Dual projections of single cholinergic and aminergic brainstem neurons to the thalamus and basal forebrain in the rat

41

Brain Research, 604 (1993) 41-52 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 18527

Dual projections of single cholinergic and aminergic brainstem neurons to the thalamus and basal forebrain in the rat B r u n o J. L o s i e r a n d K a z u e S e m b a Departments of Psychology, and Anatomy and Neurobiology, Dalhousie University, Halifax, NS (Canada) (Accepted 8 September 1992)

Key words: Basal forebrain; Thalamus; Pedunculopontine tegmental nucleus; Dorsal raphe; Acetylcholine; Serotonin; Immunohistochemistry; Retrograde labeling

Compelling evidence indicates that cholinergic basal forebrain neurons are strongly activated during waking, and concurrently thalamic spindle activity is suppressed and thalamocortical sensory transmission is facilitated. Both thalamus and basal forebrain are known to receive projections from brainstem cholinergic and aminergic neuronal pools that are involved in wake/sleep regulation. The present study addressed the question of whether single cholinergic and aminergic neurons contributed to both of these ascending projections, by using two fluorescent retrograde tracers combined with immunofluorescence. Cholinergic neurons projecting to both the basal forebrain and thalamus were found in the pedunculopontine and laterodorsal tegmental nuclei, representing an average of 8.0% of the total cholinergic cell population in these nuclei. Serotonergic neurons with dual projections were observed in the dorsal, median and caudal linear raphe nuclei, accounting for a mean of 4.7% of total serotonergic neurons in these nuclei. Relatively few noradrenergic neurons (2.0%) in the locus ceruleus projected to both target structures, and a very small subpopulation of histaminergic neurons (1.5%) in the tuberomammillary hypothalamic nucleus had dual projections. Of all brainstem neurons with dual projections, cholinergic and serotonergic neurons accounted for an overwhelming majority, with noradrenergic followed by histaminergic neurons representing the remaining minority. These data suggest that through dual projections, cholinergic and aminergic brainstem neurons can concurrently modulate the activity of neurons in the thalamus and basal forebrain during cortical arousal.

INTRODUCTION The concept of an ascending activating system of reticular origin for cortical arousal was first proposed in 1949 by Moruzzi and Magoun 29, who electrically stimulated the brainstem core and observed the replacement of a high amplitude slow wave pattern by low amplitude fast waves (activation) in the cortical electroencephalogram. Much research has since been directed at an understanding of the role of ascending projections from the brainstem in cortical arousal. In addition, the past decade has witnessed significant advances in the understanding of central cholinergic pathways41, and this, along with more recent functional data regarding the role of acetylcholine in behavioral state regulation, has brought a new perspective. In this view, basal forebrain cholinergic neurons, the major source of cortical acetylcholine, have a critical role in cortical arousal, and they receive excitatory inputs from

certain brainstem structures. Thus, the entire brainstem-basal forebrain system may be considered as a global ascending activation system for cortical arousal 4°. Anatomical data in support of this view include those at light, and, less extensively, at electron microscopic levels6k There is general agreement that the cholinergic basal nuclear complex receives inputs from a number of transmitter-specific cell populations in the brainstem, including cholinergic neurons in the mesopontine tegmentum, noradrenergic neurons in the locus ceruleus, serotonergic neurons in the raphe nuclei, dopaminergic neurons in the midbrain 6'12'15'38'43'56'6°, and histaminergic neurons in the tuberomammillary hypothalamic nucleus 31'57. Some of these have been confirmed to make synaptic contacts with cholinergic neurons 61. Furthermore, many of these transmitterspecific neurons projecting to the basal forebrain have been implicated in the regulation of behavioral state t,11,14,36'37. These anatomical findings have been

Correspondence: K. Semba, Department of Anatomy and Neurobiology, Dalhousie University, Tupper Medical Building, Halifax, NS, B3H 4H7, Canada. Fax: (1) (902) 494-1212.

42 corroborated physiologically using extracellular recordings from basal forebrain neurons combined with brainstem stimulation 43, or iontophoretic application of acetylcholine 22 and dopamine 3°. In addition to the activation of basal forebrain cholinergic neurons, cortical activation is accompanied by blockade of oscillatory activity in the thalamus and facilitation of thalamocortical transmission 51, and there is good evidence that cholinergic and aminergic neurons in the brainstem have a role in regulating neuronal activity in the thalamus. For example, acetylcholine, norepinephrine, serotonin and histamine can block the generation of thalamocortical oscillation and promote a state of excitability that is consistent with cortical arousal 23-27'32. Neurons in the reticular thalamic nucleus are responsible for the generation of thalamocortical spindle activities s°'51, and stimulation of the mesopontine tegmentum inhibits reticular thalamic neurons, an effect which is blocked by atropine s'z°. These physiological findings are consistent with anatomical evidence for cholinergic, noradrenergic, serotonergic, and histaminergic innervation of the thalamus 16. The evidence presented above strongly supports the critical role of basal forebrain in cortical activation that occurs concurrently with strong thalamocortical activation, and suggests that some of the activational inputs to the thalamus and basal forebrain arise in the same cholinergic and aminergic neuronal groups in the brainstem. It is, however, not known whether single neurons in these transmitter-specific cell groups project to both of the forebrain structures. The present study was undertaken to investigate this possibility by using two fluorescent retrograde tracers in combination with immunofluorescence. MATERIALS AND METHODS

Animals, tracer injections, and perfusion Twenty-eight male Wistar rats, 290-410 g in body weight, were used, and 8 of these rats with successful injections contributed to the quantitative data described below. All surgical procedures were performed under anesthesia with sodium pentobarbital (50 m g / k g , i.p.). Three injections were made with fluorogold (FG; Fluorochrome, 4% in saline, 0.01 txl) unilaterally in the thalamus at the following coordinates, according to the brain atlas by Paxinos and Watson34: AP = 3.1 mm, ML=2.6, DV=5.3; AP=2.4, ML=2.0, DV=5.4; AP = 1.6, ML = 1.4, DV = 5.4. These coordinates were chosen to encomlSass a large area of the thalamus, including the ventrolateral, ventral posterolateral, ventral posteromedial and posterior nuclei. Two injections

were made ipsilaterally in the basal forebrain with rhodamine latex beads (Rh, Lumafluor, 0.01 txl) at the following coordinates: AP = 1.4, ML = 2.7, DV = 8.3; AP = 0.3, ML = 2.2, DV = 7.1. These injections were aimed at clusters of cholinergic neurons in the horizontal limb of the diagonal band, the magnocellular preoptic area and the nucleus basalis magnocellularis. Seven to 14 days following tracer injections, the animals were given an overdose of sodium pentobarbital and perfused transcardially with 200 ml of 0.9% saline followed by 500 ml of a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were stored at 4°C in the same fixative for 1-3 h and then placed overnight in 15% sucrose-phosphate buffer for cryoprotection.

Immunohistochemistry Forebrains were sectioned on a freezing microtome at 30/xm, and sections were collected in 5 sets in 0.05 M Tris-buffered saline (TBS). One set of sections was mounted immediately for assessment of injection sites. Upon successful coverage of target structures, a second set of sections containing the basal forebrain was incubated with a monoclonal antibody to choline acetyltransferase (CHAT; Boehringer-Mannheim, 1 : 10), with 0.3% of Triton-X 100 and 2% normal goat serum, for 48 h at 4°C. Incubation with a secondary antibody was conducted as described below. These sections were used to examine the basal forebrain injection sites in relation to clusters of cholinergic neurons. The brainstems were also sectioned on a freezing microtome at 30 txm and collected in 5 sets in 0.05 M TBS for immunohistochemistry. Following a brief rinse in TBS, brainstem sections were incubated in primary antibodies directed to: ChAT (1 : 10), serotonin (5-HT, Incstar; 1:800), tyrosine hydroxylase (TH, East Acres; 1 : 600), and histidine decarboxylase (HDC, kindly provided by Dr. N. Inagaki; 1 : 1,000). All antibodies were raised in rabbit, with the exception of ChAT which was a rat monoclonal antibody. The specificity of the antiChAT 9 and anti-HDC antibodies 57 has previously been documented, and that of the purchased antibodies has been confirmed by the suppliers with Western blot, immunoelectrophoresis, double immunodiffusion, a n d / o r preabsorption. Incubation solutions also contained 0.3% Triton-X 100 and 2% normal goat serum. Following a 48 h incubation at 4°C, sections were rinsed for 3 × 20 min in TBS. Sections were then incubated with secondary antisera raised in goat and directed to either rabbit or rat IgG conjugated to fluorescein isothiocyanate (FITC) at a dilution of 1 : 20 with 2% normal goat serum for 1.5-2 h at room temperature. Following this incubation, sections were

43 rinsed 3 × 20 rain, and mounted on chrome-gelatincoated slides, left to air dry overnight and coverslipped with Fluoromount. Control sections were processed as above but with the omission of primary antibodies, and this resulted in no specific labeling. Microscopic examination and data analysis

Slides were examined using an Olympus BH-2 fluorescence microscope with filter blocks D M U (ultraviolet), D M I B (blue), and D M G (green), for FG, F I T C and Rh, respectively. Eight cases were analyzed for double retrograde labeling or dual projections. To determine the extent and magnitude of t h a l a m i c / F G and basal f o r e b r a i n / R h injections, a total of 36-40 sections from individual cases, mostly at 150/xm intervals, were chosen. Double retrogradely labeled neurons in the brainstem were analyzed from a sample of 22-24 sections at 240 txm intervals from the most rostral aspect of the substantia nigra to the limb of the seventh cranial nerve. Sections containing the tuberomamillary nucleus of the hypothalamus were also collected in a similar manner at 30 txm intervals. In each antigen series, counts were made of neurons that were positive for three markers, including the antigen and both of the two retrograde markers, F G and Rh, ipsilateral to the injection. In addition, antigen-positive neurons that were retrogradely labeled with either FG or Rh but not both were counted within each of the transmitter-specific cell groups containing triple-labeled neurons. Finally, the total number of antigen-positive cells in each transmitter-specific cell group was counted. All these differently labeled neurons were also plotted on matched, standardized sec-

tion drawings from Paxinos and Watson 34. On the basis of the cell counts, percentages were calculated by dividing the number of either single or double retrogradely labeled immunoreactive neurons by the number of total immunoreactive neurons in each of the nuclei containing the transmitter in question. Examining, counting and plotting double- and triple-labeled neurons required a considerable amount of exposure to fluorescence, and this necessarily caused fading of the markers, FITC in particular. As a result, a complete set of data could not usually be collected from individual cases. RESULTS I n j e c t i o n sites

In the 8 cases utilized for the analysis, all injection sites were confirmed to be within the boundaries of either the basal forebrain or the thalamic nuclei. FG injections in the thalamus were localized between level - 2 . 1 to - 4 . 3 mm from bregma according to the brain atlas by Paxinos and Watson 34 (Fig. 1A). These injections included the ventrolateral, ventral posterolateral, ventral posteromedial and posterior nuclei within the core of injections, and surrounding thalamic structures in the halo. In none of the cases analyzed did the injection invade the contralateral thalamus. In a control experiment the extent and magnitude of the spread of F G in the thalamus were analyzed after a 2-h survival following the same injection protocol. All these injections were confined within the thalamus and the extent of the spread was similar to that after 7-14 days survival.

TABLE I Percentages of cholinergic and aminergic neurons projecting to the thalamus (FG + ) and basal forebrain (Rh + ) in the brainstem

Mean +_S.D.; the number of cases indicated in parentheses Number of immunoreactive cells counted

%FG + Rh +

%FG + *

%Rh + *

Acetylcholine (CHAT)

Pedunculopontine tegmental nucleus Laterodorsal tegmental nucleus Combined

120-185 (5) 80-140 (5) 195-325 (5)

9.0_+ 6.1 (5) 6.8 +_ 2.8 (5) 8.0 _+ 3.9 (5)

33.9+_19.6 (5) 28.6 +_14.5 (5) 31.4 + 15.1 (5)

19.6_+1.2 (2) 12.3_+7.4 (3) 20.9 +_9.5 (2)

80-235 (4) 570-860 (2) 320-860 (5) 805-990 (2)

2.6 +_ 1.9 (4) 11.1 +_13.3 (2) 2.8_+ 2.7 (5) 4.7 +_ 4.9 (2)

8.2 +_ 8.8 (3) 3.7 _+ 0.0 (1) 7.1 _+ 5.0 (5) 3.3 +_ 0.0 (1)

8.9 _+5.4 (2) 4.4 +_0.0 (1) 4.1 +_2.9 (3) 4.3 _+0.0 (1)

70-150 (3)

2.0+_ 0.6 (3)

9.3+_ 6.4 (3)

2.8_+0.0 (1)

100 (2)

1.5 +_ 0.7 (2)

1.5 _+ 0.7 (2)

1.5 -t-_0.7 (2)

Serotonin

Caudal linear raphe nucleus Median raphe nucleus Dorsal raphe nucleus Combined Noradrenaline (TH)

Locus eeruleus Histamine (HDC)

Tuberomammillary nucleus * Including triple labeled (FG + Rh +) neurons.

44 Rh injections in the basal forebrain were also similar across all 8 cases analyzed, and were localized from level - 0 . 8 to - 2 . 8 from bregma (Fig. 1B). The Rh deposit, generally smaller than that with FG, was localized to the horizontal limb of the diagonal band, magnocellular preoptic area, substantia innominata, and nucleus basalis magnocellularis (identified by ChAT staining) and immediate surrounding areas. Cases in which a spill of the tracer was observed along the micropipette tract were eliminated from the analyses. Triple-labeled neurons Double retrogradely labeled neurons were found in a number of neurotransmitter-specific nuclei in the brainstem. E x a m p l e s of CHAT- and 5 - H T - i m munoreactive neurons that were labeled with both retrograde tracers are shown in Fig. 2. Double retrogradely labeled neurons which were not immunoreactive for C h A T were observed within the boundaries of ChAT-positive cell populations in the brainstem. However, their occurrences were rare and further analysis was not warranted. No double retrogradely labeled immunonegative neurons were observed within the other transmitter cell groups examined. Neurons containing both retrograde markers made up a mean of 8.0%, and up to 13.5% of the entire ChAT-positive neuronal population in the mesopontine tegmentum (Table I). The mean percentages for the pedunculopontine and laterodorsal tegmental nuclei were similar, 9.0 and 6.8%, respectively ( P > 0.1, t-test). These triple-labeled neurons were distributed in a uniform manner within each of these nuclei (Fig. 3 C - G ) . The pedunculopontine tegmental nucleus, however, contained more of these triple-labeled neurons since there were more ChAT-positive neurons in that nucleus than the laterodorsal tegmental nucleus (Table I). ChAT-immunoreactive neurons accounted for a significant portion (30-70%) of the total population of immunoreactive neurons labeled with both retrograde markers (Table II). Double retrogradely labeled 5-HT-positive neurons were seen in the dorsal, median, and caudal linear raphe nuclei, representing a mean of 2.8%, 11.1%, and 2.6%, respectively, of the total 5-HT-immunoreactive cell population in each nucleus (Table I). Combined, a mean of 4.7% and up to 8.1% of the 5-HT-im-

munoreactive neurons in these raphe nuclei were labeled with both retrograde tracers, and these neurons were distributed more or less evenly in each of the three raphe nuclei (Fig. 3 B - G ) . These triple-labeled neurons accounted for 2 0 - 6 5 % of the total population of triple labeled neurons (Table II). Neurons positive for both F G and Rh comprised a m e a n of 2.0% and up to 2.7% of the total TH-immunoreactive neuronal population in the locus ceruleus (Table I, Fig. 3G,H). Only a mean of 1.5% and up to 2.0% of HDC-positive neurons in the tuberomammillary hypothalamic nucleus were labeled with both F G and Rh (Table I), and these were found predominantly in the lateral subnucleus of the tuberomammillary hypothalamic nucleus (Fig. 3A). Double retrogradely labeled TH- and HDC-positive neurons accounted for only small proportions of total triple-labeled neurons, with less than 10% represented by TH-positive neurons in the locus ceruleus, and less than 5% accounted for by HDC-immunoreactive neurons in the t u b e r o m a m millary nucleus (Table II). Single retrogradely labeled neurons In addition to dually projecting neurons, the pedunculopontine and laterodorsal tegmental nuclei contained neurons that were retrogradely labeled with either F G or Rh alone. The percentages of these neurons were similar in the pedunculopontine and laterodorsal tegmental nuclei. Thus, combined with triple-labeled neurons, FG-labeled neurons accounted for a mean of 31.4% of the total population of CHATpositive neurons in the mesopontine tegmentum, whereas Rh-labeled neurons represented a m e a n of 20.9% (Table I). 5-HT-immunoreactive neurons retrogradely labeled from either basal forebrain or thalamus were found in the dorsal and median raphe nuclei, as well as in the caudal linear raphe nucleus. Because the present study was focused on neurons with dual projections, numerical analysis of single retrogradely labeled neurons was restricted to these raphe nuclei which contained triple-labeled neurons. Combined with triple-labeled neurons, neurons retrogradely labeled with F G from the thalamus comprised a m e a n of 8.2%, 3.7%, and 7.1% of 5HT-positive neurons in the caudal linear, the median, and the dorsal raphe nuclei, respectively,

Fig. 1. Representative examples (case 10) of FG injections in the thalamus (A) and Rh latex beads injections in the basal forebrain (B). The core of each injection is indicated by stippling. In B, immunohistochemicallyidentified cholinergic neurons in the same sections are shown with large dots (one dot = one cell). The number to the right of each drawing indicates the distance in mm from bregma according to the brain atlas by Paxinos and Watson341 3, third ventricle; ac, anterior commissure; CP, caudate putamen; f, fornix; ic, internal capsule; ot, optic tract; PO, posterior thalamic nucleus; Rt, reticular thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus.

45

A

B.....~.%.

0.3

~1_.

0.8

-0.8

_1.3

~~0,, \ /.-2.1

•. •

i

6"

I .-1.3

-2.8

, -3.6

-4.3

-2.3

.8

46

47 whereas Rh-labeled neurons made up 8.9%, 4.4%, and 4.1%, respectively, of the 5-HT-positive cell population in each nucleus (Table I). FG- and Rh-labeled neurons represented a mean of 3.3% and 4.3%, respectively, of the total 5-HT-positive cells in these three raphe nuclei combined.

Single retrogradely labeled TH-positive (presumably noradrenergic) neurons were seen in the locus ceruleus. Combined with triple-labeled neurons, FG-labeled neurons made up a mean of 9.3% of TH-immunoreactive neurons, whereas 2.8% were Rh-retrogradely labeled (Table I). TH-immunoreactive neurons retro-

A

D

B

IC

0.7

C

PPT~ / \ \

MaR <~!b C:>

~[ /

Fig. 3. Distribution of triple-labeled neurons in a representative case (case 10). 3, oculomotor nucleus; 4, trochlear nucleus; 5, motor trigeminal nucleus; A5, A5 noradrenergic cell group; A7, A7 noradrenergic cell group; B9, B9 serotonergic cell group; CG, central gray; cp, cerebral peduncle; CR, caudal linear raphe nucleus; DR, dorsal raphe nucleus; DT, dorsal tegmental nucleus; fr,. fasciculus retroflexus; IC inferior colliculus; IMLF, interstitial nucleus of the medial longitudinal fasciculus; LC, locus ceruleus; LDT, laterodorsal tegmental nucleus; MG, medial geniculate nucleus; MHb, medial habenula; ml, medial lemniscus; mlf, medial longitudinal fasciculus; MnR, medial raphe nucleus; mt, mammillothalamic tract; PB, parabrachial nucleus; PnC, pontine reticular nucleus, caudal; PPT pedunculopontine tegmental nucleus; Pr5, principal sensory trigeminal nucleus; py, pyramidal tract; RMg, raphe magnus; RPn, nucleus raphe pontis; SC, superior colliculus; scp, superior cerebellar peduncle; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; SO, superior olive; STh, subthalamic nucleus; TM, tuberomammillary hypothalamic nucleus; xscp, decussation of superior cerebellar peduncle.

Fig. 2. Examples of triple-labeled neurons. A neuron in the pedunculopontine tegmental nucleus (arrow) is retrogradely labeled with F G from the thalamus (A) and with Rh from the basal forebrain (B), and is also immunoreactive for ChAT (C). A neuron in the dorsal raphe nucleus (arrow) was retrogradely labeled with FG (D) and Rh (E), and was serotonin immunoreactive (F). A neuron indicated by an arrowhead in (D) was immunoreactive for serotonin (F), but not retrogradely labeled from the basal forebrain (E). Scale bar = 100/xm.

48

F

t

A7

J

0

.

•

CHAT+

FG+

m

CHAT+

Rh+

•

CHAT+

FG+ Rh+

®

5HT+

FG+

tD

5HT +

Rh +

•

5HT+

FG+

,o,

TH +

FG +

•

TH+

FG+

Rh+

*

HDC+

FG+

Rh+

Rh+

8

Fig. 3 (continued).

gradely labeled with Rh from the basal forebrain were also seen in the ventral midbrain, in particular, in the ventral tegmental area, as well as in the ventrolateral tegmentum (A5). The tuberomammillary hypothalamic nucleus contained a number of HDC-immunoreactive neurons with both F G and Rh, but no HDC-positive neurons with only one retrograde marker were observed (Table I). DISCUSSION The principal findings of the present study are as follows. Subpopulations of cholinergic (mean of 8.0%) and aminergic (mean of 1.5-4.7%) neurons in the brainstem had branching axons which innervated both the basal forebrain and thalamus. These neurons were located in the pedunculopontine and laterodorsal tegmental nuclei (cholinergic), the dorsal, median, and

caudal linear raphe nuclei (serotonergic), the locus ceruleus (noradrenergic), and the tuberomammillary hypothalamic nucleus (histaminergic). O f these, cholinergic and serotonergic neurons accounted for the overwhelming majority, whereas noradrenergic neurons represented a small fraction, and histaminergic neurons accounted for a still smaller portion of all dually projecting neurons. It should be noted that the number of dually projecting neurons most likely represented an underestimation, since neither the thalamus nor the basal forebrain was completely filled by the retrograde tracers. We have found that an average of 8% of cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei have collateralizing axons innervating both the thalamus and basal forebrain. Woolf and Butcher 6° reported that approximately 10% of cholinergic neurons were retrogradely labeled from restricted

49 TABLE II Breakdown by different brainstem regions of cholinergic and aminergic neurons projecting to both the thalamus and basal forebrain in two representatiue cases

The total number of double retrogradely labeled neurons was 140 for case 10 and 32 for case 12. Values are given as a % of the total number of double-labeled neurons Case 10

Case 12

24.3

37.5

7.1 31.4

31.3 68.8

1.4 17.1 45.7 64.3

6.3 - * 15.6 21.9

2.9

6.3

1.4

3.1

Acetylcholine (CHAT)

Pedunculopontine tegmental nucleus Laterodorsal tegmental nucleus Total Serotonin (5 HT)

Caudal linear raphe nucleus Median raphe nucleus Dorsal raphe nucleus Total Noradrenaline (TH)

Locus ceruleus Histamine (HDC)

Tuberomammillarynucleus * The count was lost due to fading.

regions of the thalamus and the basal forebrain, namely, the anterior thalamic area and the medial septumvertical limb of the diagonal band. Mesopontine tegmental cholinergic neurons also innervate both the cerebral cortex and the reticular thalamic nucleus 17. Neurons in the laterodorsal tegmental nucleus project to both the olfactory bulb and the mediodorsal nucleus of the thalamus, although the cholinergic nature of this projection was not confirmed 7. Collateralization has also been seen with thalamic projections and descending projections to the pontine reticular formation, in up to 20% of the cholinergic cell population 42. It remains to be investigated whether single cholinergic neurons in the mesopontine tegmentum give rise to projections to more than two structures in the brain, which is not an unlikely possibility considering a role of these neurons in behavioral state regulation 44'52. The present data confirmed the thalamic innervation by mesopontine tegmental cholinergic neurons, and these are in general agreement with previous findings using a single retrograde marker and immunohistochemistry 17'33'38'45'46'53'6°. Quantitatively, an average of over 30% of mesopontine tegmental cholinergic neurons were retrogradely labeled from the thalamus, consistent with a previous report also with the retrograde tracer, F G 42. Compared with the thalamic projection, fewer cholinergic neurons, an average of 20.9%, projected to the cholinergic basal forebrain complex. This cholinergic projection has previously been described 38'43'6°. Quantitatively, Jones and Cuello 15 have

reported that 5 - 1 5 % of mesopontine tegmental cholinergic neurons were retrogradely labeled following a single injection with wheatgerm agglutinin-conjugated horseradish peroxidase in the substantia i n n o m i n a t a nucleus basalis magnocellularis. The present and these data suggest that single cholinergic neurons innervate part, but not the entire extent, of the cholinergic basal nuclear complex. This is consistent with topographic projections of cholinergic neurons in the mesopontine tegmentum to the basal forebrain 43. The proportion of serotonergic neurons projecting to both the basal forebrain and thalamus was relatively small (4.7%). However, these dually projecting serotonergic neurons represented a large portion of the dually projecting brainstem neurons, ranking roughly on a par with cholinergic dually projecting neurons, and greatly exceeding those dually projecting neurons that were catecholaminergic or histaminergic. The presence of serotonin neurons projecting to both the thalamus and basal forebrain adds to the list of other known examples of divergently projecting serotonin neurons 13. Immunohistochemical studies have indicated that both the basal forebrain and the thalamus contain dense 5-HT-immunoreactive neuropi147. Despite this rich serotonergic innervation, however, we have found that relatively few brainstem serotonergic neurons contributed afferents to the basal forebrain, the thalamus or both. This paradox may be explained by the fact that tracer injections did not cover the entire structures, and also by the likelihood that each of these serotonergic neurons has extensive terminal fields, contributing to the apparently dense neuropil. In the present study, serotonergic neurons projecting to the basal forebrain were seen in the rostral serotonergic cell groups, including the dorsal and median raphe nuclei as well as the caudal linear raphe nucleus. The projections from the dorsal and median raphe nuclei are consistent with early reports using autoradiography on projections from the median and dorsal raphe to the diagonal band region in rat 2 and cat 4, as well as more recent observations based on retrograde tracing and immunohistochemistry on projections of serotonergic neurons in the rostral group to both rostral and caudal parts of the cholinergic basal nuclear complex in the rat 6A5'43'56. We have shown that a small percentage of noradrenergic neurons in the locus ceruleus projects to both the basal forebrain and the thalamus, and that these dually projecting neurons comprise less than 10% of all aminergic and cholinergic cells projecting to both structures. These findings corroborate previous findings of the projections of locus ceruleus neurons to either the basal forebrain 15'43 or the thalamus 16. Quan-

50 titatively, 38% of noradrenergic neurons has been reported to project to the basal forebrain 15. In contrast, our data, although based on only one case, indicate much lower incidences, about 2.8%. The reason for this discrepancy is not clear. In addition to the locus ceruleus, noradrenergic neurons in the A5 group in the ventrolateral tegmenturn have been reported to project to the basal forebrain aS, and this was also confirmed in the present study. Single noradrenergic neurons in the A5 group, however, did not appear to project to both basal forebrain and thalamus. A small number of histaminergic neurons was found to project to both the thalamus and basal forebrain. This corroborates and extends previous findings on the histaminergic systems in the brain. Histaminergic neurons are located in the tuberomammillary nucleus of the hypothalamus, which is the sole source of neuronal histamine in the brain 39'48'58. These neurons have widespread projections in the brain including all areas of the cerebral cortex, the septum and the thalamus 48'58, and affect a wide variety of activities including brain metabolism and energy, locomotor activity and arousal states39, 58.

Functional significance Axonal bifurcation or collateralization of single neurons is not a rare occurrence in the central nervous system. Previously reported incidences may be classified into two types on the basis of organization 3'21'35. One type of bifurcating projection has both divergent ascending and descending projections with ipsi- a n d / o r contralateral connections. The second type has a unilateral projection in either an ascending or a descending direction, and with multiple destinations at different levels. In either type, the information conveyed by a neuron with a branching axon would reach a larger territory than its non-bifurcating, non-collateralizing counterpart. Such arrangements would be more expedient and efficacious than two separate neurons transmitting identical information. However, when separate sets of information need to be conveyed to separate targets, neurons with bifurcating axons cannot fulfill 'this task. Thus, neurons with branching axons may be considered to serve in global transmission, whereas singly projecting cells may be involved in more focused information transmission. In this context, there are two considerations which might be relevant in understanding the significance of concurrent innervation of the basal forebrain and the thalamus by brainstem cholinergic and aminergic neurons. The first point is that the activation of collateralizing axons may not result in the same direct post-syn-

aptic effects at both sites of termination, since postsynaptic effects depend on the type of receptors present on target cells. For example, acetylcholine excites thalamocortical relay neurons 25, whereas it inhibits or hyperpolarizes reticular thalamic neurons 24. Functionally, however, because the inhibition of reticular thalamic neurons would result in blockade Of spindles which impede thalamocortical transmission, these two different actions achieve the same goal of cortical activation. In the basal forebrain, both excitatory and inhibitory actions of cholinergic agonists have been reported 18'22. Thus, it is possible that the activation of cholinergic neurons with collateralizing axons results in different post-synaptic effects in various target structures or cell populations, while ultimately leading to a common end, the activation of cerebral cortex. A second consideration relates to the firing patterns of cholinergic and aminergic neurons in the brainstem. Both noradrenergic locus ceruleus neurons and serotonergic raphe neurons have clock-Jike firing patterns, with their firing rates depending on the behavioral state 1,28'37'54. This regular firing pattern is determined by the constellation of ionic currents present, respectively, in these two groups of neurons 5'59. One implication of the regular firing pattern is that individual neurons might display little variability in their firing rates in a given behavioral state. This suggests that all noradrenergic and serotonergic neurons, respectively, may convey more or less identical information to the basal forebrain and thalamus, regardless of whether or not they innervate both structures. Furthermore, there is some evidence that the functional consequence of activation of serotonergic.input to thalamocortical relay neurons is similar to that of noradrenergic input although the two transmitters modulate both common and separate ionic currents present in these neurons 23'26'32. Thus, the dual projection of the two groups of monoaminergic neurons may be viewed as a redundant system to ensure inforhaation transmission by these monoaminergic neuronal groups. In contrast, it is not clear if a similar situation exists with respect to cholinergic or histaminergic neurons. Chronic single unit studies have reported that many neurons in the laterodorsal and pedunculopontine tegmental nuclei are more active during waking and R E M sleep than during slow wave sleep, whereas some neurons have their highest firing rate during R E M sleep and the lowest during wakillg 1°'19'49. These neurons display either phasic or tonic discharge patterns. At present, it is not known which type or types of neuron are cholinergie. Similarly, two types of neuron have been reported to be present in the posterior hypothalamus, including the region containing his-

51 taminergic neurons: one type is active during both waking and REM sleep, whereas a second type is active during waking only 55. In view of these heterogeneities and the remaining question concerning the identification of firing patterns of cholinergic and histaminergic neurons, it remains to be seen whether the information conveyed by those cholinergic or histaminergic neurons that innervate the two forebrain targets is similar to that conveyed by those neurons with a single target structure. It remains possible that the functional significance of axonal collateralization is different between noradrenergic and serotonergic compared with histaminergic and cholinergic cell groups.

ABBREVIATIONS ChAT FG FITC HDC 5-HT PB REM Rh TBS TH

choline acetyltransferase Fluorogold fiuorescein isothiocyanate histidine decarboxylase 5-hydroxytryptamine (serotonin) phosphate buffer rapid eye movement rhodamine latex beads Tris-buffered saline tyrosine hydroxylase

Acknowledgements. The authors thank Dr. N. Inagaki for a generous gift of the antisera histidine decarboxylase, Dr. Doug Rasmusson for a critical reading of an early version of the manuscript, and Ms. Janette Nason and Ms. Joan Burns for excellent technical assistance. The work was supported by the Medical Research Council, Alzheimer Society, and Scottish Rite Charitable Foundation of Canada. B.J.L. is a recipient of a Medical Research Council Studentship Award, and K.S. is an MRC Scholar.

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