EXPERIMENTAL
NEUROLOGY
65, 326-342 (1979)
Effects of Electrical Stimulation of the Lateral Habenula Single-Unit Activity of Raphe Neurons W. C. STERN, A. JOHNSON, J. D. BRONZINO,AND P.J. D. Dix Hospital, Connecticut
on
MORGANE'
Raleigh, North Carolina 27611; Trinity College, Hartford, 06106: and the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545 Received
February
20, 1979
Recent anatomical studies with horseradish peroxidase injections into the anterior raphe have demonstrated that the nucleus raphe dorsalis in the rat receives a major afferent input from the lateral habenula(LHb). The present study examined electrophysiologically the effects of electrical stimulation of the LHb on the spontaneous activity of midbrain and anterior pontine raphe units in anesthetized rats. The results showed that: (a) LHb stimulation (1 or 10 Hz, 0.5 to 1.0 mA) suppressed the activity of most raphe units, with the effects outlasting the duration of the stimulation in some instances; the raphe cells which showed periods of suppression during LHb stimulation were both those of the classical serotonin type (N = 26), characterized by slow regular baseline firing rates, and other raphe cells (N = 52) with faster baseline rates (to 60/s); (b) inhibition of unit activity was much less pronounced for non-raphe cells lateral to the midline; (c) anatomical control stimulation points dorsal to the LHb did not alter raphe unit activity; and (d) the pathway from the habenula to the raphe may involve a dorsal route. After a knife cut through the superior colliculus-central gray at the level of the interpeduncular nucleus, the effects of habenular stimulation were substantially reduced. Conversely, stimulation of the superior colliculus just posterior to the habenula (presumably containing descending fibers from the habenula) markedly suppressed raphe unit activity. In summary, the present electrophysiologic findings were consistent with the view that activation of habenular afferent fibers to the raphe exerted a major inhibitory influence on the spontaneous activity of midbrain and pontine raphe neurons. Considerably smaller effects were exerted on lateral reticular cells. A dorsal pathway may be involved in mediating the habenular effects on raphe activity. Abbreviations: LHb-lateral habenula, HRP-horseradish peroxidase. ’ This work was supported by National Science Foundation grant BNS 77- 165 12 and a grant from Burroughs Wellcome Co. A report of the findings was presented at the Society for Neuroscience, November 1978 in St. Louis, Missouri. Please address reprint requests to Dr. P. J. Morgane, Worcester Foundation, 222 Maple Avenue, Shrewsbury, MA 01545. 326 00144886/79/080326-17$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form resewed.
RAPHE
UNIT
ACTIVITY
327
INTRODUCTION The raphe region of the brain stem has been a focus of extensive anatomical, neurochemical, and electrophysiological research within the last decade. This research was stimulated by findings from neuropharmacological studies of serotonin and the histofluorescent evidence that the raphe system contains the major serotonin nuclei of the brain (10). The nuclei raphe dorsalis and medianus received special attention due to the extensive innervation of the limbic system and neocortex by axons whose cell bodies originate in those two raphe nuclei. Both lesions of the raphe and pharmacological studies with serotonin agonists and antagonists implicated the ascending pathways from the raphe in the control of various fundamental neurobehavioral states. This includes sleep and waking, anterior pituitary secretions, reproductive behavior, aggression, temperature, food intake, extrapyramidal motor functioning, epilepsy, and varisus forms of mental illness in man [for reviews see (12- 15,20,23,24,25,40)]. It is worth noting, however, that not all cell bodies in the raphe system are serotonergic (1,5), and that effects observed after lesions or stimulation of these nuclei may reflect modulation of the activity of other neurotransmitter systems. To more fully understand the factors which influence the activity of the raphe system, both serotonergic and non-serotonergic components, it is necessary to determine both the source of afferent input to the raphe and the effects these afferents exert on raphe cellular activity. Projections to the anteriorraphe have been studied with the classical lesion-degeneration methods. Prominent projections were found originating in the septal area and lateral hypothalmus (26, 39), caudate nucleus (34), cerebellum (8), and lateral habenular nucleus (6), among others. Although degeneration studies provided much information concerning the source of raphe afferents, interpretation of these studies is limited by the fact that lesions may disrupt fibers passing through the nuclei under study, hence giving a misleading impression of internuclear connectivities. Recently, afferent projections to the raphe have been studied with the horseradish peroxidase (HRP) method in which HRP is injected into the raphe dorsalis and/or medianus. The fluorescent HRP, taken up by the nerve terminals of fibers projecting to the raphe, appears in the cell bodies of the fiber of origin by retrograde axonal transport. Pasquier et al. (27) first reported in the rat with a HRP study that the raphe dorsalis received major projections from the lateral habenula, parafascicular nucleus, and in a later study, from the substantia nigra (28). Similar findings from other HRP studies in the rat and cat confirmed these major projections from the habenula and substantia nigra and also indicated significant projections from the nucleus of the solitary
328
STERN
ET AL.
tract, prefrontal cortex, am ygdaloid complex, preoptic nucleus, and the reticular formation (4), as well as from the locus coeruleus, parabrachial nuclei, and various posterior raphe nuclei (30). These three raphe-HRP studies all reported habenula-raphe connections and considered this projection to be both a major afferent source to the raphe and to be specific to the lateral habenula (LHb). The habenula did not appear to project directly to more posterior raphe nuclei (16). There was an absence of raphe projections from the medial habenula and from the region surrounding the lateral habenula. The nucleus closest to the LHb which sent afferents to the raphe was the parafascicular nucleus, situated caudoventral to the LHb. In light of these anatomical studies demonstrating afferent projections to the anterior raphe, it is important to ascertain the nature of the influence of various brain regions projecting to the raphe on the cellular activity of individual raphe neurons. Specifically, it can be determined by electrophysiological means whether or not the various afferents exert major or minor inhibitory/excitatory effects on raphe activity and what neurotransmitter system(s) are involved in mediating these effects. The present study is addressed to the former question, i.e., what is the effect of stimulation of one major afferent group of fibers, those originating from the LHb, on the activity of individual neurons in the midbrain and anterior pontine raphe nuclei. In addition, two other electrophysiological studies were conducted examining a possible pathway from the LHb to the raphe which mediates the effects of LHb stimulation on raphe activity.
METHODS General Experimental Procedures. Adult male albino Sprague-Dawley rats, averaging 300 g were used in the four acute electrophysiologic experiments described below. The rats were anesthetized with 400 mg/kg chloral hydrate, i.p., supplemented with 100 mg/kg injections during the course of 3- to 7-h stimulation-recording sessions. The rats were placed in a Kopf stereotaxic frame within a shielded cage, and a midline incision of the scalp was made. Local anesthetic injections with Xylocaine, and i.p. injections of 10 mg/kg atropine methyl bromide to reduce respiratory congestion, were used in a few preparations. Body temperature was maintained at 37°C using a rectal probe and a hot water heating pad activated by body temperature less than 37”. Small burr holes were drilled through the skull to permit lowering of concentric bipolar stainless-steel insulated stimulating electrodes, 200~pm outer diameter, 0.5mm separation of tips, into the LHb [stereotaxic coordinates AP +3290 to +4230 pm, 0.7 mm lateral and 4 mm below brain surface (IS)], posterior habenula (AP
RAPHE
UNIT
ACTIVITY
329
+3290 to +2790 pm, 0.7 mm lateral, 4.5 mm below brain surface), the midline area of superior colliculus at 1 to 2 mm posterior to the caudal end of the habenula (0.7 mm lateral, 4.5 mm below brain surface), or into the substantia nigra. [The substantia nigra data are reported elsewhere (33).] Glass micropipet recording electrodes with a tip diameter of approximately 1 pm, 2 to 6 Mfl, filled with 3 M KC1 were lowered either into the raphe system of the posterior midbrain and anterior pons [AP +0.5 to - 1 .O(3 l)] or into a region of the tegmentum lateral to the raphe, 0.7-2.0 mm off the midline. At the conclusion of the recordings, all rats were perfused with saline and then 10% buffered formalin. Frozen brain sections were cut at 32 pm and photographed for anatomic localization of the tips of the stimulating electrodes and the tracks of the recording microelectrodes. Electrical stimulation consisted of 20 to 300 s of continuous trains of bipolar pulses, 0.5 to 1.0 mA, 1 or 10 Hz, 0.1 ms per pulse. Pulses were generated by Grass Model S-8 or S-88 stimulators with their outputs connected to Grass PSIU-6 stimulus isolation units. Current passing through the stimulating electrodes was monitored in parallel on a Tektronix Model D61A oscilloscope. Extracellular single-unit activity was recorded by stereotaxically lowering the microelectrode with a Kopf hydraulic microdrive (approximately 10% of the units were recorded using a manual Narishige microdrive) into the desired anatomic site. The output of the microelectrode was fed into a Grass P16 preamplifier and then to a Tektronix Model 5103N storage oscilloscope and to a Brush universal amplifier. This amplifier output was sent to a Vetter Model A FM tape recorder, a Brush Model 220 ink-writing polygraph, an audio monitor and to a Neurolog window discriminator/programming unit for digitizing of spikes. The output of the window discriminator was recorded on the Brush polygraph and on the FM tape. Individual units were typically recorded for 3 to 30 min, with some for as long as 1 h. A typical stimulation-recording session for a given cell consisted of a minimum of 60 to 90 s of baseline, 30 to 300 s of l- or lo-Hz stimulation, a 60- to 300-s recovery/baseline period, and additional series of stimulation/recording periods using other stimulation values and/or different positions of the stimulating electrode. A range of 1 to 10 units was recorded from each rat. For some cells only data from l- or lo-Hz stimulation were obtained due to loss of the unit during recording. Data analysis of unit activity primarily consisted of: (a) Brush polygraph output of the window discriminator which provided an accurate integrated value of the spike rate during baseline and the post-stimulation recovery period. The presence of stimulation artifact in these recordings, which occurred in approximately one-half the units, precluded the use of these data for estimating firing rates during delivery of the stimulation trains. (b)
330
STERN
ET AL.
Photographic records of oscilloscope tracings of the analog spike wave forms were fed directly into the scope from the FM tape recorder, and from these photographs (actually negatives), spike rates during baseline stimulation and stimulation periods were manually obtained, along with measures of the duration of suppression/excitation following individual pulses. Data were grouped across units of a common anatomic location and common stimulating electrode site. These group values were compared by two-tailed parametric (t-test) and nonparametric (Mann-Whitney U, chi-square) tests. Specific Experiments. Experiment 1: Effects of LHb Stimulation on Raphe Unit Activity. Forty male rats had stimulating electrodes placed in the LHb, or immediately adjacent, with microelectrode recordings from 89 raphe units taken during electrical stimulation of the habenula. In five rats, control stimulation sites were also utilized in which the stimulating electrode was raised 2 mm above the habenula site. In these rats, the initial placement of the stimulating electrode in the LHb was observed to produce effects of raphe activity. Hence, the effects of stimulation of the control sites were assessed on the same raphe units in which habenula stimulation was shown to be effective. Experiment 2: Effects of LHb Stimulation on Non-raphe Unit Activity. Fifteen male rats were implanted with stimulating electrodes in the LHb and recording electrodes placed lateral to the midline at the level of the midbrain and pontine raphe (0.7 to 2.0 mm lateral to the midline). Twenty-one units were recorded from the same side of the brain as the stimulating electrode. Experiment 3: Effects of Stimulation of the Dorsal Superior ColliculusCentral Gray Posterior to the LHb on Raphe Activity. This study and the following one were addressed to the question of whether fibers projecting from the LHb to the raphe might take a dorsal pathway or descend ventroposteriorally to the interpeduncular nucleus, a major descending projection of the habenula. Forty-five raphe units were recorded from 18 rats which had collicular-central gray placements [AP +2.2 to 2.5,0.4 to 0.7 mm lateral, 4.0 to 5.0 mm below the brain surface (18)]. Experiment 4: Knife Cut through the Superior Colliculus-Central Gray. The purpose of this experiment was to provide additional evidence that the habenular projection to the raphe region responsible for the effects observed in Experiment 1 is, at least in part, a dorsal pathway through the region of the superior colliculus and dorsal central gray. Six rats had a bilateral knife cut placed at AP 1500 pm, 4 mm long (+2 mm on both sides of the midline) and extending down 4.5 to 5.0 mm below the brain surface, to the level of the aqueduct. These rats had a stimulating electrode placed in the LHb with unit recordings taken from 18 raphe units.
331
RAPHE UNIT ACTIVITY
RESULTS Experiment I. Of the 40 rats implanted with LHb stimulating electrodes, four had placements which were histologically shown to be outside the habenula, and their data were excluded from the analysis. Activity of 89 raphe units were recorded before and during stimulation of the LHb. In five rats, a control site 2 mm above the LHb was also stimulated. Comparisons of the LHb and control site results are shown in Fig. 1. This display of storage oscilloscope tracings during baseline, l-Hz stimulation (Is/sweep) and 10 Hz (100 mskweep) shows that raising the LHb stimulating electrode 2 mm abolished or greatly attenuated the suppressing effects of LHb RAISE RAT
2-E
UNIT NO
BASELINE
STIMULATION
BASELINE
Ltib ELECTRODE
2mm
STIMULATION
1.0.63
2-15
LO.75
3- 16
1.0.50
4-20
-1.0.54
4-24
-1.0.4.7
FIG. 1. Oscilloscope tracings of the effects of stimulation of the lateral habenula (LHb) (two left columns) and a control site 2 mm dorsal to LHb (two right columns) on activity of five raphe units. Each unit was recorded with stimulation tirst delivered to the LHb, then to the control site. Stimulation of the control sites produced minimal to no effects on raphe activity, whereas LHb stimulation decreased unit firings, in two cases with total suppression of activity. The time represented by a given trace is indicated below each photograph, e.g., 0.1 s x 10 (oscilloscope sweeps) = 1 s.
332
STERN ET AL.
stimulation on raphe activity. In addition, this figure illustrates the type of multisweep oscilloscope tracings from which the discharge rates and duration of postpulse effects were calculated. Table 1 describes the effects of stimulation of the LHb on the firing rate of several populations of raphe cells, defined by their baseline characteristics. These populations consisted of very slow units (discharge rates 2O/s). Units with the very slow baseline showed a significant decrease in firing rates at l-Hz (P < 0.01) and IO-Hz (P < 0.05) stimulation of the LHb. At 10 Hz, 96% of the 26 units showed temporal suppression of activity. In a few cells, the suppression was followed by a burst of firing prior to the next stimulation pulse. This burst occasionally raised the overall cell firing rate above baseline and resulted in 12% of the very slow raphe units being categorized as showing both temporal suppression and an increase in firing rate (minimum increase of 40%) at IO-Hz stimulation. The IO-Hz stimulation significantly lowered firing rates 30 to 50% for all four classes of raphe units, whereas l-Hz stimulation was mostly ineffective except for the very slow units. A mean duration of suppression averaged for all cells of a given class at IO-Hz stimulation was from 42 to 69 ms (maximum possible suppression of 100 ms). For all 89 raphe units, 88% showed a period of completely suppressed activity at 10 Hz and only 9% showed stimulation. TABLE
1
Effects of Electrical Stimulation of the Lateral Habenula on Activity of Raphe Units (Mean c SE)
Spikes Type of raphe cell
N
Baseline
Very slow baseline
22
2.7 + 0.4
SIOW baseline
22
Moderate baseline
21
Fast baseline
II
All raphe units
76
@Defined suppression b P < 0.01 c P < 0.05
Duration of suppression (ms)
per second
Percentage Suppressed
of units Stimulated”
1Hz
N
Baseline
10 HZ
I Hz
IO Hz
1 Hz
10 Hz
2.0 2 0.3b
26
2.4 + 0.3
1.7 f 0.4’
168 *39
69 +s
6-l
%
5
12
4.4 2 1.3
NEUROLOGY
65, 326-342 (1979)
Effects of Electrical Stimulation of the Lateral Habenula Single-Unit Activity of Raphe Neurons W. C. STERN, A. JOHNSON, J. D. BRONZINO,AND P.J. D. Dix Hospital, Connecticut
on
MORGANE'
Raleigh, North Carolina 27611; Trinity College, Hartford, 06106: and the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545 Received
February
20, 1979
Recent anatomical studies with horseradish peroxidase injections into the anterior raphe have demonstrated that the nucleus raphe dorsalis in the rat receives a major afferent input from the lateral habenula(LHb). The present study examined electrophysiologically the effects of electrical stimulation of the LHb on the spontaneous activity of midbrain and anterior pontine raphe units in anesthetized rats. The results showed that: (a) LHb stimulation (1 or 10 Hz, 0.5 to 1.0 mA) suppressed the activity of most raphe units, with the effects outlasting the duration of the stimulation in some instances; the raphe cells which showed periods of suppression during LHb stimulation were both those of the classical serotonin type (N = 26), characterized by slow regular baseline firing rates, and other raphe cells (N = 52) with faster baseline rates (to 60/s); (b) inhibition of unit activity was much less pronounced for non-raphe cells lateral to the midline; (c) anatomical control stimulation points dorsal to the LHb did not alter raphe unit activity; and (d) the pathway from the habenula to the raphe may involve a dorsal route. After a knife cut through the superior colliculus-central gray at the level of the interpeduncular nucleus, the effects of habenular stimulation were substantially reduced. Conversely, stimulation of the superior colliculus just posterior to the habenula (presumably containing descending fibers from the habenula) markedly suppressed raphe unit activity. In summary, the present electrophysiologic findings were consistent with the view that activation of habenular afferent fibers to the raphe exerted a major inhibitory influence on the spontaneous activity of midbrain and pontine raphe neurons. Considerably smaller effects were exerted on lateral reticular cells. A dorsal pathway may be involved in mediating the habenular effects on raphe activity. Abbreviations: LHb-lateral habenula, HRP-horseradish peroxidase. ’ This work was supported by National Science Foundation grant BNS 77- 165 12 and a grant from Burroughs Wellcome Co. A report of the findings was presented at the Society for Neuroscience, November 1978 in St. Louis, Missouri. Please address reprint requests to Dr. P. J. Morgane, Worcester Foundation, 222 Maple Avenue, Shrewsbury, MA 01545. 326 00144886/79/080326-17$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form resewed.
RAPHE
UNIT
ACTIVITY
327
INTRODUCTION The raphe region of the brain stem has been a focus of extensive anatomical, neurochemical, and electrophysiological research within the last decade. This research was stimulated by findings from neuropharmacological studies of serotonin and the histofluorescent evidence that the raphe system contains the major serotonin nuclei of the brain (10). The nuclei raphe dorsalis and medianus received special attention due to the extensive innervation of the limbic system and neocortex by axons whose cell bodies originate in those two raphe nuclei. Both lesions of the raphe and pharmacological studies with serotonin agonists and antagonists implicated the ascending pathways from the raphe in the control of various fundamental neurobehavioral states. This includes sleep and waking, anterior pituitary secretions, reproductive behavior, aggression, temperature, food intake, extrapyramidal motor functioning, epilepsy, and varisus forms of mental illness in man [for reviews see (12- 15,20,23,24,25,40)]. It is worth noting, however, that not all cell bodies in the raphe system are serotonergic (1,5), and that effects observed after lesions or stimulation of these nuclei may reflect modulation of the activity of other neurotransmitter systems. To more fully understand the factors which influence the activity of the raphe system, both serotonergic and non-serotonergic components, it is necessary to determine both the source of afferent input to the raphe and the effects these afferents exert on raphe cellular activity. Projections to the anteriorraphe have been studied with the classical lesion-degeneration methods. Prominent projections were found originating in the septal area and lateral hypothalmus (26, 39), caudate nucleus (34), cerebellum (8), and lateral habenular nucleus (6), among others. Although degeneration studies provided much information concerning the source of raphe afferents, interpretation of these studies is limited by the fact that lesions may disrupt fibers passing through the nuclei under study, hence giving a misleading impression of internuclear connectivities. Recently, afferent projections to the raphe have been studied with the horseradish peroxidase (HRP) method in which HRP is injected into the raphe dorsalis and/or medianus. The fluorescent HRP, taken up by the nerve terminals of fibers projecting to the raphe, appears in the cell bodies of the fiber of origin by retrograde axonal transport. Pasquier et al. (27) first reported in the rat with a HRP study that the raphe dorsalis received major projections from the lateral habenula, parafascicular nucleus, and in a later study, from the substantia nigra (28). Similar findings from other HRP studies in the rat and cat confirmed these major projections from the habenula and substantia nigra and also indicated significant projections from the nucleus of the solitary
328
STERN
ET AL.
tract, prefrontal cortex, am ygdaloid complex, preoptic nucleus, and the reticular formation (4), as well as from the locus coeruleus, parabrachial nuclei, and various posterior raphe nuclei (30). These three raphe-HRP studies all reported habenula-raphe connections and considered this projection to be both a major afferent source to the raphe and to be specific to the lateral habenula (LHb). The habenula did not appear to project directly to more posterior raphe nuclei (16). There was an absence of raphe projections from the medial habenula and from the region surrounding the lateral habenula. The nucleus closest to the LHb which sent afferents to the raphe was the parafascicular nucleus, situated caudoventral to the LHb. In light of these anatomical studies demonstrating afferent projections to the anterior raphe, it is important to ascertain the nature of the influence of various brain regions projecting to the raphe on the cellular activity of individual raphe neurons. Specifically, it can be determined by electrophysiological means whether or not the various afferents exert major or minor inhibitory/excitatory effects on raphe activity and what neurotransmitter system(s) are involved in mediating these effects. The present study is addressed to the former question, i.e., what is the effect of stimulation of one major afferent group of fibers, those originating from the LHb, on the activity of individual neurons in the midbrain and anterior pontine raphe nuclei. In addition, two other electrophysiological studies were conducted examining a possible pathway from the LHb to the raphe which mediates the effects of LHb stimulation on raphe activity.
METHODS General Experimental Procedures. Adult male albino Sprague-Dawley rats, averaging 300 g were used in the four acute electrophysiologic experiments described below. The rats were anesthetized with 400 mg/kg chloral hydrate, i.p., supplemented with 100 mg/kg injections during the course of 3- to 7-h stimulation-recording sessions. The rats were placed in a Kopf stereotaxic frame within a shielded cage, and a midline incision of the scalp was made. Local anesthetic injections with Xylocaine, and i.p. injections of 10 mg/kg atropine methyl bromide to reduce respiratory congestion, were used in a few preparations. Body temperature was maintained at 37°C using a rectal probe and a hot water heating pad activated by body temperature less than 37”. Small burr holes were drilled through the skull to permit lowering of concentric bipolar stainless-steel insulated stimulating electrodes, 200~pm outer diameter, 0.5mm separation of tips, into the LHb [stereotaxic coordinates AP +3290 to +4230 pm, 0.7 mm lateral and 4 mm below brain surface (IS)], posterior habenula (AP
RAPHE
UNIT
ACTIVITY
329
+3290 to +2790 pm, 0.7 mm lateral, 4.5 mm below brain surface), the midline area of superior colliculus at 1 to 2 mm posterior to the caudal end of the habenula (0.7 mm lateral, 4.5 mm below brain surface), or into the substantia nigra. [The substantia nigra data are reported elsewhere (33).] Glass micropipet recording electrodes with a tip diameter of approximately 1 pm, 2 to 6 Mfl, filled with 3 M KC1 were lowered either into the raphe system of the posterior midbrain and anterior pons [AP +0.5 to - 1 .O(3 l)] or into a region of the tegmentum lateral to the raphe, 0.7-2.0 mm off the midline. At the conclusion of the recordings, all rats were perfused with saline and then 10% buffered formalin. Frozen brain sections were cut at 32 pm and photographed for anatomic localization of the tips of the stimulating electrodes and the tracks of the recording microelectrodes. Electrical stimulation consisted of 20 to 300 s of continuous trains of bipolar pulses, 0.5 to 1.0 mA, 1 or 10 Hz, 0.1 ms per pulse. Pulses were generated by Grass Model S-8 or S-88 stimulators with their outputs connected to Grass PSIU-6 stimulus isolation units. Current passing through the stimulating electrodes was monitored in parallel on a Tektronix Model D61A oscilloscope. Extracellular single-unit activity was recorded by stereotaxically lowering the microelectrode with a Kopf hydraulic microdrive (approximately 10% of the units were recorded using a manual Narishige microdrive) into the desired anatomic site. The output of the microelectrode was fed into a Grass P16 preamplifier and then to a Tektronix Model 5103N storage oscilloscope and to a Brush universal amplifier. This amplifier output was sent to a Vetter Model A FM tape recorder, a Brush Model 220 ink-writing polygraph, an audio monitor and to a Neurolog window discriminator/programming unit for digitizing of spikes. The output of the window discriminator was recorded on the Brush polygraph and on the FM tape. Individual units were typically recorded for 3 to 30 min, with some for as long as 1 h. A typical stimulation-recording session for a given cell consisted of a minimum of 60 to 90 s of baseline, 30 to 300 s of l- or lo-Hz stimulation, a 60- to 300-s recovery/baseline period, and additional series of stimulation/recording periods using other stimulation values and/or different positions of the stimulating electrode. A range of 1 to 10 units was recorded from each rat. For some cells only data from l- or lo-Hz stimulation were obtained due to loss of the unit during recording. Data analysis of unit activity primarily consisted of: (a) Brush polygraph output of the window discriminator which provided an accurate integrated value of the spike rate during baseline and the post-stimulation recovery period. The presence of stimulation artifact in these recordings, which occurred in approximately one-half the units, precluded the use of these data for estimating firing rates during delivery of the stimulation trains. (b)
330
STERN
ET AL.
Photographic records of oscilloscope tracings of the analog spike wave forms were fed directly into the scope from the FM tape recorder, and from these photographs (actually negatives), spike rates during baseline stimulation and stimulation periods were manually obtained, along with measures of the duration of suppression/excitation following individual pulses. Data were grouped across units of a common anatomic location and common stimulating electrode site. These group values were compared by two-tailed parametric (t-test) and nonparametric (Mann-Whitney U, chi-square) tests. Specific Experiments. Experiment 1: Effects of LHb Stimulation on Raphe Unit Activity. Forty male rats had stimulating electrodes placed in the LHb, or immediately adjacent, with microelectrode recordings from 89 raphe units taken during electrical stimulation of the habenula. In five rats, control stimulation sites were also utilized in which the stimulating electrode was raised 2 mm above the habenula site. In these rats, the initial placement of the stimulating electrode in the LHb was observed to produce effects of raphe activity. Hence, the effects of stimulation of the control sites were assessed on the same raphe units in which habenula stimulation was shown to be effective. Experiment 2: Effects of LHb Stimulation on Non-raphe Unit Activity. Fifteen male rats were implanted with stimulating electrodes in the LHb and recording electrodes placed lateral to the midline at the level of the midbrain and pontine raphe (0.7 to 2.0 mm lateral to the midline). Twenty-one units were recorded from the same side of the brain as the stimulating electrode. Experiment 3: Effects of Stimulation of the Dorsal Superior ColliculusCentral Gray Posterior to the LHb on Raphe Activity. This study and the following one were addressed to the question of whether fibers projecting from the LHb to the raphe might take a dorsal pathway or descend ventroposteriorally to the interpeduncular nucleus, a major descending projection of the habenula. Forty-five raphe units were recorded from 18 rats which had collicular-central gray placements [AP +2.2 to 2.5,0.4 to 0.7 mm lateral, 4.0 to 5.0 mm below the brain surface (18)]. Experiment 4: Knife Cut through the Superior Colliculus-Central Gray. The purpose of this experiment was to provide additional evidence that the habenular projection to the raphe region responsible for the effects observed in Experiment 1 is, at least in part, a dorsal pathway through the region of the superior colliculus and dorsal central gray. Six rats had a bilateral knife cut placed at AP 1500 pm, 4 mm long (+2 mm on both sides of the midline) and extending down 4.5 to 5.0 mm below the brain surface, to the level of the aqueduct. These rats had a stimulating electrode placed in the LHb with unit recordings taken from 18 raphe units.
331
RAPHE UNIT ACTIVITY
RESULTS Experiment I. Of the 40 rats implanted with LHb stimulating electrodes, four had placements which were histologically shown to be outside the habenula, and their data were excluded from the analysis. Activity of 89 raphe units were recorded before and during stimulation of the LHb. In five rats, a control site 2 mm above the LHb was also stimulated. Comparisons of the LHb and control site results are shown in Fig. 1. This display of storage oscilloscope tracings during baseline, l-Hz stimulation (Is/sweep) and 10 Hz (100 mskweep) shows that raising the LHb stimulating electrode 2 mm abolished or greatly attenuated the suppressing effects of LHb RAISE RAT
2-E
UNIT NO
BASELINE
STIMULATION
BASELINE
Ltib ELECTRODE
2mm
STIMULATION
1.0.63
2-15
LO.75
3- 16
1.0.50
4-20
-1.0.54
4-24
-1.0.4.7
FIG. 1. Oscilloscope tracings of the effects of stimulation of the lateral habenula (LHb) (two left columns) and a control site 2 mm dorsal to LHb (two right columns) on activity of five raphe units. Each unit was recorded with stimulation tirst delivered to the LHb, then to the control site. Stimulation of the control sites produced minimal to no effects on raphe activity, whereas LHb stimulation decreased unit firings, in two cases with total suppression of activity. The time represented by a given trace is indicated below each photograph, e.g., 0.1 s x 10 (oscilloscope sweeps) = 1 s.
332
STERN ET AL.
stimulation on raphe activity. In addition, this figure illustrates the type of multisweep oscilloscope tracings from which the discharge rates and duration of postpulse effects were calculated. Table 1 describes the effects of stimulation of the LHb on the firing rate of several populations of raphe cells, defined by their baseline characteristics. These populations consisted of very slow units (discharge rates 2O/s). Units with the very slow baseline showed a significant decrease in firing rates at l-Hz (P < 0.01) and IO-Hz (P < 0.05) stimulation of the LHb. At 10 Hz, 96% of the 26 units showed temporal suppression of activity. In a few cells, the suppression was followed by a burst of firing prior to the next stimulation pulse. This burst occasionally raised the overall cell firing rate above baseline and resulted in 12% of the very slow raphe units being categorized as showing both temporal suppression and an increase in firing rate (minimum increase of 40%) at IO-Hz stimulation. The IO-Hz stimulation significantly lowered firing rates 30 to 50% for all four classes of raphe units, whereas l-Hz stimulation was mostly ineffective except for the very slow units. A mean duration of suppression averaged for all cells of a given class at IO-Hz stimulation was from 42 to 69 ms (maximum possible suppression of 100 ms). For all 89 raphe units, 88% showed a period of completely suppressed activity at 10 Hz and only 9% showed stimulation. TABLE
1
Effects of Electrical Stimulation of the Lateral Habenula on Activity of Raphe Units (Mean c SE)
Spikes Type of raphe cell
N
Baseline
Very slow baseline
22
2.7 + 0.4
SIOW baseline
22
Moderate baseline
21
Fast baseline
II
All raphe units
76
@Defined suppression b P < 0.01 c P < 0.05
Duration of suppression (ms)
per second
Percentage Suppressed
of units Stimulated”
1Hz
N
Baseline
10 HZ
I Hz
IO Hz
1 Hz
10 Hz
2.0 2 0.3b
26
2.4 + 0.3
1.7 f 0.4’
168 *39
69 +s
6-l
%
5
12
4.4 2 1.3
