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Previous Article | Next Article
Volume 16, Number 21, Issue of November 1, 1996 pp. 7010-7020
Copyright ©1996 Society for NeuroscienceModulation of Forebrain Electroencephalographic Activity in Halothane-Anesthetized Rat via Actions of Noradrenergic
-Receptors within the Medial Septal Region
Craig W. Berridge1, Sarah J. Bolen2, Michael S. Manley2, and Stephen L. Foote2 1 Psychology Department, University of Wisconsin, Madison, Wisconsin 53706-1611, and 2 Psychiatry Department, University of California, San Diego, La Jolla, California 92093
ABSTRACT
The locus coeruleus (LC)-noradrenergic system modulates forebrain electroencephalographic (EEG) activity in halothane-anesthetized rat. For example, unilateral enhancement of LC neuronal activity increases cortical EEG (ECoG) and hippocampal EEG (HEEG) indices of arousal bilaterally (Berridge and Foote, 1991
). Conversely, bilateral suppression of LC discharge activity increases EEG measures of sedation (Berridge et al., 1993b
-receptors (Berridge and Foote, 1991
Two candidate sites at which LC efferents could influence ECoG and HEEG are the medial septum/vertical limb of the diagonal band of Broca (MS) and the substantia innominata/nucleus basalis of Meynert (SI). To determine whether norepinephrine mediates such actions within either of these regions, the EEG effects of small infusions of a
These results indicate that under these experimental conditions, noradrenergic efferents, presumably arising from LC, modulate forebrain EEG state via actions at
Key words: norepinephrine; medial septum; arousal; EEG;
INTRODUCTION
Distinct amplitude and frequency patterns in forebrain electroencephalographic (EEG) activity constitute major defining characteristics of behavioral states (Timo-Iaria et al., 1970
One system posited to participate in the modulation of behavioral state and state-dependent processes is the noradrenergic nucleus locus coeruleus (LC) and its efferents (for review, see Foote et al., 1983
In previous studies in halothane-anesthetized rats, microelectrode recordings were used to guide placement of small (35-150 nl) peri-LC infusions of drugs and verify that they altered LC discharge levels. This approach avoids many of the problems associated with techniques available previously for manipulation of the LC-noradrenergic system such as lesions, electrical stimulation, and local infusions guided solely by stereotaxic coordinates (see Berridge and Foote, 1991
The site(s) at which LC efferents act to modulate EEG state remains to be elucidated. Among the candidate sites are portions of the basal forebrain containing the medial septum/vertical limb of the diagonal band of Broca (collectively referred to as MS) and the substantia innominata/nucleus basalis of Meynert (SI). MS and SI receive a dense noradrenergic innervation (Segal, 1976
MATERIALS AND METHODS
Animals and surgery. Male Sprague Dawley rats (Charles River, Wilmington, MA), weighing 280-350 gm, were anesthetized with halothane using a face mask. A tracheotomy was then performed, and halothane (0.75-1.25% in air) was administered via this route for the duration of the experiment. The animal was placed in a stereotaxic instrument with the incisor bar set 11.5 mm below ear bar zero. Body temperature was maintained at 36-38°C.Drugs/intratissue infusions. In the majority of cases, 26 gauge guide cannulae were then positioned over areas of interest and cemented into place with dental acrylic. MS cannulae were inserted at an angle of 4° from vertical (within the coronal plane) to minimize infusion-induced damage to fibers of passage that travel along the medial aspect of MS. In some cases (e.g., mapping studies), a cannula was cemented to a plastic holder held in a micromanipulator. This facilitated making infusions into multiple sites within a given subject. Infusions were made via a 33 gauge needle that was inserted into the cannula and extended 3 mm beyond its ventral tip. This needle was slightly beveled to minimize travel of fluid up the length of the needle. The infusion needle was attached to PE20 tubing via a 26 gauge stainless steel sleeve glued to the needle. The other end of the tubing was attached to a 10 µl syringe, the plunger of which was advanced using a microprocessor-controlled infusion pump (Harvard Apparatus, South Natick, MA). Infusions consisted of 150 nl given over a 60 sec period. If no effect of drug or vehicle was observed within 10 min of the first infusion, a second infusion was given. This strategy was based on previous studies using peri-LC infusions to alter LC neuronal discharge activity (Berridge and Foote, 1991
EEG recording and analyses. Bipolar surface-to-depth electrodes were used to record ECoG (anterior, +3.0; lateral, ±1.5) and HEEG (anterior,
4.8; lateral, ±2.5; ventral
Statistical analyses. For MS ISO infusions, ECoG and HEEG absolute power and relative power for each frequency bandwidth were analyzed using a paired t test with Bonferoni correction to compare pre- and postinfusion segments. A one-way, repeated-measures ANOVA, followed by the Duncan's multiple-range test, was used to statistically assess EEG recovery in the subset of MS ISO animals for which spontaneous recovery data were collected. Repeated-measures, one-way ANOVA was used to assess EEG effects of bilateral MS vehicle and timolol infusions.
Histology. After each experiment, the animal was deeply anesthetized and then perfused with 50 ml of 4% formaldehyde. The brain was removed and placed in perfusion solution. After a minimum of 24 hr, the brain was frozen and 40 µm sections were cut and collected through MS and other areas in which infusions were made. The sections were stained with neutral red dye for subsequent examination of the infusion sites. The distribution of Pontamine Sky Blue dye was noted in both freshly sectioned and stained tissue.
Dopamine
Sections were incubated in the following solutions for the indicated times for immunocytochemical localization of DBH: (1) 0.3% hydrogen peroxide in 0.1 M PB, 30 min; (2) 1% BSA, 10% normal horse serum (Sigma) in 0.1 M Tris-buffered saline (TBS) containing 0.3% Triton X-100, 60 min; (3) mouse anti-DBH (Chemicon International, Temecula, CA), diluted 1:1500, in TBS containing 2% normal horse serum, 0.1% BSA, and 0.3% Triton X-100, 24 hr at 4°C with constant shaking; (4) horse anti-mouse IgG (Vector Labs, Burlingame, CA) diluted 1:300 in the same diluent as in (3), 60 min; (5) avidin-biotin-peroxidase complex (ABC Elite, Vector Labs), diluted 1:200, in TBS-0.1% BSA, 60 min; and (6) substrate solution containing 0.05% diaminobenzidine tetrahydrochloride (Sigma), 0.0004% nickel chloride, and 0.0002% hydrogen peroxide in 0.1 M Tris buffer for four 10 min rinses. All incubations were performed at room temperature unless otherwise indicated. Three 10 min rinses in the appropriate buffers were performed between steps. Sections were dehydrated in ascending concentrations of ethanol, then cleared in xylenes. For dark-field photomicrography, sections were additionally incubated in chloroform-ethanol (50:50 v/v) for 30 min before clearing in xylenes. Tissue sections were coverslipped with DPX (BDH Laboratory Supplies, Poole, England) then photographed using a Nikon Optiphot microscope with a 4× objective.
Data selection criteria. Data from a particular animal were included in the analyses for all cases, and in only those cases in which EEG electrode placements were accurate, EEG recordings were electrically adequate, and placement of the infusion needle could be anatomically verified.
RESULTS
General observations
In previous studies, we observed that LC-induced changes in EEG state are reliable and robust when observed during a stable, carefully maintained, and appropriate level of anesthesia (Berridge and Foote, 1991In preliminary studies (n = 35), various infusion volumes and concentrations of ISO were used. In these studies, EEG responses were observed, and spread of dye from the infusion site was assessed. Dye was readily visible at sectioning of fixed tissue. After a 150 nl infusion, the spread of dye was limited to a radius of ~600-800 µm from the infusion site. This radius was somewhat smaller in the medial-lateral direction where the midline and lateral ventricles appeared to form barriers across which the dye did not readily penetrate. Thus, dye was usually visible only in the region between midline and the lateral ventricles and was confined to an area defined by the most anterior and posterior aspects of MS. Dye rarely spread as far dorsally as the lateral septum. These observations, together with the EEG effects of these infusions, suggested that an infusion volume of ~150 nl was adequate to ensure spread of drug throughout MS while minimizing its spread beyond this region. Doses substantially below that used in these studies appeared to elicit less consistent EEG effects.
Effects of unilateral ISO and glutamate infusions into MS on ECoG and HEEG
Unilateral infusions of ISO (150 nl) into MS (Fig. 1) (n = 21) resulted in a shift from slow-wave, large-amplitude to high-frequency, low-amplitude activity in ECoG and the appearance of nearly pure theta activity in HEEG (Fig. 2). These EEG responses were observed only when infusions were placed within a radius of ~500 µm of MS (see Figs. 1, 3). In ~30% of the cases, two infusions (separated by 10 min) were required to elicit this effect (see Materials and Methods). The EEG responses were observed bilaterally, within 3-8 min of termination of the infusion, and persisted for 5-90 min (median = 16 min), as determined from visual inspection. Comparable EEG responses were observed with repeated ISO infusions. Response duration and magnitude appeared to most closely correlate with the level of anesthesia, assessed by intensity and duration of tail pinch-induced ECoG desynchronization, rather than with the specific location of the infusion site within MS. Within the limits of the temporal resolution of the analyses (~1 sec), HEEG and ECoG responses were generally observed simultaneously. However, in ~50% of the experiments, the onset of HEEG responses appeared to occur 1-5 sec before the onset of the ECoG responses. Glutamate infusions into MS elicited an activation of both ECoG and HEEG similar to that observed after ISO infusions (n = 7). However, the latency to onset of the EEG responses was much shorter after glutamate infusions, ranging from before termination of the infusion to 30 sec after termination of the infusion. Comparable EEG responses were observed after repeated glutamate infusions. In nine experiments, two 150 nl infusions of vehicle were made before effective infusions of ISO (n = 5) or glutamate (n = 4) (see below). In all cases, vehicle infusions had no obvious effects on either HEEG or ECoG (data not shown).
Fig. 1. Photomicrograph of a neutral red-stained coronal section showing the position of an effective ISO MS infusion site from a typical experiment (large arrow). The infusion needle was lowered into MS at an angle of 4° from vertical, and a 150 nl infusion of ISO was made over 1 min. The most ventral extent of the needle track is shown in this section. The lateral ventricle on the same side is indicated by a small arrow. AC, Anterior commissure. Original magnification, 25×. Scale bar, 1 mm.
[View Larger Version of this Image (151K GIF file)]
Fig. 2. Raw ECoG and HEEG traces and PSA from pre- and postinfusion periods of a typical MS ISO experiment. A 40 sec raw EEG trace taken from the 60 sec interval from which the PSA was computed is shown above each power spectrum. The most striking postinfusion changes are the decrease in power of the slowest frequencies in ECoG and the appearance of theta (3-4 Hz) activity in HEEG. Shading indicates the theta frequency band (2.3-6.9 Hz) in the HEEG power spectra.
[View Larger Version of this Image (28K GIF file)]
Fig. 3. Schematic diagram depicting effective and ineffective MS (A) and SI (B) ISO infusion sites. A depicts sites at which 150 nl ISO infusions made within and around the region of MS were either effective or ineffective at inducing activation of forebrain EEG. B indicates sites at which ISO infusions (either 150 or 450 nl) were made into SI. In all cases, SI ISO infusions were ineffective at eliciting EEG activation. Shaded boxes indicate sites at which ISO infusions elicited forebrain EEG activation, whereas solid circles indicate sites at which they did not. AC, Anterior commissure; CC, corpus callosum; CP, caudate-putamen; GP, globus pallidus; I, internal capsule; LS, lateral septum; LV, lateral ventricle; MS, medial septum; NA, nucleus accumbens; SI, substantia innominata. Each level within A or B is separated by 250 µm, with the most anterior section on the left (modified from Swanson, 1992
[View Larger Version of this Image (28K GIF file)]
PSA of the MS ISO-induced changes in EEG
PSA demonstrated statistically significant effects of unilateral MS ISO infusions on ECoG and HEEG consistent with the above-described qualitative observations (Fig. 2; Table 1). In ECoG (n = 20) (in one case, it was not possible to obtain an electrically adequate ECoG recording), the primary effect was a significant decrease in absolute and relative power of the lowest frequency band (0.3-2.3 Hz). There were also significant increases in the relative power of the 20-30, 30-40, and 40-50 Hz bands. In HEEG (n = 21), ISO induced a decrease in the absolute and relative power of the 0.3-2.3 Hz frequency band and increased the absolute and relative power of the theta-activity frequency band (2.3-6.8 Hz). There was a tendency for increased absolute and relative power of the higher-frequency bands. However, this was statistically significant only for absolute power of the 30.0-40.0 Hz band and relative power of the 20.0-30.0 Hz band. Because of prolonged drug-induced responses or additional experimental manipulations, recovery epochs were analyzed only in a subset of the experiments (n = 13). Absolute and relative power of recovery EEG epochs for all frequency bands in both ECoG and HEEG did not significantly differ from preinfusion values (data not shown).
Table 1. Effects of MS ISO infusions on EEG: mean absolute and relative power of postinfusion segments of ECoG and HEEG expressed as percentage of preinfusion means
Frequency ECoG HEEG Absolute Relative Absolute Relative 0.3 -2.3 53 ± 6** 76 ± 5** 67 ± 5** 64 ± 4** 2.3 -6.8 82 ± 5 114 ± 8 116 ± 5* 118 ± 4** 6.8 -13.0 79 ± 6 107 ± 8 95 ± 4 105 ± 4 13.0 -20.0 78 ± 5 108 ± 8 96 ± 4 102 ± 5 20.0 -30.0 87 ± 5 119 ± 10* 109 ± 5 117 ± 2** 30.0 -40.0 88 ± 5 123 ± 11* 114 ± 5** 125 ± 10 40.0 -50.0 91 ± 8 132 ± 13** 104 ± 3 117 ± 10 Absolute and relative power expressed as percentages of preinfusion means (±SEM) for ECoG (n = 20) and HEEG (n = 21) for the specified frequency bands. *p < 0.05, **p < 0.01 significantly different from preinfusion mean determined by t test with Bonferoni correction (n = 20). Effects of ISO infusions made outside the region of MS on ECoG and HEEG
To determine whether the MS ISO-induced changes in forebrain EEG resulted from diffusion and subsequent action of ISO outside of MS, ISO infusions were made into the following regions: (1) the striatum, at a similar distance from the lateral ventricle as the MS infusions (n = 6); (2) the region immediately lateral to the effective MS infusion sites (n = 6); and (3) the lateral septum (n = 4) (see Fig. 3). These infusions were made in a subset of the experiments described above using a within-subjects design. ISO infusions were made into one or two of the regions listed above outside of MS before making a final, effective infusion into MS. This design was chosen to minimize the possibility that negative effects of control-site injections could arise from factors other than those directly related to the issue of anatomical site of action such as level of anesthesia or other physiological concerns. In all cases, if no effects were observed within 10 min after an infusion, a second infusion was performed. If no effects were observed within 15 min after the second infusion, the infusion was considered to be ineffective.Infusions into striatum, 200-800 µm lateral to the lateral ventricle, did not elicit noticeable changes in either ECoG or HEEG. This indicates that diffusion of ISO into the ventricular system is not responsible for EEG activation observed after ISO infusions into MS. For infusions into the nucleus accumbens and lateral septum, only infusions closest to MS elicited EEG activation (see Fig. 3).
Effects of ISO and glutamate infusions into SI on ECoG and HEEG
In nine cases, 150 nl (n = 6) or 450 nl (n = 3) infusions of ISO were made into SI (see Fig. 3). In all cases, these infusions did not noticeably alter baseline HEEG or ECoG as determined by visual inspection (Fig. 4). The ineffectiveness of SI ISO infusions in altering EEG state might result from insufficient infusion volume or properties unique to this experimental preparation that interfere with the normal EEG modulatory action of SI neurons. To assess this possibility, infusions of glutamate (150 nl) were made into SI. As in previous studies (Metherate et al., 1992
Fig. 4. Effects of ISO and glutamate (Glu) infusions made into SI on ECoG and HEEG. A, ECoG and HEEG before and after a 150 nl infusion of glutamate into SI. These infusions elicited robust activation of both ECoG and HEEG as demonstrated by the reduction of slow-wave activity in ECoG and the induction of theta-activity in HEEG. B, ECoG and HEEG taken from same experiment depicted in A before and after a 450 nl infusion of ISO.
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Effects of bilateral MS infusions of the
To determine the degree to which endogenous NE contributes to the maintenance of forebrain activation, the effects of bilateral infusions of theUnder these conditions, bilateral infusion of timolol (3.75 µg/150 nl in each hemisphere) consistently resulted in the alteration of both ECoG and HEEG activity (Fig. 5). This concentration was chosen on the basis of pilot studies that demonstrated inconsistent EEG effects at concentrations substantially below this. ECoG changes consisted of a shift from predominantly desynchronized (high-frequency, low-amplitude) to large-amplitude, slow-wave activity. In the HEEG, theta-dominated activity was replaced with mixed-frequency activity. The onset of these EEG responses occurred within 3-30 min (median = 10 min) after the termination of the infusions. The latency between the initial and the maximal EEG responses, as estimated from visual inspection of the EEG trace, ranged from 2 to 10 min. The duration of these EEG responses ranged from 60 min to >120 min. In four of the eight experiments, the first obvious signs of recovery were observed before 120 min. However, in two of these cases, additional manipulations were then made, precluding the use of these animals in statistical analyses of EEG recovery epochs. In the remaining four experiments, recovery was not observed within 120 min after the infusions.
Fig. 5. Effects of bilateral MS timolol infusions on ECoG and HEEG. Representative 40 sec epochs of ECoG and HEEG traces taken from a 5 min epoch used to compute PSA values in Table 2. Displayed are data taken before any infusions (Pre-Infusion), 15 min after bilateral MS vehicle infusions (Post-Vehicle), and 15 min after bilateral timolol infusions (Post-Timolol). Whereas bilateral vehicle infusions did not noticeably affect either ECoG or HEEG, bilateral timolol infusions increased the occurrence of slow-wave, large-amplitude activity in ECoG and substantially decreased the presence of theta activity in HEEG.
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Bilateral vehicle infusions were made in seven of the above experiments 30-45 min before bilateral timolol infusions, following a within-subjects design. In these experiments, 15-30 min after bilateral vehicle infusions, the needles were removed and filled with timolol and reinserted into the cannulae. Fifteen minutes after insertion of the timolol-containing needles, an infusion was made into each hemisphere.
In all cases, vehicle infusions did not have consistent obvious effects on either ECoG or HEEG (see Fig. 5, Table 2). Unilateral infusion of a comparable dose (10-20 µg) of timolol directly into the lateral ventricles had no obvious effects on either ECoG or HEEG (data not shown). In four cases, the infusion needles were not successfully positioned bilaterally within MS (see Fig. 6). In all of these cases, at least one of the infusion needles was positioned in the most posterior extent of MS, at the level of the decussation of the anterior commissure, in close proximity to portions of the bed nucleus of the stria terminalis. In these four experiments, no obvious effects on either ECoG or HEEG were observed (data not shown).
Table 2. Effects of bilateral MS timolol infusions on EEG: mean absolute and relative power of ECoG and HEEG expressed as percentage of prevehicle infusion means
Frequency Absolute power Relative power Vehicle Timolol Vehicle Timolol ECoG 0.3 -2.3 142 ± 38 346 ± 70** 111 ± 13 170 ± 12** 2.3 -6.8 127 ± 21 205 ± 33** 109 ± 5 105 ± 5 6.8 -13.0 100 ± 8 129 ± 10 94 ± 9 71 ± 5** 13.0 -20.0 94 ± 6 122 ± 9 91 ± 9 67 ± 6** 20.0 -30.0 92 ± 3 100 ± 4 94 ± 12 59 ± 7** 30.0 -40.0 93 ± 4 78 ± 11 95 ± 13 55 ± 7** 40.0 -50.0 98 ± 7 93 ± 5 94 ± 14 54 ± 6** HEEG 0.3 -2.3 117 ± 11 138 ± 7** 113 ± 8 152 ± 8** 2.3 -6.8 100 ± 3 72 ± 5** 98 ± 5 79 ± 3** 6.8 -13.0 109 ± 11 98 ± 7 104 ± 8 106 ± 5 13.0 -20.0 101 ± 5 102 ± 5 98 ± 3 111 ± 3** 20.0 -30.0 103 ± 4 99 ± 3 101 ± 3 110 ± 3* 30.0 -40.0 100 ± 4 87 ± 4* 98 ± 6 97 ± 5 40.0 -50.0 100 ± 5 81 ± 4** 100 ± 7 92 ± 5 Absolute and relative power of the subset of experiments (n = 7) for which 5 min preinfusion, postvehicle, and post-timolol EEG epochs were collected. Data are expressed as percentages of preinfusion means (±SEM) for ECoG and HEEG. *p < 0.05, **p < 0.01 significantly different from preinfusion mean. Statistical significance determined using a one-way ANOVA followed by Duncan's multiple-range test. 
Fig. 6. Schematic diagram depicting effective and ineffective bilateral timolol infusion sites. Each symbol pair represents the left and right infusion sites for an individual experiment. Infusions placed bilaterally within the general region of MS increased ECoG and HEEG indices of sedation (EFFECTIVE). Cases in which either one infusion or both infusions were placed outside of MS did not alter ECoG/HEEG activity patterns (INEFFECTIVE). See Figure 3 legend for abbreviations. Schematic depicts three levels through MS separated by 250 µm (modified from Swanson, 1992
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PSA of the bilateral MS timolol-induced changes in EEG
PSA was conducted on a subset of animals (n = 7) for which 5 min prevehicle, postvehicle, and post-timolol EEG epochs were collected (see Table 2). EEG segments were taken immediately before vehicle infusions, 15 min after bilateral vehicle infusions, and 15-30 min after bilateral timolol infusions. Post-timolol epochs containing maximum EEG responses were selected on the basis of visual inspection. PSA demonstrated statistically significant effects of bilateral MS timolol infusions, but not vehicle, on ECoG and HEEG activity consistent with the qualitative observations described above. Thus, in ECoG, absolute power of the 0.3-2.3 and 2.3-6.8 Hz frequency bands was significantly increased. Relative power of the 0.3-2.3 Hz band was also increased, whereas relative power of the five highest frequency bands was decreased. In HEEG, absolute power and relative power were increased in the 0.3-2.3 Hz band and decreased in the 2.3-6.8 Hz (theta) frequency band. Absolute power in the 30.0-40.0 and 40.0-50.0 Hz frequency bands was decreased. A small, statistically significant increase in relative power of the 13.0-20.0 and 20.0-30.0 Hz frequency bands was also observed.DBH immunoreactivity in MS
The distribution of noradrenergic fibers in the MS region was examined using immunohistochemical staining for DBH. At the anterior-posterior level at which the largest number of successful infusions were made, the highest concentration of DBH-like immunoreactive (DBH-LI) fibers was contained within the general region of MS (see Fig. 7). Within this area, DBH-LI was contained within moderately long, vertically or diagonally oriented fibers intermingled with shorter, randomly oriented, highly varicose terminal branches. Some vertically oriented thick fibers that ran parallel to the midline exhibited large round varicosities and occasional large terminal boutons. Laterally, toward the edge of the medial septum-diagonal band complex and just beyond, a rich network of highly varicose DBH fibers was observed. At the level shown in Figure 7, a low to moderate density of fibers was seen in the region between the lateral edge of the medial septum and the nucleus accumbens core, probably contained within the shell of the nucleus accumbens. The density of these fibers rapidly diminished anteriorally.
Fig. 7. Dark-field photomicrograph of a coronal section through MS prepared for immunocytochemical localization of DBH. Note the high density of DBH-positive fibers in the vicinity of the medial septum-diagonal band area compared with the much lower density of fibers laterally (compare with Fig. 1 showing a typical infusion site). M, midline. Original magnification, 40×. Scale bar, 0.5 mm.
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At slightly more rostral levels (~0.5 mm rostral to Fig. 7), DBH-LI fibers were even more clearly concentrated within the boundaries of the medial septum and vertical limb of the diagonal band of Broca. The majority of DBH-positive fibers were vertically or diagonally oriented and exhibited occasional round or fusiform varicosities. Intermixed with these were a few highly varicose fibers with no obvious preferred orientation. Moving laterally toward the anterior commissure, only a few DBH-LI fibers were observed. In the anterior nucleus accumbens and the striatum, only rare DBH-LI fibers of passage were observed (data not shown). In more caudal sections, (~0.6 mm caudal to Fig. 7) DBH-LI fibers were more widely distributed. MS exhibited many fine varicose DBH fibers with nearly random orientation, but the density of DBH-LI increased dramatically in the vicinity of the anterior commissure and the bed nucleus of the stria terminalis (data not shown).
EEG effects of atropine
In four experiments, the effects of the cholinergic antagonist atropine (50 mg/kg, i.v.) on MS ISO- and MS glutamate-induced ECoG and HEEG activation were examined. After intravenous saline infusions, ISO and glutamate infusions into MS elicited robust EEG activation. Ten minutes after intravenous atropine administration, neither ISO nor glutamate elicited EEG activation (data not shown). Atropine also blocked tail pinch-induced EEG activation. Thus, as described for urethane-anesthetized rats (Vanderwolf and Robinson, 1981
DISCUSSION
These results indicate that a region of the basal forebrain, situated either within or immediately lateral to MS, is a site at which NE acts to modulate forebrain EEG in the halothane-anesthetized rat through actions atA number of observations suggest that the drug-induced changes in EEG stem from actions at
Site of action
A number of observations indicate that the infusion-induced changes in ECoG and HEEG stem from action of the infused drugs within the general region of MS. First, the relatively small infusion volume (150 nl) imposes a limit on the distance over which physiologically active drug concentrations will be maintained. Second, ISO infusions placed outside the immediate vicinity of MS did not alter EEG, indicating that EEG effects were not attributable to diffusion of drug into the ventricular system or into the core of the nucleus accumbens (Zaborsky et al., 1985) (for review, see Deutch et al., 1993MS, as defined here, is an anatomically complex area containing neurons located within the medial septum, the vertical limb of the diagonal band of Broca, the islands of Calleja, portions of the lateral preoptic area, and the shell region of the nucleus accumbens. Infusions placed as far laterally to the shell region of the nucleus accumbens as effective MS infusions were placed medially did not elicit changes in EEG state. This suggests that the shell region of the accumbens is not the site at which
Potential circuitry underlying bilateral, simultaneous ECoG, and HEEG responses
Both SI and MS receive a dense innervation from LC (see above) and both have been implicated in the regulation of forebrain EEG. SI influences ECoG (Belardetti et al., 1977Noradrenergic receptors located within SI other than
Within the limits of temporal resolution (~1-2 sec), near simultaneous activation of ECoG and HEEG was observed after ISO and glutamate infusions into MS as well as glutamate infusions into SI. A number of possible substrates could support the coordinated activation of cortical and HEEG activity. First, reciprocal projections linking cortex and hippocampus could provide an anatomical substrate through which such coordination could be achieved (Van Hoesen et al., 1972
Cholinergic systems
Cholinergic neurons located within MS have been implicated in the regulation of HEEG theta activity (for review, see Buzsaki et al., 1983In urethane-anesthetized rats, systemic administration of the cholinergic antagonist atropine elicits a profound suppression of EEG activation (Vanderwolf and Robinson, 1981
2-agonists, which act to inhibit LC neuronal activity and NE release (De Sarro et al., 1987
Conclusions
Previous studies demonstrated that selective enhancement of LC discharge rates increases EEG measures of arousal (Berridge and Foote, 1991The anesthetized preparation offers distinct advantages for experimentally addressing the question of whether, and at what site, noradrenergic systems modulate EEG state. This preparation facilitates the following: (1) electrically adequate EEG recordings; (2) stable baseline EEG activity; and (3) performance of small infusions in multiple brain sites using a within-subjects design. However, the use of the anesthetized preparation substantially limits the degree to which inferences can be made concerning modulation of behavioral state. This concern has motivated the studies conducted in the unanesthetized rat, which are described in the accompanying article (Berridge and Foote, 1996
FOOTNOTES
Received May 7, 1996; revised Aug. 14, 1996; accepted Aug. 19, 1996.
This work was supported by Public Health Service Grant MH40008 (S.L.F.) and a grant from the University of Wisconsin Graduate School (C.W.B.).
Correspondence should be addressed to Dr. Craig W. Berridge, Psychology Department, University of Wisconsin, 1202 West Johnson Street, Madison, WI 53706-1611.
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