indicated in Fig. 1. A comparison of neuronal activity in the record of Fig. 1 reveals that the firing rate remains essentially unchanged before and following stimulation to the locus coeruleus. Stimulation parameters are provided in the legends. Figure 2 presents line drawings of transverse brainstem sections from Rat 28 with locations of the stimulating and recording sites for the unit shown in Fig. 1. The absence of a stimulation effect in the experiment with Rat 28 was apparent from an analysis of repeated tests such as the one illustrated above. These results are representative of what was obtained in similar experiments. When the oscilloscope is set for a very slow sweep speed as was used in Fig. 1, individual stimulus artifacts merge on
Transport Procedure
Injections of 0.05 ~1 of horseradish peroxidase solution (Sigma Type VI, 30% in saline) were made through micropipettes (80 pm tip diameter) in the nucleus raphe dorsalis of 20 adult rats. After a 24 hr survival period the animals were anesthetized deeply and perfused intracardially first with saline followed by 1% paraformaldehyde, 4% glutaraldehyde fixative buffered with phosphate at pH 7.2. The brains were removed and cut immediately (40 pm thick sections), and processed according to a procedure already described [ 131. Detailed
FIG. 1. Pre- and poststimulation firing of a dorsal raphe unit recorded from Rat 28. Stimulation parameters are 80 PA per pulse delivered at 4 Hz for a 3.0 set train. Each pulse is a 0.2 msec biphasic square wave. Calibrations: 5 set and 50 NV. Pre- and poststimulation tiring rates: 0.9 discharges per sec. The stimulation location is identified by the large white artifact showing clearly above the unit spike height. Individual artifacts have merged because of the slow sweep speed of the oscilloscope. It is obvious that the stimulation did not change the baseline firing of the neuron.
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IV
FIG. 2. Scale drawings of transverse sections from Rat 28. The figure on the left shows the location of the tip of the stimulating electrode near the locus coeruleus (dark area indicated by the arrow). The right hand figure shows the position of the recording microelectrode in the nucleus raphe dorsalis. The open circle indicated by the arrow represents the location of the tip of the microelectrode. These. electrode locations are associated with the record presented in Fig. 1. Abbreviations: LC, locus coeruleus; MLF, fasciculus longitudinalis medialis, MV, radix mesencephalica nervi trigemini; N IV, nucleus trochlearis; TAC, tuberculum acusticum; VIII, nervus statoacusticus
the oscilloscope screen during trains of stimuli, thereby obscuring unit discharges between individual stimulus pulses. To handle this problem faster sweep speeds were used on repeated playback of the data from the tape recorder to allow inspection of the unit activity during long trains of stimulation. When carried out in this fashion, inspection of firing rates during stimulation confirmed that the unit activity was unchanged by locus coeruleus stimulation while long stimulus trains were in progress. Figure 3 presents poststimuius time histograms of three units recorded from Rat 66. The locations of the stimulation and recording sites will be detailed in Figs. 4 through 8. The upper histogram is from a unit located just dorsal to the nucleus raphe dorsalis in the substantia grisea centralis, the middle histogram is from a unit within the nucleus raphe dorsalis, and the lower histogram is from a unit within the decussation of the brachium conjunctivum. Inspection of these histograms gives evidence that the firing rates in these units were unaffected by stimulation to the locus coeruleus. Although not shown in Fig. 3, single stimuli to the locus coeruleus were also tested for their effect on the dorsal raphe unit and the histogram revealed no evidence of an influence. Also, trains of stimuli were administered to the iocus coeruleus while recording from these two Fig. 3 units located above and below the nucleus raphe dorsalis, and again there was no effect. These data are representative of our results with poststimulus time histograms. Thus, averaging the data did not bring out any effects too subtle to be seen in individual tests of the locus coeruleus stimulation on the unit firing rates in the region of the dorsal raphe. Figure 4 presents line drawings of sagittal sections used to analyze the placements in Rat 66. Figure 4A shows the location of the locus coeruleus in histological Section 33,
and Fig. 43 shows the termination of the track of the sti~lulating electrode in Section 37. For reference, an inset drawing of the stimulating electrode is shown with poles in black. The electrode is drawn to the same scale as the rest of Fig. 4. Since the sections were 40 Mm in thickness, approximately 120 pm intervened between the illustrated locations. Photomicrographs of the corresponding sections are presented in Figs. 5 and 6. Figures 4C, 4D, 7 and 8 illustrate the locations of the cells which produced the recordings in Fig. 3. Figure 4C illustrates the section in which a dye mark (open circle) was located. Figure 7 presents a photomicrograph showing the location of the dye mark illustrated in Fig. 4C. The actual dye mark is only visible under high magnification. The track shown above the dye mark in Fig. 4C is actually in Section 66 shown in Fig. 4D, and has been localized and traced into Fig. 4C by superimposing the outline of the two drawings. This was done to illustrate how the line drawings were used to confirm relationships in the histological sections. By referring to records of the microdrive readings, the sites of the two units recorded at points above the final tip excursion (located at the dye mark) were determined. The unit locations are illustrated with the open circles in Fig. 40. An enlargement of the corresponding section is presented in Fig. 8 with the unit locations marked by arrows. Anatomical landmarks, particularly the cerebral aqueduct, verify the midsagittal location. Neurons in the region of the central gray bordering the nucleus raphe dorsalis often showed steady, nonburst~ng firing patterns with rates of approximat~iy 7 to 20 per second. Figures 9, 10 and 11 present one such unit which showed an entrainment during the first 10 stimulations of the locus coeruleus, but the relationship was not sustained with further stimuli. The location of the stimulating and
ANDERSON
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ET AL.
STIMULATION TO 1 OCUS EOERUL EUS
SWEEPS
.2msec
115yA +
10 Hz +
FIG. 3. Poststimulus time histograms of the firing of three units are presented. The upper histogram is from a central gray unit located just dorsal to the nucleus raphe dorsalis (firing rate: 6.0 discharges per set). The middle histogram is from a dorsal raphe unit (firing rate: 1.8 discharges per set). The lower histogram is from a unit located in the decussation of the brachium conjunctivum (firing rate: 5.0 discharges per set). The upper two ~~istograms are from the units localized in Fig. 4D and 8. The Iowest histogram is from a unit located at the dye mark shown in Fig. 4C and Fig. 7. The total number of counts are indicated on the ordinate and each address on the abscissa represents 10 msec. The number of sweeps for each histogram is indicated on the right. Stimulation values are indicated on the figure. Location of the stimuli are indicated by the heavy arrows and by the stimulation artifacts which are clear and have been truncated at the top. Stimulation parameters were changed to explore for the possibility that certain stimulus values would influence the firing rate. All units were tested with single stimuli and with trains, but none of the parameters were effective. recording electrodes are given in Fig. 10 and 11. No additional observations of this kind were made with other units. Another ph~nonlenon seen with central gray units is illustrated in Fig.
12 per sec. The electrical stimulation of the locus coeruleus did not influence the activity of these cells. Seven cerebellar Purkinje cells in the vermian lobule were studied, but no influence from locus coeruleus stimulation could be verified with these units. Another source of influence on two of the fast, steady central gray units was found in exploratory experiments
when the stimulating electrode was placed in the substantia nigra. One example of the observed inhibition is presented together with a poststimulus time histogram in Fig. 13. The electrode locations are depicted in Figs. 14 and 15. Following stimulation to the substantia nigra with single stimulus pairs, slowing was observed for about 500 msec. The latency of onset was somewhat variable, but was generally about 100 msec. The effect occurred with great reliability. After specific injections of horseradish peroxidase in the nucleus raphe dorsalis, cells were sparsely labeled in the locus coeruleus. Figure 16 shows an example of a labeled cell in the main portion of the locus coeruleus. One other animal showed comparable transport of this kind. In all other cases there was no evidence of retrograde transport to focus coeruleus cells. Thus, the morphological data complement the e~ectrophysiolo~ical findings. We report here only the retrograde transport results concerning the locus coeruieus-dorsal raphe connections. Detailed subscriptions of our results indicating afferents to nucleus raphe dorsalis from other regions have been described elsewhere [ IO,1 11.
The results of the electrophysiological tests suggest that the hypothesized direct projections from locus coeruieus to the nucleus raphe dorsalis do not exist. This conclusion is
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FIG. 4. Line drawings of crucial brain sections used to analyze the electrode placements for Rat 66. The units locations described in C and D below correspond to the poststimulus time histograms in Fig. 3. (A) Section 33 with the descending electrode track in the cerebellum and the locus coeruleus (LC) visible. The substantia grisea centralis (SGC) and the genu of the seventh nerve (g VII) are indicated. (B) The termination of the stimulating electrode track is presented, together with an inset drawing of the electrode to the same scale. This is Section 37. Sections were cut at a thickness of 40 p. (C) The open circle below the fasciculus longitudinalis medialis (MLF) represents the location of the dye mark indicating the tip of the microelectrode which is situated in the decussation of the brachium conjunctivum. The track seen above the dye mark was superimposed on this drawing of Section 61., but was actually found in Section 66. This was done to illustrate how the line drawings were used to confirm relationships in the histological sections. (D) Section 66 containing the track of the microelectrode. The two open circles indicate locations of units. Their location was obtained from microdrive readings and from the dye mark location illustrated in part C of this figure.
substantiated by the morphological studies performed with horseradish peroxidase injections into nucleus raphe dorsalis. Additional support is p:ovided from studies utilizing the Fink-Heimer technique in which no evidence of terminal degeneration in the nucleus raphe dorsalis was shown following lesions in the locus coeruleus [ 14,181 Moreover, other authors [22] have reported results in agreement with our preliminary electrophysiological data fll. Reconciliation of the interpretations of the fluorescence studies with the results contraindicating a locus coeruleusdorsal raphe pathway are possible. Loizou [ 71 indicated
that his lesions of the region of the locus coeruleus may have extended deep ventrally from the nucleus. Thus, noradrener~c pathways collecting into the dorsal noradrenergic bundle from sources other than A6 may have been interrupted. This interpretation implies that some other source of noradrenergic fibers may reach the nucleus raphe dorsalis. Descriptions of the origins of the monoaminergic pathways are not incompatible with this interpretation [ 211 . Moreover, other neuronal cell groups located around the locus coeruleus have been shown to project to the nucleus raphe dorsalis. Specifically, projections from the para-
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ANDERSON ET AL
FIG. 5. A photomicrograph of the brain section (No. 33) which the drawing shown in Fig. 4.4 is based upon. This and other sections are stained with cresyl violet. The stimulating electrode track can be seen in the cerebellum. Notice that it is pointed toward the locus coeruleus (indicated by the arrow).
FIG. 6. A photomicrograph of Section 37 from Rat 66. The drawing shown in Fig. 4B is based upon this section. The location of the tip of the stimulating electrode is between the points of the arrows.
LOCUS
COERULEUS-DORSAL
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STUDIES
FIG. 7. Photomicrograph corresponding to the drawing in Fig. 4C. The arrow points to the location where the dye marker indicated the final extent of travel of the microelectrode, i.e., the brachium conjunctivum. The unit activity obtained from the indicated position is presented in h’ig. 3 as the bottom poststimulus time histogram.
FIG. 8. The location of the arrows show where the units were as determined by relating the location of the dye mark (Fig. 7) to the travel distance on the microdrive. This is Section 66 from Rat 66, and the arrow point locations correspond to the open circles shown in Fig. 4D. The uppermost poststimulus time histogram in Fig. 3 is taken from the more dorsal neuron. The more ventral unit is in the nucleus raphe dorsalis. The middle histogram in Fig. 3 is from this neuron.
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mo SWEECS
l- 10
11.40
Zl- 30
31 - 40
FIG. 9. Unit recordings from Rat 60. Each trace represents 10 sweeps of the storage oscilloscope superimposed for stimulus location. The stimulus parameters are shown on the figure. Temporal location of stimulation is indicated by the arrow. Single 0.2 msec stimuli were administered at the arrow and the oscilloscope made one sweep each time just as a stimulation was to occur. (Firing rate: 12 discharges per set). During the first 10 stimulations of the locus coeruleus an entrainment of the unit discharge occurred with a latency of about 80 msec. However, the relationship was not sustained with further stimulation. The anatomical locations of the electrodes are presented in Figs. 10 and 11.
FIG. 10. Photomicrograph of a section from Rat 60 illustrating the location of the stimulating electrode. The lesion marking the tip of this electrode is clearly visible (indicated by the arrow), as is the track passing through the locus coeruleus. There were four tips on this particular electrode at 0.2 mm intervals, indicating that the locus coeruleus clearly was stimulated. The corresponding electrophysiological record is in Fig. 9.
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FIG. Il. Photomicrograph containing the microelectrode track for Rat 60. The pointer indicates the unit location determined relative to the iontophoretically produced dye mark for the tip location of the microelectrode. The unit appears to be in the central gray on the midline caudal to the nucleus raphe dorsalis. This neuron provided the record presented in Fig. 9.
brachial nuclei reach the nucleus raphe dorsalis [ IO,1 I]. Thus, these connections should be taken into account when considering any relationship between this dorsal tegmental region and the nucleus raphe dorsalis. It should be noted in addition that, while recordings in the present study were made primarily upon midline portions of dorsal raphe, Chu and Bloom [3] report that the noradrenergic terminations occur only in the most lateral portions of nucleus raphe dorsalis. This lateral location for the appearance of monoaminergic fluorescence in the central gray is substantiated from earlier [Zl] and later research [ 9, I 5 1. Our neurophysiological evidence suggests an influence from the locus coeruleus upon the central gray neurons. This result is in good conformation with the fluorescence studies summarized above. The oscillatory firing pattern initiated in these units by stimulation of Iocus coeruleus suggests that more than one input pathway must be involved. The inhibitory effects on central gray units obtained from stimulation of the substantia nigra provide additional evidence of complex inputs to this region. One possibiiity is that fibers of the habenulo-dorsal raphe pathway were captivated [lo], although evidence for projections from substantia nigra directly to raphe dorsalis exist [11,12]. Although generalization from our small sample should be conservative, the absence of an influence from the stimulation of’ the locus coeruleus upon the majority of the neurons recorded from in the region of the central gray in
this study suggests that the morphological correlates of this finding may be somewhat sparse. In addition, the complexity of the area must be considered in making interpretations relative to it, as was noted earlier in this discussion. In spite of the small number of ceils involved, mention should be made of the fact that we did not see inhibition in cerebellar Purkinje cells as others have when stimulating in the locus coeruleus [5,20]. Perhaps the resolution of this discrepancy is related to the fact that the recordings in our study were made in a cerebellar lobule very remote from the one used in the earlier investigations. In conclusion, it may be said that, while the assumption made by Jouvet [61 regarding direct locus coeruleus to dorsal raphe projections was clearly the most parsimonious for interpreting the evidence pointing to a noradrenergicserotonergic interaction, it now seems necessary to revise this concept. Obviously, complex interacting brainstem chemical systems are involved in the regulation of the states of vigilance. The mechanisms of these interactions remain to be determined.
For their helpful of this investigation Macrides, William technical assistance Berman, Mr. Morris Marks.
discussions during the neurophysiological phase we wish to thank Drs. Steven Fredman, Foteos M. Youngs and Mr. Steven Schneider. For with histology we thank Mr. Howard M. Feinstein, Ms. Sharon A. Madden and Mr. David
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FIG. 12. Firing pattern of one central gray unit located just lateral to the dorsal raphe nucleus is shown during the administration of stimulus trains to the locus coeruleus. Three separate test sequences are represented in the A, B, and C portions of the figure. Within each of these sections of the figure the second sweep follows immediately after the first Stimulus locations are marked by the arrows. (A) The application of stimulation at 2 Hz to the locus coeruleus results in immediate slowing by about 25%. The return of the prest~mu~ation firing rate can be seen. (8) A train of four pulses again results in slowing. Termination of stimulation after the train of four pulses results in a rebeound, f&lowed by a return to the prestimulation firing rate. Additional stimulation in the second sweep of part B again results in slowing which lasts for about 1.5 sec. (C) A longer train of stimulation results in an initial slowing followed by a rebound (at about 1.5 set from stimulation onset) to a rate faster than before stimulation, even though the stimulation is continuing. The termination of this train in the second sweep results in a more sustained rebound. (Calibration: 0.5 set and 150 ,uV).
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J
L
i
SEC
msec SlNbLE SHOCKS TO SUBSTANTIA NIGRA +
CpO:RNTS
ADDRESS
60 SWEEPS IOmsec per address
I SEC FIG. 13. Poststimulus time histogram for Rat 59. Stimulating and recording electrode sites are shown in Figs. 14 and 15, respectively. The inset photograph of the present figure shows a single sweep of the unit fining with the stimulus artifact indicated by the arrow. Note the different time bases for the photograph of the single unit firing and the poststimulus time histogram. In the inset photograph can be seen the slowing of the firing rate for a period of about 200 msec occurring with a latency of about 100 msec following stimulation. The corresponding slowing in the histogram is indicated by the triangular pointer. In single sweeps the latency of the inhibition seemed slightly variable, but the effect was consistent. l:rom the histogram the inhibition is seen to have a reliable latency of 130 msec and a duration of 60 msec.
FIG. 14. Photomicrograph showing the location of the stimulating electrode in Rat 59. The arrow points to the location of electrode tip. The darkly stained nucleus at the electrode termination is the substantia nigra. The corresponding eiectrophysioiogical recording is presented in Fig. 13.
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FIG. 15. Photomicrograph showing the location of the recording microelectrode in Rat 59. The pointer indicates the location of the microelectrode tip. This unit was about 160 pm off of the midline in the central gray. The corresponding electrophysiological record is in Fig. 13.
FIG. 16. Photomicrograph illustrating the very sparse indications for retrograde transport of horseradish peroxidase to the locus coeruleus when injected into the nucleus raphe dorsalis. The margins of the locus coeruleus are indicated by the arrows. To the upper left in the figure can be seen the brachium conjunctivum (BC), and to the right can be seen the fourth ventricle (V IV) and the cerebellum (CER). Notice that only one cell in the locus coeruleus is labeled. (Initial magnification X100).
ET AL.
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Vol. 2, pp. 209-221, 1977. Copyright 63 ANKHO in any form reserved. Printed in the U.S.A.
International
Inc.
Locus Coeruleus-to-Dorsal Raphe Input Examined by Electrophysiological and Morphological Methods’ C. D. ANDERSON,’
Worcester Foundation
D. A. PASQUIER,
for l?xperimental
W. B. FORBES AND P. J. MORGANE
SioIogy,
222 Maple Avenue,
Shrewsbury,
MA 01545
(Received 4 May 1977) ANDERSON, C. D., D. A. PASQUIER, W. B. FORBES AND P. J. MORGANE. f.ocus coerufeus-to-dorsonl raphe input by electrophysiologicaf and morphological methods. BRAIN RES. BULL. 2(3) 209-221, 1971. - In order to examine the hypothesis that the locus coeruleus (LC) projects directly to the nucleus raphe dorsalis (DR), electrical stimulation was applied to the LC of rats while recording from single neurons in the region of the DR. Slow firing units of the DR were not influenced by the stimulation, although faster firing units in the nearby substantia grisea centralis (SGC) were. These latter cells become oscillatory in their firing rates during LC stimulation. In parallel studies a retrograde transport technique was employed to obtain morphological evidence regarding projections to DR. Placements of horseradish peroxidase precisely in the DR resulted in very sparse labeling in the LC, although positive transport occurred to other areas. The results indicate that the LC does not project directly to slow firing DR neurons, but does influence faster firing cells in the region of the XC, probably by complex routes. Suggestions are made for the integration of these findings with earlier fluorescence studies.
examined
Dorsal raphe
Single units
Locus coeruleus
Horseradish peroxidase
‘l‘hc objective of the present investigation was to examine the locus coeruleus-dorsal raphe relationship by studies of neurophysiological influences on single neurons in the nucleus raphe dorsalis and inimediately surrounding areas from electrical stimulation of the locus coeruleus, and by morphological studies of retrograde cell labeling in the locus coeruleus following transport of horseradish peroxidase injected into the nucleus raphe dorsalis.
AS ONE suggestion to help account for the many sources of evidence linking serotonergic and noradrenergic actions in the regulation of sleep states, Jouvet has proposed that projections from the locus coeruleus may reach the nucleus raphe dorsalis and exert an inhibitory control there [6], Neuronal cell groups more remote from the locus coeruleus than the nucleus raphe dorsalis are known to receive connections from the locus coeruleus. Direct projections from the locus coeruleus have been reported to produce noradrener~c inhibition in cerebellar Purkinje cells [ 5,201 and in hippocampal pyramidal cells [ 161. Retrograde transport of horseradish peroxidase has provided additional evidence of locus coeruleus projections to the hippocampus oftherat [17] andthecat [13]. Morphological evidence for projections from the locus coeruleus to the nucleus raphe dorsalis has been provided by Loizou through fluorescence studies in the rat [7]. By noting locations with reduced fluorescence in material from animals with lesions of the locus coeruleus (relative to material from control animals), Loizou inferred that the locus coeruleus projects to the nucleus raphe dorsalis. Chu and Bloom [3] observed enhancement of green fluorescence in the ventrolateral portion of the nucleus raphe dorsalis following more rostra1 unilateral hemisections of the dorsal mesencephalon in the cat. Thus, considerable support for the hypothesis of direct locus coeruleus-todorsal raphe projections issues from these fluorescence studies. ’ Supported by NSF Grant BNS 74-02620
Brainstem relations
METHOD
Electrophysiologicu2
Procedui-e
A series of 60 adult male Long-Evans rats were used for the electrophysiological experiments. Chloral hydrate has been the anesthesia of choice in studies of the nucleus raphe dorsalis because it is known to minimally affect firing rates there [2,8]. However, it is possible that circuits leading from the locus coeruleus to the nucleus raphe dorsalis could be affected by the anesthesia. Therefore, in order to make our experiments more general, 35 rats were anesthetized under chloral hydrate (350 mg/kg) and 25 under urethane (I 200 mg/kg). Glass micropipettes of 5 to 12 fin resistance (tip diameter less than 1 urn) were filled with 2 M NaCl solution for use in recording single neurons. The saline filler was saturated with a Procion Red dye for iontophoretic delivery to mark tip locations. Potentials were fed into a conventional high impedance amplifying system with the
and Supplemental Support under Small College Grant Program.
2 Also at Providence College, Providence, RI.
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210 output monitored via a speaker and an oscilloscope and recorded on magnetic tape for subsequent analysis. Stimulating electrodes were bipolar stainless steel with approximately 0.5 mm tip separation. Stimulation was delivered from constant-current isolation units. Stimulus pulses were biphasic, lasting 0.2 msec for each pulse pair, and delivered singly or in trains of 1 to 11 sec. Frequencies of trains were from 4 to 50 Hz, but were usually 10 HZ as used in earlier analyses of locus coeruleus stimulation effects on other areas [ 5,201. Stimulation currents were generally in the 90- 150 PA range to avoid current spread associated with the proximity of stimulating and recording sites. A further objective in the use of these current parameters was to avoid activation of nuclei outside the locus coeruleus [ 5,201 The surgical approach was made through the cerebellum in order to avoid the venous sinuses. The stimulation electrode was located by standard stereotaxic procedures (coordinates: P 1.8, L 1.1, V 6.5 from lambda [4] ). Then the recording microelectrode was moved to the vicinity of the nucleus raphe dorsalis (coordinates: A 0.8, L 0.0, V 7.0 from lambda [4] ), and a motor-driven hydraulic microdrive was used to search for units with raphe-like characteristics. The rectal temperature was maintained near 37.S’C by a circulating-water heating pad. Following each experiment the microelectrode placement was marked by iontophoretically driving a small quantity of dye from the tip by a cathodal current increased slowly from IO to 40 PA over a 30 min period while the current was monitored to avoid blocking. In some animals the stimulating electrode location was marked by passing a small anodal current (300psec pulses, 200 PA, 200 Hz) for one min. In all experiments histological identification of both stimulating and recording sites were routinely obtained using Nissl methods. For the analysis of the unit data stored on magnetic tapes, firing rates before and following stimulation of the locus coeruleus were evaluated by using ink recorder write-outs of Schmidt trigger outputs, photographs of storage oscilloscope displays, and poststimulus time histograms. The latter were produced on an Ortec system which collects up to 128 counts in each of 255 bins. Comparisons between pre- and poststimulation firing rates were made by counts of the number of discharges during two four-second periods: the first, immediately prior to stimulation, and the second, immediately following it. Repeated samples of firing data were made on each unit; the samples varied in number from 8 to 40 depending upon experimental conditions. These data were analyzed by determining the number of pre- minus poststimulation differences that were positive (or negative) and applying a sign test to evaluate for significance [ 191 A two-tailed test and 0.05 confidence interval were adopted as the criteria for significance. Retrograde
ET AL.
description of the material and examples showing representative injection sites have been reported previously [lo]. The material was studied under bright- and dark-field illumination. Control injections were made in the regions around the nucleus raphe dorsalis and in the cerebellum. RESULTS
The requirement of histological confirmation in both electrode sites limited the data analysis to 35 units recorded in 14 animals. Of these 35 units, only 13 met the neurophysiological criteria for dorsal raphe units: firing rates not exceeding 3 per set and absence of bursting [ 2,8]. Using post-stimulus time histograms, together with online monitoring, visual study of storage oscilloscope displays, and statistical study of ink recorder writeouts of Schmidt trigger outputs, we did not see any consistent effects of locus coeruleus stimulation upon the activity of 13 units recorded from verified sites in the nucleus raphe dorsalis. None of the data analyses reached the 0.05 confidence level when the sign test was applied as described in the method section. Figure 1 is an illustration of a recording from a unit in the nucleus raphe dorsalis of Rat 28. During this recording electrical stimulation was applied to the locus coeruleus as indicated in Fig. 1. A comparison of neuronal activity in the record of Fig. 1 reveals that the firing rate remains essentially unchanged before and following stimulation to the locus coeruleus. Stimulation parameters are provided in the legends. Figure 2 presents line drawings of transverse brainstem sections from Rat 28 with locations of the stimulating and recording sites for the unit shown in Fig. 1. The absence of a stimulation effect in the experiment with Rat 28 was apparent from an analysis of repeated tests such as the one illustrated above. These results are representative of what was obtained in similar experiments. When the oscilloscope is set for a very slow sweep speed as was used in Fig. 1, individual stimulus artifacts merge on
Transport Procedure
Injections of 0.05 ~1 of horseradish peroxidase solution (Sigma Type VI, 30% in saline) were made through micropipettes (80 pm tip diameter) in the nucleus raphe dorsalis of 20 adult rats. After a 24 hr survival period the animals were anesthetized deeply and perfused intracardially first with saline followed by 1% paraformaldehyde, 4% glutaraldehyde fixative buffered with phosphate at pH 7.2. The brains were removed and cut immediately (40 pm thick sections), and processed according to a procedure already described [ 131. Detailed
FIG. 1. Pre- and poststimulation firing of a dorsal raphe unit recorded from Rat 28. Stimulation parameters are 80 PA per pulse delivered at 4 Hz for a 3.0 set train. Each pulse is a 0.2 msec biphasic square wave. Calibrations: 5 set and 50 NV. Pre- and poststimulation tiring rates: 0.9 discharges per sec. The stimulation location is identified by the large white artifact showing clearly above the unit spike height. Individual artifacts have merged because of the slow sweep speed of the oscilloscope. It is obvious that the stimulation did not change the baseline firing of the neuron.
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IV
FIG. 2. Scale drawings of transverse sections from Rat 28. The figure on the left shows the location of the tip of the stimulating electrode near the locus coeruleus (dark area indicated by the arrow). The right hand figure shows the position of the recording microelectrode in the nucleus raphe dorsalis. The open circle indicated by the arrow represents the location of the tip of the microelectrode. These. electrode locations are associated with the record presented in Fig. 1. Abbreviations: LC, locus coeruleus; MLF, fasciculus longitudinalis medialis, MV, radix mesencephalica nervi trigemini; N IV, nucleus trochlearis; TAC, tuberculum acusticum; VIII, nervus statoacusticus
the oscilloscope screen during trains of stimuli, thereby obscuring unit discharges between individual stimulus pulses. To handle this problem faster sweep speeds were used on repeated playback of the data from the tape recorder to allow inspection of the unit activity during long trains of stimulation. When carried out in this fashion, inspection of firing rates during stimulation confirmed that the unit activity was unchanged by locus coeruleus stimulation while long stimulus trains were in progress. Figure 3 presents poststimuius time histograms of three units recorded from Rat 66. The locations of the stimulation and recording sites will be detailed in Figs. 4 through 8. The upper histogram is from a unit located just dorsal to the nucleus raphe dorsalis in the substantia grisea centralis, the middle histogram is from a unit within the nucleus raphe dorsalis, and the lower histogram is from a unit within the decussation of the brachium conjunctivum. Inspection of these histograms gives evidence that the firing rates in these units were unaffected by stimulation to the locus coeruleus. Although not shown in Fig. 3, single stimuli to the locus coeruleus were also tested for their effect on the dorsal raphe unit and the histogram revealed no evidence of an influence. Also, trains of stimuli were administered to the iocus coeruleus while recording from these two Fig. 3 units located above and below the nucleus raphe dorsalis, and again there was no effect. These data are representative of our results with poststimulus time histograms. Thus, averaging the data did not bring out any effects too subtle to be seen in individual tests of the locus coeruleus stimulation on the unit firing rates in the region of the dorsal raphe. Figure 4 presents line drawings of sagittal sections used to analyze the placements in Rat 66. Figure 4A shows the location of the locus coeruleus in histological Section 33,
and Fig. 43 shows the termination of the track of the sti~lulating electrode in Section 37. For reference, an inset drawing of the stimulating electrode is shown with poles in black. The electrode is drawn to the same scale as the rest of Fig. 4. Since the sections were 40 Mm in thickness, approximately 120 pm intervened between the illustrated locations. Photomicrographs of the corresponding sections are presented in Figs. 5 and 6. Figures 4C, 4D, 7 and 8 illustrate the locations of the cells which produced the recordings in Fig. 3. Figure 4C illustrates the section in which a dye mark (open circle) was located. Figure 7 presents a photomicrograph showing the location of the dye mark illustrated in Fig. 4C. The actual dye mark is only visible under high magnification. The track shown above the dye mark in Fig. 4C is actually in Section 66 shown in Fig. 4D, and has been localized and traced into Fig. 4C by superimposing the outline of the two drawings. This was done to illustrate how the line drawings were used to confirm relationships in the histological sections. By referring to records of the microdrive readings, the sites of the two units recorded at points above the final tip excursion (located at the dye mark) were determined. The unit locations are illustrated with the open circles in Fig. 40. An enlargement of the corresponding section is presented in Fig. 8 with the unit locations marked by arrows. Anatomical landmarks, particularly the cerebral aqueduct, verify the midsagittal location. Neurons in the region of the central gray bordering the nucleus raphe dorsalis often showed steady, nonburst~ng firing patterns with rates of approximat~iy 7 to 20 per second. Figures 9, 10 and 11 present one such unit which showed an entrainment during the first 10 stimulations of the locus coeruleus, but the relationship was not sustained with further stimuli. The location of the stimulating and
ANDERSON
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ET AL.
STIMULATION TO 1 OCUS EOERUL EUS
SWEEPS
.2msec
115yA +
10 Hz +
FIG. 3. Poststimulus time histograms of the firing of three units are presented. The upper histogram is from a central gray unit located just dorsal to the nucleus raphe dorsalis (firing rate: 6.0 discharges per set). The middle histogram is from a dorsal raphe unit (firing rate: 1.8 discharges per set). The lower histogram is from a unit located in the decussation of the brachium conjunctivum (firing rate: 5.0 discharges per set). The upper two ~~istograms are from the units localized in Fig. 4D and 8. The Iowest histogram is from a unit located at the dye mark shown in Fig. 4C and Fig. 7. The total number of counts are indicated on the ordinate and each address on the abscissa represents 10 msec. The number of sweeps for each histogram is indicated on the right. Stimulation values are indicated on the figure. Location of the stimuli are indicated by the heavy arrows and by the stimulation artifacts which are clear and have been truncated at the top. Stimulation parameters were changed to explore for the possibility that certain stimulus values would influence the firing rate. All units were tested with single stimuli and with trains, but none of the parameters were effective. recording electrodes are given in Fig. 10 and 11. No additional observations of this kind were made with other units. Another ph~nonlenon seen with central gray units is illustrated in Fig.
12 per sec. The electrical stimulation of the locus coeruleus did not influence the activity of these cells. Seven cerebellar Purkinje cells in the vermian lobule were studied, but no influence from locus coeruleus stimulation could be verified with these units. Another source of influence on two of the fast, steady central gray units was found in exploratory experiments
when the stimulating electrode was placed in the substantia nigra. One example of the observed inhibition is presented together with a poststimulus time histogram in Fig. 13. The electrode locations are depicted in Figs. 14 and 15. Following stimulation to the substantia nigra with single stimulus pairs, slowing was observed for about 500 msec. The latency of onset was somewhat variable, but was generally about 100 msec. The effect occurred with great reliability. After specific injections of horseradish peroxidase in the nucleus raphe dorsalis, cells were sparsely labeled in the locus coeruleus. Figure 16 shows an example of a labeled cell in the main portion of the locus coeruleus. One other animal showed comparable transport of this kind. In all other cases there was no evidence of retrograde transport to focus coeruleus cells. Thus, the morphological data complement the e~ectrophysiolo~ical findings. We report here only the retrograde transport results concerning the locus coeruieus-dorsal raphe connections. Detailed subscriptions of our results indicating afferents to nucleus raphe dorsalis from other regions have been described elsewhere [ IO,1 11.
The results of the electrophysiological tests suggest that the hypothesized direct projections from locus coeruieus to the nucleus raphe dorsalis do not exist. This conclusion is
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FIG. 4. Line drawings of crucial brain sections used to analyze the electrode placements for Rat 66. The units locations described in C and D below correspond to the poststimulus time histograms in Fig. 3. (A) Section 33 with the descending electrode track in the cerebellum and the locus coeruleus (LC) visible. The substantia grisea centralis (SGC) and the genu of the seventh nerve (g VII) are indicated. (B) The termination of the stimulating electrode track is presented, together with an inset drawing of the electrode to the same scale. This is Section 37. Sections were cut at a thickness of 40 p. (C) The open circle below the fasciculus longitudinalis medialis (MLF) represents the location of the dye mark indicating the tip of the microelectrode which is situated in the decussation of the brachium conjunctivum. The track seen above the dye mark was superimposed on this drawing of Section 61., but was actually found in Section 66. This was done to illustrate how the line drawings were used to confirm relationships in the histological sections. (D) Section 66 containing the track of the microelectrode. The two open circles indicate locations of units. Their location was obtained from microdrive readings and from the dye mark location illustrated in part C of this figure.
substantiated by the morphological studies performed with horseradish peroxidase injections into nucleus raphe dorsalis. Additional support is p:ovided from studies utilizing the Fink-Heimer technique in which no evidence of terminal degeneration in the nucleus raphe dorsalis was shown following lesions in the locus coeruleus [ 14,181 Moreover, other authors [22] have reported results in agreement with our preliminary electrophysiological data fll. Reconciliation of the interpretations of the fluorescence studies with the results contraindicating a locus coeruleusdorsal raphe pathway are possible. Loizou [ 71 indicated
that his lesions of the region of the locus coeruleus may have extended deep ventrally from the nucleus. Thus, noradrener~c pathways collecting into the dorsal noradrenergic bundle from sources other than A6 may have been interrupted. This interpretation implies that some other source of noradrenergic fibers may reach the nucleus raphe dorsalis. Descriptions of the origins of the monoaminergic pathways are not incompatible with this interpretation [ 211 . Moreover, other neuronal cell groups located around the locus coeruleus have been shown to project to the nucleus raphe dorsalis. Specifically, projections from the para-
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ANDERSON ET AL
FIG. 5. A photomicrograph of the brain section (No. 33) which the drawing shown in Fig. 4.4 is based upon. This and other sections are stained with cresyl violet. The stimulating electrode track can be seen in the cerebellum. Notice that it is pointed toward the locus coeruleus (indicated by the arrow).
FIG. 6. A photomicrograph of Section 37 from Rat 66. The drawing shown in Fig. 4B is based upon this section. The location of the tip of the stimulating electrode is between the points of the arrows.
LOCUS
COERULEUS-DORSAL
RAPHE
STUDIES
FIG. 7. Photomicrograph corresponding to the drawing in Fig. 4C. The arrow points to the location where the dye marker indicated the final extent of travel of the microelectrode, i.e., the brachium conjunctivum. The unit activity obtained from the indicated position is presented in h’ig. 3 as the bottom poststimulus time histogram.
FIG. 8. The location of the arrows show where the units were as determined by relating the location of the dye mark (Fig. 7) to the travel distance on the microdrive. This is Section 66 from Rat 66, and the arrow point locations correspond to the open circles shown in Fig. 4D. The uppermost poststimulus time histogram in Fig. 3 is taken from the more dorsal neuron. The more ventral unit is in the nucleus raphe dorsalis. The middle histogram in Fig. 3 is from this neuron.
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ANDERSON ET AL.
216
mo SWEECS
l- 10
11.40
Zl- 30
31 - 40
FIG. 9. Unit recordings from Rat 60. Each trace represents 10 sweeps of the storage oscilloscope superimposed for stimulus location. The stimulus parameters are shown on the figure. Temporal location of stimulation is indicated by the arrow. Single 0.2 msec stimuli were administered at the arrow and the oscilloscope made one sweep each time just as a stimulation was to occur. (Firing rate: 12 discharges per set). During the first 10 stimulations of the locus coeruleus an entrainment of the unit discharge occurred with a latency of about 80 msec. However, the relationship was not sustained with further stimulation. The anatomical locations of the electrodes are presented in Figs. 10 and 11.
FIG. 10. Photomicrograph of a section from Rat 60 illustrating the location of the stimulating electrode. The lesion marking the tip of this electrode is clearly visible (indicated by the arrow), as is the track passing through the locus coeruleus. There were four tips on this particular electrode at 0.2 mm intervals, indicating that the locus coeruleus clearly was stimulated. The corresponding electrophysiological record is in Fig. 9.
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FIG. Il. Photomicrograph containing the microelectrode track for Rat 60. The pointer indicates the unit location determined relative to the iontophoretically produced dye mark for the tip location of the microelectrode. The unit appears to be in the central gray on the midline caudal to the nucleus raphe dorsalis. This neuron provided the record presented in Fig. 9.
brachial nuclei reach the nucleus raphe dorsalis [ IO,1 I]. Thus, these connections should be taken into account when considering any relationship between this dorsal tegmental region and the nucleus raphe dorsalis. It should be noted in addition that, while recordings in the present study were made primarily upon midline portions of dorsal raphe, Chu and Bloom [3] report that the noradrenergic terminations occur only in the most lateral portions of nucleus raphe dorsalis. This lateral location for the appearance of monoaminergic fluorescence in the central gray is substantiated from earlier [Zl] and later research [ 9, I 5 1. Our neurophysiological evidence suggests an influence from the locus coeruleus upon the central gray neurons. This result is in good conformation with the fluorescence studies summarized above. The oscillatory firing pattern initiated in these units by stimulation of Iocus coeruleus suggests that more than one input pathway must be involved. The inhibitory effects on central gray units obtained from stimulation of the substantia nigra provide additional evidence of complex inputs to this region. One possibiiity is that fibers of the habenulo-dorsal raphe pathway were captivated [lo], although evidence for projections from substantia nigra directly to raphe dorsalis exist [11,12]. Although generalization from our small sample should be conservative, the absence of an influence from the stimulation of’ the locus coeruleus upon the majority of the neurons recorded from in the region of the central gray in
this study suggests that the morphological correlates of this finding may be somewhat sparse. In addition, the complexity of the area must be considered in making interpretations relative to it, as was noted earlier in this discussion. In spite of the small number of ceils involved, mention should be made of the fact that we did not see inhibition in cerebellar Purkinje cells as others have when stimulating in the locus coeruleus [5,20]. Perhaps the resolution of this discrepancy is related to the fact that the recordings in our study were made in a cerebellar lobule very remote from the one used in the earlier investigations. In conclusion, it may be said that, while the assumption made by Jouvet [61 regarding direct locus coeruleus to dorsal raphe projections was clearly the most parsimonious for interpreting the evidence pointing to a noradrenergicserotonergic interaction, it now seems necessary to revise this concept. Obviously, complex interacting brainstem chemical systems are involved in the regulation of the states of vigilance. The mechanisms of these interactions remain to be determined.
For their helpful of this investigation Macrides, William technical assistance Berman, Mr. Morris Marks.
discussions during the neurophysiological phase we wish to thank Drs. Steven Fredman, Foteos M. Youngs and Mr. Steven Schneider. For with histology we thank Mr. Howard M. Feinstein, Ms. Sharon A. Madden and Mr. David
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FIG. 12. Firing pattern of one central gray unit located just lateral to the dorsal raphe nucleus is shown during the administration of stimulus trains to the locus coeruleus. Three separate test sequences are represented in the A, B, and C portions of the figure. Within each of these sections of the figure the second sweep follows immediately after the first Stimulus locations are marked by the arrows. (A) The application of stimulation at 2 Hz to the locus coeruleus results in immediate slowing by about 25%. The return of the prest~mu~ation firing rate can be seen. (8) A train of four pulses again results in slowing. Termination of stimulation after the train of four pulses results in a rebeound, f&lowed by a return to the prestimulation firing rate. Additional stimulation in the second sweep of part B again results in slowing which lasts for about 1.5 sec. (C) A longer train of stimulation results in an initial slowing followed by a rebound (at about 1.5 set from stimulation onset) to a rate faster than before stimulation, even though the stimulation is continuing. The termination of this train in the second sweep results in a more sustained rebound. (Calibration: 0.5 set and 150 ,uV).
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J
L
i
SEC
msec SlNbLE SHOCKS TO SUBSTANTIA NIGRA +
CpO:RNTS
ADDRESS
60 SWEEPS IOmsec per address
I SEC FIG. 13. Poststimulus time histogram for Rat 59. Stimulating and recording electrode sites are shown in Figs. 14 and 15, respectively. The inset photograph of the present figure shows a single sweep of the unit fining with the stimulus artifact indicated by the arrow. Note the different time bases for the photograph of the single unit firing and the poststimulus time histogram. In the inset photograph can be seen the slowing of the firing rate for a period of about 200 msec occurring with a latency of about 100 msec following stimulation. The corresponding slowing in the histogram is indicated by the triangular pointer. In single sweeps the latency of the inhibition seemed slightly variable, but the effect was consistent. l:rom the histogram the inhibition is seen to have a reliable latency of 130 msec and a duration of 60 msec.
FIG. 14. Photomicrograph showing the location of the stimulating electrode in Rat 59. The arrow points to the location of electrode tip. The darkly stained nucleus at the electrode termination is the substantia nigra. The corresponding eiectrophysioiogical recording is presented in Fig. 13.
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FIG. 15. Photomicrograph showing the location of the recording microelectrode in Rat 59. The pointer indicates the location of the microelectrode tip. This unit was about 160 pm off of the midline in the central gray. The corresponding electrophysiological record is in Fig. 13.
FIG. 16. Photomicrograph illustrating the very sparse indications for retrograde transport of horseradish peroxidase to the locus coeruleus when injected into the nucleus raphe dorsalis. The margins of the locus coeruleus are indicated by the arrows. To the upper left in the figure can be seen the brachium conjunctivum (BC), and to the right can be seen the fourth ventricle (V IV) and the cerebellum (CER). Notice that only one cell in the locus coeruleus is labeled. (Initial magnification X100).
ET AL.
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STUDIES REFERENCES
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