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Studies on the collateralization of some basal forebrain and mesopontine tegmental projection systems… Jourdain, Anne 1988

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STUDIES ON THE COLLATERALIZATION OF SOME BASAL FOREBRAIN AND MESOPONTINE TEGMENTAL PROJECTION SYSTEMS IN THE RAT By Anne J o u r d a i n B.A., Concordia U n i v e r s i t y , 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES GRADUATE PROGRAM IN NEUROSCIENCE We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1988 ® Anne J o u r d a i n , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Neuroscience The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Many basal forebrain and mesopontine tegmental cholinergic projection systems tend to overlap i n t h e i r o r i g i n s . This raises the p o s s i b i l i t y that these projection systems are c o l l a t e r a l i z e d to innervate divergent areas. In experiment one, the degree to which basal forebrain and mesopontine tegmental neurons that innervate the r e t i c u l a r thalamic nucleus have axons that c o l l a t e r a l i z e to innervate the cortex as well was examined with a retrograde fluorescence l a b e l i n g method combined with immunohistochemistry. A s i g n i f i c a n t portion of the labeled neurons i n the region of the nucleus basalis magnocellularis and pedunculopontine tegmental nucleus projecting to the r e t i c u l a r thalamic nucleus were observed to be also labeled (double-labeled) following i n t r a c o r t i c a l tracer i n j e c t i o n s . Many of these double-labeled neurons displayed choline acetyltransferase choline acetyltransferase immunoreactivity. I t was also shown that numerous basal forebrain neurons that innervated the r e t i c u l a r thalamic nucleus contained the calcium-binding protein, parvalbumin. These neurons tended to be located more r o s t r a l l y than the ChAT immunoreactive neurons; primarily i n the region of the ventral pallidum. There was some in d i c a t i o n that parvalbumin-containing neurons i n the basal forebrain that innervate the r e t i c u l a r thalamic nucleus also have axons that branch to innervate the cortex. F i n a l l y , none of the basal forebrain neurons innervating the r e t i c u l a r thalamic nucleus was found to contain somatostatin. In experiment two, the degree to which basal forebrain neurons have axons that c o l l a t e r a l i z e to innervate the interpeduncular nucleus and hippocampus was examined with retrograde fluorescence labeling methods. Labeled neurons projecting to both of these limbic structures were observed only occasionally. Comparison of the d i s t r i b u t i o n of single labeled neurons innervating each of these structures revealed that within the region of o r i g i n , i n the horizontal limb of the diagonal band, neurons innervating the interpeduncular nucleus tended to be located d o r s a l l y to those innervating the hippocampus. The r e s u l t s of these experiments are discussed i n r e l a t i o n to t h e i r anatomical and functional implications toward a greater understanding of the basal forebrain and mesopontine cholinergic and non-cholinergic projection systems. i v Table of Contents GENERAL INTRODUCTION 1 EXPERIMENT ONE 12 I n t r o d u c t i o n 12 Methods 16 Animals 16 S t e r e o t a x i c S u r g i c a l Procedures 19 F l u o r e s c e n t T r a c e r H i s t o l o g y and M i c r o s c o p i c Examination 23 Immunohistochemical Procedures 26 R e s u l t s 28 I n j e c t i o n S i t e s 28 D i s t r i b u t i o n of S i n g l e - L a b e l e d C e l l s 29 D i s t r i b u t i o n of Double-Labeled C e l l s 43 Immunohistochemical Analyses 54 D i s c u s s i o n 68 T e c h n i c a l C o n s i d e r a t i o n s 6 C o l l a t e r a l i z e d P r o j e c t i o n s I n n e r v a t i n g the RTN and Cortex 73 Co n c l u s i o n s 76 EXPERIMENT TWO 78 I n t r o d u c t i o n 78 Methods 82 V R e s u l t s 83 I n j e c t i o n S i t e s 83 P a t t e r n of Retrograde L a b e l i n g 86 D i s c u s s i o n 90 GENERAL DISCUSSION 92 Anatomical C o n s i d e r a t i o n s 92 F u n c t i o n a l C o n s i d e r a t i o n s 93 REFERENCES 96 L i s t of T a b l e s v i Table 1. S t e r e o t a x i c Coordinates of C o r t i c a l I n j e c t i o n s 22 Table 2. Summary of the D i s t r i b u t i o n of S i n g l y and Doubly Labeled Neurons i n the B a s a l F o r e b r a i n and Mesopontine Tegmentum 48 v i i L i s t of F i g u r e s F i g u r e 1. Schematic I l l u s t r a t i o n of H y p o t h e t i c a l B a s a l F o r e b r a i n and Mesopontine Tegmental P r o j e c t i o n s C o l l a t e r a l i z i n g t o Innervate the R e t i c u l a r Thalamic Nucleus and Cortex 15 F i g u r e 2. Schematic I l l u s t r a t i o n of the I n j e c t i o n P r o t o c o l s 18 F i g u r e 3. Three-Dimensional R e c o n s t r u c t i o n of a T y p i c a l I n j e c t i o n S i t e i n the R e t i c u l a r Thalamic Nucleus 31 F i g u r e 4. Photomicrographs I l l u s t r a t i n g I n j e c t i o n S i t e s i n the R e t i c u l a r Thalamic Nucleus 33 F i g u r e 5. Photomicrographs I l l u s t r a t i n g the D i s t r i b u t i o n of Labeled Neurons i n The Nucleus B a s a l i s M a g n o c e l l u l a r i s t h a t Innervate the R e t i c u l a r Thalamic Nucleus and Cortex 38 F i g u r e 6. Photomicrographs I l l u s t r a t i n g the M o r p h o l o g i c a l C h a r a c t e r i s t i c s of B a s a l F o r e b r a i n Neurons R e t r o g r a d e l y Labeled F o l l o w i n g I n j e c t i o n i n the R e t i c u l a r Thalamic Nucleus 41 F i g u r e 7. R e p r e s e n t a t i v e D i s t r i b u t i o n of Labeled Neurons Through S i x Coronal Planes of the B a s a l F o r e b r a i n F o l l o w i n g I n j e c t i o n i n the R e t i c u l a r Thalamic Nucleus and Cortex 45 F i g u r e 8. R e p r e s e n t a t i v e D i s t r i b u t i o n of Labeled Neurons Through S i x Coronal Planes of the Mesopontine Tegmentum F o l l o w i n g I n j e c t i o n i n the R e t i c u l a r Thalamic Nucleus and Cortex 51 F i g u r e 9. Photomicrographs of Double-Labeled Neurons i n the Nucleus B a s a l i s M a g n o c e l l u l a r i s and i n the Pedunculopontine Tegmental Nucleus 53 F i g u r e 10. C o l o r Photomicrograph of a Double-Labeled Neuron i n the Nucleus B a s a l i s M a g n o c e l l u l a r i s 56 F i g u r e 11 C o l o r Photomicrograph of a Double-Labeled Neuron i n the Pedunculopontine Tegmental Nucleus 58 F i g u r e 12 Photomicrographs I l l u s t r a t i n g a S i n g l e L a b e l e d Neuron t h a t Innervate the R e t i c u l a r Thalamic Nucleus and Cortex t h a t was a l s o C hAT-Positive 61 i x F i g u r e 13. Photomicrographs I l l u s t r a t i n g Labeled Neurons t h a t Innervate the R e t i c u l a r Thalamic Nucleus t h a t were a l s o Immunoreactive w i t h Parvalbumin 64 F i g u r e 14. Photomicrographs I l l u s t r a t i n g a Parvalbumin-Immunoreactive Neuron t h a t Innervate both the R e t i c u l a r Thalamic Nucleus and Cortex 66 F i g u r e 15. Schematic I l l u s t r a t i o n of a H y p o t h e t i c a l B a s a l F o r e b r a i n P r o j e c t i o n t h a t Innervate both the Inte r p e d u n c u l a r Nucleus and the Hippocampus 81 F i g u r e 16. Photomicrograph I l l u s t r a t i n g a T y p i c a l I n j e c t i o n S i t e i n the Int e r p e d u n c u l a r Nucleus 85 F i g u r e 17. R e p r e s e n t a t i v e D i s t r i b u t i o n of Labeled Neurons Through Three Coronal Planes of the B a s a l F o r e b r a i n F o l l o w i n g I n j e c t i o n s i n the Interpeduncular Nucleus and Hippocampus • 89 X Acknowledgements Dr. H.C. F i b i g e r s u p e r v i s e d t h i s t h e s i s . I am g r a t e f u l t o him f o r p r o v i d i n g me w i t h t h e o p p o r t u n i t y o f w o r k i n g i n h i s l a b . I am i n d e b t e d t o Dr. S.R. V i n c e n t and Dr. K.B. B a i m b r i d g e who have g e n e r o u s l y a l l o w e d me t o use t h e i r a n t i b o d i e s and m i c r o s c o p e and who have g i v e n me p r e c i o u s a d v i c e s . To Kazue, I w i l l always be g r a t e f u l f o r h e r p a t i e n c e i n t e a c h i n g me t h e a r t o f neuroanatomy. I w i s h her w e l l i n a f u t u r e endeavors. Chui-Se, P a u l , Maureen and L o r i have been s p e c i a l f r i e n d s , m e r c i f o r e v e r y t h i n g . To t h e o t h e r members o f t h e Kinsmen's Lab, thank you a l l , and a s p e c i a l m e r c i t o P e t e r f o r h i s c o n t a g i o u s e n t h u s i a s m . S i ce n ' a v a i t e t e de l ' a i d e e t s u p p o r t de ma f a m i l l e , c e t t e t h e s e n ' a u r a i t j a m a i s vu j o u r . M e r c i du f o n d du c o e u r . A A l a i n , pour t o u t , pour a v o i r c r u , pour a v o i r r e v e , pour a v o i r gagne...par-dessus t o u t . A nous deux. 1 GENERAL INTRODUCTION Two r e l a t i v e l y r e c e n t developments have been p i v o t a l i n g e n e r a t i n g the impetus f o r a d e t a i l e d a n a l y s i s of the anatomical d i s t r i b u t i o n and o r g a n i z a t i o n of c e n t r a l c h o l i n e r g i c neurons. The f i r s t i s the development of r e l i a b l e markers t h a t a l l o w d i r e c t v i s u a l i z a t i o n of c e n t r a l c h o l i n e r g i c neurons. Immunohistochemical methods f o r c h o l i n e a c e t y l t r a n s f e r a s e (ChAT), the s y n t h e t i c enzyme f o r a c e t y l c h o l i n e ( C o z z a r i and Hartman, 1980; Levey e t a l . , 1981; E c k e n s t e i n and Thoenen, 1982), t o g e t h e r w i t h a pharmacohistochemical procedure f o r the i d e n t i f i c a t i o n of a c e t y l c h o l i n e s t e r a s e (AChE), the d e g r a d a t i v e enzyme f o r a c e t y l c h o l i n e (Satoh e t a l . , 1983; Mesulam e t a l . , 1983), are c u r r e n t l y the most r e l i a b l e methods f o r the v i s u a l i z a t i o n of c e n t r a l c h o l i n e r g i c neurons (see F i b i g e r , 1982). The p r o g r e s s generated by the development of these methods i s i l l u s t r a t e d by the l a r g e number of r e c e n t c h o l i n e r g i c mapping s t u d i e s i n the mammalian b r a i n t h a t f o l l o w e d (Kimura e t a l . , 1981; Satoh and F i b i g e r , 1985a; 1985b; Satoh e t a l . , 1983; Ichikawa and H i r a t a , 1986; Houser e t a l . , 1983; Mesulam e t a l . , 1983; V i n c e n t and Re i n e r , 1987; Wainer e t a l . , 1984; McGeer e t a l . , 1984). The second development p e r t a i n s t o the body of evidence i m p l i c a t i n g a c e t y l c h o l i n e i n a wide v a r i e t y of behaviours i n c l u d i n g l e a r n i n g and memory (Bartus e t a l . , 1987). The 2 f u n c t i o n a l s i g n i f i c a n c e of c h o l i n e r g i c mechanisms, as p a r t i c u l a r l y i l l u s t r a t e d by t h e i r proposed involvement i n the pathogenesis of Alzheimer's d i s e a s e (Coyle e t a l . , 1983; McGeer e t a l . , 1984), i l l u s t r a t e s the need f o r a comprehensive understanding of the anatomy of c h o l i n e r g i c systems i n the mammalian b r a i n . Although t h e r e are s t i l l u n r e s o l v e d q u e s t i o n s , knowledge on the d i s t r i b u t i o n s and p r o j e c t i o n s of major c h o l i n e r g i c n u c l e a r groups i n the mammalian b r a i n has reached a g e n e r a l l e v e l of consensus. C h o l i n e r g i c complexes c o n t a i n e d i n the b a s a l f o r e b r a i n on the one hand, and those c o n t a i n e d i n the mesopontine tegmentum on the other hand, are two p o p u l a t i o n s of c h o l i n e r g i c neurons about which reasonable consensus has been achieved over the l a s t decade. The v e n t r a l p o r t i o n of the t e l e n c e p h a l o n , or b a s a l f o r e b r a i n , c o n t a i n s an important p o p u l a t i o n of c h o l i n e r g i c neurons t h a t extends from the l e v e l of the o l f a c t o r y t u b e r c l e r o s t r a l l y t o the l e v e l of the subthalamic nucleus c a u d a l l y ( F i b i g e r , 1982), c o i n c i d i n g w i t h the r o s t r a l p o l e of the medial septum and the caudal end of the globus p a l l i d u s r e s p e c t i v e l y , as d e s c r i b e d i n the c a t b r a i n (Vincent and Reiner, 1987). The d i s t r i b u t i o n of c h o l i n e r g i c neurons through the b a s a l f o r e b r a i n encompasses v a r i o u s r e g i o n s , i n c l u d i n g the medial septum and the v e r t i c a l and h o r i z o n t a l limbs of the d i a g o n a l band r o s t r a l l y ; more c a u d a l l y , c h o l i n e r g i c neurons have been a s s o c i a t e d w i t h the m a g n o c e l l u l a r p r e o p t i c area, v e n t r a l p a l l i d u m , s u b s t a n t i a 3 innominata, nucleus b a s a l i s m a g n o c e l l u l a r i s and the nucleus of the ansa l e n t i c u l a r i s (Wainer e t a l . , 1984; V i n c e n t e t a l . , 1986). An important concept i n understanding the d i s t r i b u t i o n and o r g a n i z a t i o n of c h o l i n e r g i c neurons w i t h i n the b a s a l f o r e b r a i n p e r t a i n s to t h e i r proposed columnar arrangement (Satoh e t a l . , 1983; Wainer e t a l . , 1984) which has r e c e n t l y r e c e i v e d e m p i r i c a l support w i t h t h r e e - d i m e n s i o n a l r e c o n s t r u c t i o n s u s i n g a d i g i t a l microscopy system (Schwaber e t a l . , 1987). Such a columnar o r g a n i z a t i o n i m p l i e s t h a t c h o l i n e r g i c neurons are d i s t r i b u t e d along a continuum through the r o s t r o - c a u d a l extent of the b a s a l f o r e b r a i n , and as such, the term " r o s t r a l c h o l i n e r g i c column of the b a s a l f o r e b r a i n " and " c h o l i n e r g i c b a s a l n u c l e a r complex" have been proposed to account f o r t h i s o r g a n i z a t i o n (Satoh e t a l . , 1983; Schwaber e t a l . , 1987; V i n c e n t e t a l . , 1986). C o n s i s t e n t w i t h the p r i n c i p l e of columnar o r g a n i z a t i o n , i s the r e a l i z a t i o n t h a t the d i s t r i b u t i o n of c h o l i n e r g i c neurons extends beyond the l i m i t s of the t r a d i t i o n a l boundaries of the r e g i o n s i t encompasses (Schwaber e t a l . , 1987). Another important aspect i n the d i s t r i b u t i o n of b a s a l f o r e b r a i n c h o l i n e r g i c neurons p e r t a i n s to the evidence demonstrating the neurochemical h e t e r o g e n e i t y of b a s a l f o r e b r a i n neurons. Hence, c h o l i n e r g i c neurons g e n e r a l l y c o n s t i t u t e a neuronal sub p o p u l a t i o n w i t h i n a g i v e n r e g i o n s i n c e they are o f t e n i n t e r m i n g l e d or i n c l o s e p r o x i m i t y w i t h n o n - c h o l i n e r g i c neurons (Schwaber e t a l . , 1987; Wainer e t a l . , 1984; Rye e t a l . , 1984). For example, i n the r e g i o n of the h o r i z o n t a l limb of the d i a g o n a l band, GABAergic 4 neurons are i n t e r m i n g l e d w i t h c h o l i n e r g i c neurons (Brashear e t a l . , 1986). P h y s i o l o g i c a l evidence f o r h e t e r o g e n e i t y of c o r t i c a l l y p r o j e c t i n g b a s a l f o r e b r a i n neurons has a l s o been o b t a i n e d (Reiner e t a l . , 1987; Aston-Jones e t a l . , 1985). The r o s t r a l c h o l i n e r g i c column of the b a s a l f o r e b r a i n i n n e r v a t e s a wide range of t e l e n c e p h a l i c s t r u c t u r e s , i n c l u d i n g the c o r t e x (Lehmann e t a l . , 1980; Rye e t a l . , 1984), the hippocampus (Woolf e t a l . , 1984; Amaral and Kurz, 1985), the amygdala ( C a r l s e n e t a l . , 1985; Nagai e t a l . , 1982) and the o l f a c t o r y b ulb (Zaborszky e t a l . , 1986). In a d d i t i o n , b a s a l f o r e b r a i n c h o l i n e r g i c neurons have been shown to i n n e r v a t e the r e t i c u l a r t h a l a m i c nucleus of the diencephalon (Hallanger e t a l . , 1987) and the h a b e n u l o - i n t e r p e d u n c u l a r system of the mesencephalon (Woolf and Butcher, 1985; C o n t e s t a b i l e e t a l . , 1987). As knowledge on the d i s t r i b u t i o n and o r g a n i z a t i o n of b a s a l f o r e b r a i n c h o l i n e r g i c neurons p r o g r e s s e s , i t i s becoming i n c r e a s i n g l y d i f f i c u l t t o c l a s s i f y c h o l i n e r g i c n u c l e a r subgroups of the b a s a l f o r e b r a i n on the b a s i s of the o r i g i n s of t h e i r r e s p e c t i v e p r o j e c t i o n s such as proposed by the ( c h l -ch4) nomenclature of Mesulam e t a l . (1983). Hence, t h i s c l a s s i f i c a t i o n scheme does not r e f l e c t s u f f i c i e n t l y w e l l the e x t e n s i v e o v e r l a p i n the o r i g i n s of many of the c h o l i n e r g i c p r o j e c t i o n systems of the b a s a l f o r e b r a i n (Schwaber e t a l . , 1987). For example, i t has been demonstrated t h a t c h o l i n e r g i c neurons d i s t r i b u t e d throughout the r o s t r o - c a u d a l e x t e n t of the b a s a l f o r e b r a i n i n n e r v a t e the neocortex (Saper,1984) and 5 amygdala (Woolf and Butcher, 1985). Furthermore, the l o c a l i z a t i o n of c h o l i n e r g i c neurons i n n e r v a t i n g the neocortex c o i n c i d e s w i t h t h a t of amygdalopetal c h o l i n e r g i c neurons ( C a r l s e n e t a l . , 1985). A second major p o p u l a t i o n of c h o l i n e r g i c neurons i s found i n the mesencephalic and pontine tegmentum where i t i s predominantly a s s o c i a t e d w i t h the l a t e r o d o r s a l tegmental nucleus, c a u d a l l y , and the pedunculopontine tegmental nucleus, r o s t r a l l y (Woolf and Butcher, 1986; Mesulam e t a l . , 1983; Satoh e t a l . , 1983). In many r e s p e c t s , the o r g a n i z a t i o n of mesopontine c h o l i n e r g i c neurons i s s i m i l a r t o t h a t of b a s a l f o r e b r a i n c h o l i n e r g i c neurons. Hence, these c h o l i n e r g i c neurons are a l s o arranged i n a l o n g i t u d i n a l column, "the ca u d a l c h o l i n e r g i c column" (see Satoh e t a l . , 1983), which extends from the c a u d a l p o l e of the s u b s t a n t i a n i g r a , r o s t r a l l y , t o the a n t e r i o r p a r t of the lo c u s c e r u l e u s , c a u d a l l y (Satoh e t a l . , 1983; Armstrong e t a l . , 1983; Mesulam e t a l . , 1983; V i n c e n t e t a l . , 1986). Furthermore, mesopontine c h o l i n e r g i c neurons have a l s o been shown to be i n t e r m i n g l e d w i t h n o n - c h o l i n e r g i c c e l l s such as c a t e c h o l a m i n e - c o n t a i n i n g neurons i n the c a t (Jones and Beaudet, 1987; V i n c e n t and Re i n e r , 1987). The ascending c h o l i n e r g i c p r o j e c t i o n s a r i s i n g from the ca u d a l c h o l i n e r g i c column i n n e r v a t e numerous r e g i o n s i n c l u d i n g l i m i t e d r e g i o n s of the c o r t e x ( V i n c e n t e t a l . , 1983; Woolf and Butcher, 1986; Satoh and F i b i g e r , 1986), the thalamus 6 (Sofroniew e t a l . , 1985; Satoh and F i b i g e r ; 1986); the i n t e r p e d u n c u l a r nucleus (Woolf and Butcher; 1985), the s u p e r i o r c o l l i c u l u s (Beninato and Spencer, 1986) and the b a s a l f o r e b r a i n (Woolf and Butcher; 1986; Jones and Beaudet, 1987; Semba e t a l . , 1988a). I n c r e a s i n g numbers of anatomical i n v e s t i g a t i o n s have sought to analyze i n g r e a t e r d e t a i l the o r g a n i z a t i o n of c e n t r a l c h o l i n e r g i c systems; f o c u s i n g on such q u e s t i o n s as neuropeptide c o - l o c a l i z a t i o n (Vincent e t a l . , 1983), t o p o g r a p h i c a l o r g a n i z a t i o n (Saper, 1984), course t r a j e c t o r y (Ichikawa and H i r a t a , 1986) and axonal c o l l a t e r a l i z a t i o n ( B i g l e t a l . , 1982). As a l l u d e d to above, when comparing the d i s t r i b u t i o n of v a r i o u s b a s a l f o r e b r a i n or mesopontine c h o l i n e r g i c p r o j e c t i o n systems, t h e r e i s c o n s i d e r a b l e o v e r l a p i n the o r i g i n s of a number of independent p r o j e c t i o n s . I t has been suggested t h a t such o v e r l a p i n the o r i g i n s of c h o l i n e r g i c p r o j e c t i o n systems may be i n d i c a t i v e of axonal c o l l a t e r a l i z a t i o n i n t h a t i n d i v i d u a l c h o l i n e r g i c neurons may have axons t h a t branch to i n n e r v a t e d i v e r g e n t r e g i o n s ( F i b i g e r , 1982). Most s t u d i e s t h a t have examined the degree of c o l l a t e r a l i z e d p r o j e c t i o n s o r i g i n a t i n g i n the r o s t r a l c h o l i n e r g i c column of the b a s a l f o r e b r a i n have focused on the subset of c h o l i n e r g i c neurons i n n e r v a t i n g the neocortex. In both the r a t and primate b r a i n , s t u d i e s u s i n g a r e t r o g r a d e d o u b l e - l a b e l i n g technique w i t h f l u o r e s c e n t probes (Saper, 7 1984; L u s k i n and P r i c e , 1982; P r i c e and S t e r n , 1983; Walker e t a l . , 1985) or combined w i t h an AChE pharmacohistochemistry procedure ( B i g l e t a l . , 1982) have r e v e a l e d t h a t o n l y a s m a l l p r o p o r t i o n (0-5%) of c h o l i n e r g i c b a s a l f o r e b r a i n neurons t h a t p r o j e c t to the neocortex have axons t h a t branch to i n n e r v a t e d i v e r g e n t c o r t i c a l areas. C o n s i s t e n t w i t h these r e s u l t s are the data o b t a i n e d by way of an anterograde t r a c t - t r a c i n g method (Lamour e t a l . , 1984), and a n t i d r o m i c a c t i v a t i o n (Aston-Jones e t a l . , 1985). However, i n s e v e r a l of these s t u d i e s , i n d i v i d u a l c o r t i c a l l y p r o j e c t i n g b a s a l f o r e b r a i n neurons thought t o be c h o l i n e r g i c were observed to have axons t h a t branched e x t e n s i v e l y w i t h i n a g i v e n c o r t i c a l area (Lamour e t a l . , 1984; P r i c e and S t e r n , 1983; Aston-Jones e t a l . , 1985). In c o n t r a s t , u s i n g r e t r o g r a d e d o u b l e - l a b e l i n g techniques, McKinney e t a l . (1983) and Levine and c o l l a b o r a t o r s (Boylan e t a l . , 1986; Adams et a l . , 1986) have shown t h a t a s i g n i f i c a n t p r o p o r t i o n (15-30%) of c o r t i c a l l y p r o j e c t i n g b a s a l f o r e b r a i n neurons b e l i e v e d to be c h o l i n e r g i c , have axons t h a t c o l l a t e r a l i z e t o i n n e r v a t e d i v e r g e n t c o r t i c a l areas i n the r a t and c a t b r a i n r e s p e c t i v e l y . The p o s s i b i l i t y t h a t other subpopulations of b a s a l f o r e b r a i n c h o l i n e r g i c neurons may have axons t h a t c o l l a t e r a l i z e t o i n n e r v a t e s u b c o r t i c a l areas has been s p o r a d i c a l l y i n v e s t i g a t e d . A s m a l l percentage of b a s a l f o r e b r a i n neurons i n the r a t b r a i n have been observed t o branch f o l l o w i n g p a i r e d i n j e c t i o n s of d i f f e r e n t f l u o r e s c e n t t r a c e r s i n the o l f a c t o r y bulb and p i r i f o r m c o r t e x ( L u s k i n and 8 P r i c e , 1982), amygdala and a l l o c o r t e x or neocortex ( C a r l s e n e t a l . , 1985), o l f a c t o r y b ulb and hippocampus (Okoyama e t a l . , 1987), as w e l l as i n the hippocampus and neocortex of the primate b r a i n ( K o l i a t s o s e t a l . , 1987). In c o n t r a s t , a s i g n i f i c a n t number of b a s a l f o r e b r a i n neurons have been found to send c o l l a t e r a l s to the r e t i c u l a r nucleus of the thalamus and the p e r i b r a c h i a l area, but not to the p e r i b r a c h i a l area and other t h a l a m i c n u c l e i or c o r t e x of the primate b r a i n as demonstrated w i t h r e t r o g r a d e d o u b l e - l a b e l i n g f l u o r e s c e n c e (Parent e t a l . , 1987). S i m i l a r l y , i n the c a t b r a i n , branched p r o j e c t i o n s a r i s i n g from the nucleus b a s a l i s m a g n o c e l l u l a r i s have been shown to terminate i n the neocortex and head of the caudate nucleus ( F i s h e r e t a l . , 1985) on the one hand, and i n the r e t i c u l a r t h a l a m i c and mediodorsal thalamic n u c l e i on the other hand ( S t e r i a d e e t a l . , 1987). However, the chemical i d e n t i t y of these c o l l a t e r a l i z e d p r o j e c t i o n s was not assessed i n the l a t t e r s t u d i e s . F u r t h e r evidence f o r branched p r o j e c t i o n s o r i g i n a t i n g i n the b a s a l f o r e b r a i n has a l s o been o b t a i n e d u s i n g p h y s i o l o g i c a l approaches. For example, h a l f of the b a s a l f o r e b r a i n neurons t h a t were a n t i d r o m i c a l l y a c t i v a t e d from the amygdala c o u l d a l s o be a c t i v a t e d from the f i m b r i a of the r a t , s u g g e s t i n g t h a t t h e r e i s a c o l l a t e r a l i z e d p r o j e c t i o n from the b a s a l f o r e b r a i n to the amygdala and hippocampus (Dutar e t a l . , 1986). In c o n t r a s t , i n the primate b r a i n , the degree of c o l l a t e r a l i z a t i o n t o the amygdala and hippocampus from b a s a l f o r e b r a i n neurons was found to be r e l a t i v e l y low ( 2 % ) , as r e v e a l e d by r e t r o g r a d e d o u b l e - l a b e l i n g f l u o r e s c e n c e 9 ( K o l i a t s o s e t a l . , 1987). F i n a l l y , n o n - c h o l i n e r g i c neurons of the r a t d i a g o n a l band have been shown by r e t r o g r a d e double-l a b e l i n g a n a l y s i s combined w i t h AChE h i s t o c h e m i s t r y to i n n e r v a t e s i m u l t a n e o u s l y the median raphe and i n t e r p e d u n c u l a r nucleus ( S t r a t f o r d and W i r t s h a f t e r , 1987). Evidence f o r c o l l a t e r a l i z e d p r o j e c t i o n s a r i s i n g from c h o l i n e r g i c neurons of the mesopontine tegmentum i s l e s s c o n t r o v e r s i a l . In a r e t r o g r a d e t r i p l e - l a b e l i n g study combining f l u o r e s c e n t r e t r o g r a d e probes w i t h ChAT immunohistochemistry, Woolf and Butcher (1985) o b t a i n e d evidence f o r c o l l a t e r a l i z e d p r o j e c t i o n s to the a n t e r i o r t h a l a m i c area and s e p t a l - d i a g o n a l band complex (10%), mediodorsal t h a l a m i c nucleus and l a t e r a l hypothalamic area ( 8 % ) , subthalamus and p o s t e r i o r t h a l a m i c area (18%) or l a t e r a l g e n i c u l a t e nucleus (12%), and s u b s t a n t i a n i g r a and caudate-putamen ( 5 % ) , from c h o l i n e r g i c neurons c o n t a i n e d i n both the pedunculopontine tegmental nucleus and l a t e r o d o r s a l tegmental nucleus of the r a t . In another study, a h i g h percentage (50%) of presumed c h o l i n e r g i c neurons i n the l a t e r o d o r s a l tegmental nucleus were double l a b e l e d f o l l o w i n g i n j e c t i o n s of f l u o r e s c e n t t r a c e r s i n the r e t i c u l a r and mediodorsal t h a l a m i c n u c l e i , thus s t r o n g l y s u g g e s t i n g t h a t t h i s p r o j e c t i o n system i s s i g n i f i c a n t l y c o l l a t e r a l i z e d i n both the r a t and c a t b r a i n (Cornwall and P h i l l i p s o n , 1987; S t e r i a d e e t a l . , 1987). 10 I t has been concluded t h a t the p r o j e c t i o n s d e r i v e d from c h o l i n e r g i c neurons c o n t a i n e d i n the b a s a l f o r e b r a i n are g e n e r a l l y n o n - c o l l a t e r a l i z e d ( C a r l s e n e t a l . , 1985) whereas those a r i s i n g from c h o l i n e r g i c neurons of the mesopontine tegmentum tend to c o l l a t e r a l i z e e x t e n s i v e l y (Woolf and Butcher, 1985). However, i n the case of the p r o j e c t i o n systems of the r o s t r a l c h o l i n e r g i c column of the b a s a l f o r e b r a i n , t h i s c o n c l u s i o n i s perhaps premature i n view of the s m a l l number of s u b c o r t i c a l p r o j e c t i o n systems t h a t have been examined to date. In a d d i t i o n , the a b i l i t y t o d e t e c t c o l l a t e r a l i z e d p r o j e c t i o n s i s d i r e c t l y r e l a t e d to the s e n s i t i v i t i e s and l i m i t a t i o n s of the t r a c t - t r a c i n g methods used. For example, i t i s p o s s i b l e t h a t i n some s t u d i e s the s i z e of the i n j e c t i o n s i t e d i d not cover o p t i m a l l y the t e r m i n a l f i e l d s of the c o l l a t e r a l i z e d p r o j e c t i o n s thereby r e s u l t i n g i n a u n d e r e s t i m a t i o n of branched p r o j e c t i o n s . T h i s can a l s o be the case i n p h y s i o l o g i c a l s t u d i e s w i t h a n t i d r o m i c a c t i v a t i o n s i n c e the sampling p o p u l a t i o n of neurons i s o f t e n s m a l l and a n t i d r o m i c a c t i v a t i o n can a l s o f a i l a t p o i n t s of b i f u r c a t i o n . For example, i n the study of Aston-Jones e t a l . (1985) the sampling p o p u l a t i o n used f o r a n t i d r o m i c a c t i v a t i o n accounted f o r l e s s than 10% of the t o t a l number of c e l l s s t u d i e d . Another f a c t o r which seems to be of importance p e r t a i n s to p o t e n t i a l s p e c i e s d i f f e r e n c e s i n the o r g a n i z a t i o n of b a s a l f o r e b r a i n p r o j e c t i o n systems as i l l u s t r a t e d from s t u d i e s i n the c a t b r a i n (see above). Such t e c h n i c a l l i m i t a t i o n s and p o s s i b l e s p e c i e s d i f f e r e n c e s may be a t the 11 b a s i s of the d i s c r e p a n c i e s between the v a r i o u s s t u d i e s reviewed e a r l i e r , and f u r t h e r emphasize the need f o r a d d i t i o n a l data b e f o r e f i r m c o n c l u s i o n s can be reached. Notwithstanding the o b s e r v a t i o n s i n the c a t b r a i n and the r e s u l t s of McKinney e t a l . (1983), i t would appear t h a t s u f f i c i e n t d ata have been o b t a i n e d by d i f f e r e n t l a b o r a t o r i e s to support the c o n c l u s i o n t h a t most c h o l i n e r g i c neurons c o n t a i n e d i n the b a s a l f o r e b r a i n have r a t h e r r e s t r i c t e d p r o j e c t i o n s to the neocortex. However, such a c o n c l u s i o n cannot at p r e s e n t be extended to other b a s a l f o r e b r a i n p r o j e c t i o n systems i n n e r v a t i n g s u b c o r t i c a l areas, i n view of the p a u c i t y and i n c o n s i s t e n c y of the data c u r r e n t l y a v a i l a b l e . Hence, each p r o j e c t i o n system of the r o s t r a l c h o l i n e r g i c column t h a t appears a l i k e l y c a ndidate f o r c o l l a t e r a l i z a t i o n on the b a s i s of s i g n i f i c a n t anatomical o v e r l a p , or f u n c t i o n a l r e l a t i o n s h i p s should be examined. A c c o r d i n g l y , the f o l l o w i n g s t u d i e s sought to determine the degree of axonal c o l l a t e r a l i z a t i o n f o r s e v e r a l p r o j e c t i o n systems o r i g i n a t i n g i n the b a s a l f o r e b r a i n or mesopontine tegmentum by way of r e t r o g r a d e d o u b l e - l a b e l i n g methods. These s t u d i e s a l s o i n c l u d e d immunohistochemical analyses i n an e f f o r t t o i d e n t i f y the n e u r o t r a n s m i t t e r content of any neurons t h a t showed axonal c o l l a t e r a l i z a t i o n . 12 EXPERIMENT 1 INTRODUCTION On the b a s i s of r e c e n t anatomical o b s e r v a t i o n s , the c h o l i n e r g i c n u c l e a r groups c o n t r i b u t i n g to the i n n e r v a t i o n of the thalamus, and i n p a r t i c u l a r t o the r e t i c u l a r t h a l a m i c nucleus (RTN), are a t t r a c t i v e c a n didates f o r the examination of c o l l a t e r a l i z a t i o n . Numerous s t u d i e s t h a t have combined t r a c t - t r a c i n g methods wit h ChAT immunohistochemistry or AChE h i s t o c h e m i s t r y have r e v e a l e d t h a t the p r i n c i p a l source of c h o l i n e r g i c i n p u t to v a r i o u s t h a l a m i c n u c l e i i n the mammalian b r a i n i s d e r i v e d from the c a u d a l c h o l i n e r g i c column, i . e . t h e pedunculopontine and l a t e r o d o r s a l tegmental n u c l e i (Sofroniew e t a l . , 1985; Satoh and F i b i g e r , 1986; Woolf and Butcher, 1986; Mesulam e t a l . , 1983). R e c e n t l y , however, i n both the r a t and c a t b r a i n , c h o l i n e r g i c neurons c o n t a i n e d i n the b a s a l f o r e b r a i n , and p a r t i c u l a r l y i n the nucleus b a s a l i s m a g n o c e l l u l a r i s , have been shown to p r o v i d e a major i n p u t t o the RTN i n a d d i t i o n to the w e l l e s t a b l i s h e d c h o l i n e r g i c a f f e r e n t s d e r i v e d from the mesopontine tegmentum (Levey e t a l . , 1987; H a l l a n g e r e t a l . , 1987; S t e r i a d e e t a l . , 1987). Of p a r t i c u l a r i n t e r e s t i s the r e a l i z a t i o n t h a t i n the r a t thalamus, the RTN appears unique i n t h a t i t i s a p p a r e n t l y the s o l e r e c i p i e n t of a major c h o l i n e r g i c i n p u t from the b a s a l f o r e b r a i n (Hallanger e t a l . , 1987), whereas i n the c a t b r a i n , b a s a l f o r e b r a i n c h o l i n e r g i c neurons have been shown to terminate i n the mediodorsal 13 thalamic nucleus as w e l l ( S t e r i a d e e t a l . , 1987). I t i s a l s o i n t e r e s t i n g t o note t h a t a c c o r d i n g to the s t u d i e s of H a l l a n g e r and c o l l a b o r a t o r s (Hallanger e t a l . , 1987; Levey e t a l . , 1987), the p r o p o r t i o n of b a s a l f o r e b r a i n and mesopontine tegmental c h o l i n e r g i c neurons i n n e r v a t i n g the r a t RTN accounts f o r approximately 25-50% of the t o t a l number of r e t r o g r a d e l y l a b e l e d c e l l s observed i n these r e g i o n s . The chemical s i g n a t u r e of the remaining neurons i n n e r v a t i n g the RTN i s unknown. The l o c a l i z a t i o n of the b a s a l f o r e b r a i n and mesopontine tegmental c h o l i n e r g i c neurons i n n e r v a t i n g the RTN appears to c o i n c i d e w i t h the l o c a l i z a t i o n of c h o l i n e r g i c c o r t i c o p e t a l neurons as d e s c r i b e d i n the r a t b r a i n (Vincent e t a l . , 1983; Lehmann e t a l . , 1980, Saper and Loewy, 1982; Mesulam e t a l . , 1983). On the b a s i s of the s i g n i f i c a n t o v e r l a p i n the o r i g i n s of the c h o l i n e r g i c p r o j e c t i o n s to the RTN and c o r t e x , the f o l l o w i n g study examined whether axons of i n d i v i d u a l c h o l i n e r g i c neurons o r i g i n a t i n g i n the b a s a l f o r e b r a i n on the one hand, and i n the mesopontine tegmentum on the other hand, send c o l l a t e r a l s t h a t i n n e r v a t e both the c o r t e x and RTN of the r a t b r a i n . F i g u r e 1 shows a schematic r e p r e s e n t a t i o n of the h y p o t h e t i c a l c o l l a t e r a l i z e d p r o j e c t i o n s under study. Furthermore, the f o l l o w i n g study a l s o i n c l u d e d s e v e r a l immunohistochemical experiments i n an attempt to c h a r a c t e r i z e the n e u r o t r a n s m i t t e r content of these neurons. 14 F i g u r e 1. A s chemat i c r e p r e s e n t a t i o n of h y p o t h e t i c a l p r o j e c t i o n s a r i s i n g i n the b a s a l f o r e b r a i n on the one hand, and mesopont ine tegmentum on the o the r hand, t h a t b ranch t o i n n e r v a t e bo th the r e t i c u l a r t ha l am i c nuc leus and c o r t e x ( do t ted l i n e s ) . Note t h a t the t r a j e c t o r i e s o f the f i b e r s as i l l u s t r a t e d do not co r re spond t o a c t u a l t r a j e c t o r i e s . A b b r e v i a t i o n s : c x , c o r t e x ; r t n , r e t i c u l a r t h a l a m i c n u c l e u s ; p p t , peduncu lopon t ine tegmenta l n u c l e u s ; b f , b a s a l f o r e b r a i n . (mod i f i ed s a g g i t a l p l a t e from Paxinos and Watson, 1986). 15 METHODS The r e t r o g r a d e f l u o r e s c e n c e d o u b l e - l a b e l i n g procedure i n v o l v e s , as d e s c r i b e d by Van der Kooy e t a l . (1978), two fluorochromes which emit f l u o r e s c e n c e w i t h d i f f e r e n t s p e c t r a l c h a r a c t e r i s t i c s . Each t e r m i n a l f i e l d of the p r o j e c t i o n systems under study i s i n j e c t e d w i t h one of the two t r a c e r s (see F i g u r e 2) . Neurons t h a t f l u o r e s c e a t both e x c i t a t i o n wavelengths (double-labeled) are thought t o have r e t r o g r a d e l y t r a n s p o r t e d the t r a c e r s v i a d i v e r g e n t axonal c o l l a t e r a l s . F l u o r o - g o l d (FG), propidium i o d i d e (PI) and t r u e b l u e (TB) were the f l u o r e s c e n t dyes chosen f o r t h i s experiment. These dyes are known to be r e a d i l y t r a n s p o r t e d r e t r o g r a d e l y , are r e s i s t a n t t o f a d i n g by f l u o r e s c e n t i l l u m i n a t i o n , e x h i b i t l i t t l e tendency t o d i f f u s e out of the c e l l bodies and are compatible w i t h immunohistochemical procedures ( S k i r b o l l e t a l . , 1984; Schmued and F a l l o n , 1986). Animals Male Wistar r a t s (U.B.C Animal Breeding Center) weighing 250-300g were used. Upon a r r i v a l , the animals were allowed t o accommodate to t h e i r new environment f o r at l e a s t one day p r i o r t o surgery. The animals were housed, both b e f o r e and a f t e r surgery, i n groups of two or f o u r and maintained on a 12 hour l i g h t \ d a r k c y c l e w i t h f r e e access t o food and water. Figure 2. A schematic representation of the i n j e c t i o n protocols (see text for d e t a i l s ) . Abbreviations: PI, propidium iodide; FG, fluoro-gold; cx, cortex; rtn; r e t i c u l a r thalamic nucleus. (modified coronal plate from Paxinos and Watson, 1986). 28 19 Stereotaxic Surgical Procedures Each animal was anesthetized with sodium pentobarbital (Nembutal; 50mg/kg, i.p.) and placed i n a stereotaxic instrument (David Kopf Instruments; T i j i n g a , CA, USA). Supplemental doses of anesthetic were administered when required. A l l animals received a single i n j e c t i o n of FG (Fluorochrome, Englewood, CO, USA) i n the RTN of the r i g h t hemisphere. A 4% solution of FG dissolved i n 0.9% saline was prepared. The intracerebral infusion of the tracer was performed by iontophoresis (ByTech, Vancouver, B.C., Can.). The parameters used for the iontophoretic infusion of FG were determined p r i o r to the experiment. A single infusion with an anodal DC current of 2.6-2.8 u.A administered at 6 second i n t e r v a l s for a period of 10 minutes was chosen. These eje c t i o n parameters generally resulted i n a small i n j e c t i o n of FG with s a t i s f a c t o r y retrograde transport yet produced l i t t l e necrosis and minimal d i f f u s i o n of the tracer from the s i t e of i n j e c t i o n or along the micropipette track. Glass c a p i l l a r y tubing (outer diameter: 1.5mm) was pulled on the same day of the surgery and broken at the t i p to a diameter of 20uxn-30ura. The micropipettes were then f i l l e d by c a p i l l a r y action with the FG solution, b a c k f i l l e d with 0.9% saline and maintained i n a humid environment u n t i l surgery. 20 The RTN of the r i g h t hemisphere was approached a t an angle from the l e f t hemisphere (see F i g u r e 2). The s t e r e o t a x i c parameters used i n t h i s experiment were d e r i v e d from the a t l a s of Paxinos and Watson (1986). The heads of the animals were p o s i t i o n e d so t h a t the s u r f a c e of the s k u l l was f l a t ; the i n c i s o r bar was s e t 3.3mm below the i n t e r a u r a l l i n e . The co o r d i n a t e s used were as f o l l o w : 1.4mm p o s t e r i o r t o bregma, 1.0mm l a t e r a l t o the m i d l i n e and 6.4mm v e n t r a l t o dura, w i t h an angle of 24° o f f the v e r t i c a l plane. F o l l o w i n g the i n f u s i o n of the t r a c e r , the m i c r o p i p e t t e s were l e f t i n p l a c e f o r 10-15 minutes. During the same s u r g i c a l s e s s i o n o r , on some o c c a s i o n s , on the day f o l l o w i n g the FG i n f u s i o n , 15 animals (group 1) r e c e i v e d m u l t i p l e i n f u s i o n s of PI (C a i Biochem; La J o l l a , CA, USA; Sigma; S t - L o u i s , MO, USA) i n t o the neocortex i p s i l a t e r a l t o the FG i n f u s i o n . A 10% s o l u t i o n of PI d i s s o l v e d i n d i s t i l l e d water by s o n i c a t i o n was prepared. A one u l s y r i n g e (Hamilton Co., Reno, NV, USA) was used t o i n j e c t a volume of 0.2 - 0 . 4 u l / i n j e c t i o n of PI over a p e r i o d of 5-10 minutes. F o l l o w i n g the i n f u s i o n , the s y r i n g e was l e f t i n p l a c e f o r 5-10 minutes to al l o w the t r a c e r to d i f f u s e . M u l t i p l e c o r t i c a l i n j e c t i o n s were performed t o maximize r e t r o g r a d e l a b e l i n g i n the b a s a l f o r e b r a i n and mesopontine tegmentum. Each animal r e c e i v e d 4-9 i n j e c t i o n s i n t o s e v e r a l d i f f e r e n t c o r t i c a l f i e l d s (See Table 1 f o r c o o r d i n a t e s ) . To c o n t r o l f o r p o s s i b l e l a b e l i n g of t r a n s e c t e d f i b e r s of passage, many of the animals r e c e i v e d d i f f e r e n t combinations of c o r t i c a l i n f u s i o n s . Table 1. L i s t of s t e r e o t a x i c c o o r d i n a t e s t h a t were chosen f o r the c o r t i c a l i n j e c t i o n s . A b b r e v i a t i o n s : A-P, a n t e r o - p o s t e r i o r plane; L-M, l a t e r o - m e d i a l plane; D-V, d o r s o - v e n t r a l plane. (Based on Paxinos and Watson, 1986). 22 Table 1. S t e r e o t a x i c c o o r d i n a t e s ( i n nun) f o r c o r t i c a l i n j e c t i o n s C o r t i c a l t a r g e t A-P L-M D-V (from bregma) (from dura) agr a n u l a r i n s u l a r +3.7 -3.4 -2.8 f r o n t a l +3.7 -1.6 -0.8 c i n g u l a t e \ i n f r a l i m b i c +3.2 -0.6 -2.6 f r o n t a l \ c i n g u l a t e +2.7 -1. -1.3 f r o n t a l \ p a r i e t a l +2.7 -4.0 -1.2 g r a n u l a r i n s u l a r \ a g ranular i n s u l a r +1.7 -5.0 -3.4 f r o n t a l -2.3 -1.4 -0.6 r e t r o s p l e n i a l -3.8 -1.4 -0.4 r e t r o s p l e n i a l -4.3 -1.6 -0.4 23 However, a l l animals r e c e i v e d i n j e c t i o n s of the t r a c e r i n t o the f r o n t a l and medial p r e f r o n t a l c o r t e x . A second group of animals (n=4) was prepared a c c o r d i n g to the same procedure as above except t h a t TB (Sigma; S t - L o u i s , MO, USA) was used f o r the c o r t i c a l i n j e c t i o n s . A 4% s o l u t i o n of TB d i s s o l v e d i n d i s t i l l e d water by s o n i c a t i o n was prepared. The i n j e c t i o n procedure and i n f u s i o n parameters were i d e n t i c a l to those used f o r the i n j e c t i o n of PI wi t h the e x c e p t i o n t h a t adjacent s e c t i o n s were r i n s e d w i t h 0.0001% ethidium bromide (EB; Sigma; S t - L o u i s , MO, USA) which p r o v i d e s a r ed c o u n t e r s t a i n under f l u o r e s c e n t i l l u m i n a t i o n . T h i s c o u n t e r s t a i n i n g method allowed f o r a more p r e c i s e c y t o a r c h i t e c t o n i c l o c a l i z a t i o n of TB r e t r o g r a d e l y l a b e l e d c e l l s . F i n a l l y , as a c o n t r o l group, f o u r r a t s w i t h m i s p l a c e d FG i n j e c t i o n s were analyzed. F l u o r e s c e n t T r a c e r H i s t o l o g y and M i c r o s c o p i c Examination F o l l o w i n g i n j e c t i o n s of the t r a c e r s , the animals were allowed t o s u r v i v e f o r a minimum of 4 days. T h i s p o s t - s u r g e r y s u r v i v a l p e r i o d has been shown to be s u f f i c i e n t f o r adequate FG and PI r e t r o g r a d e t r a n s p o r t and l a b e l i n g of c e l l bodies ( S k i r b o l l e t a l . , 1984; Schmued and F a l l o n , 1986). At the end of the p o s t - s u r g e r y s u r v i v a l p e r i o d , each animal was deeply a n e s t h e t i z e d w i t h c h l o r a l hydrate (400mg/kg, i.p.) and then t r a n s c a r d i a l l y p e r f u s e d w i t h an i n i t i a l 50-100ml bolus of 0.9% 24 s a l i n e f o l l o w e d by 400ml of 4% paraformaldelyde (PFA) i n 0.1 M phosphate b u f f e r (pH 7.4) ad m i n i s t e r e d through a p e r i s t a l t i c pump a t a slow r a t e over a 20-30 minute p e r i o d . The b r a i n s were removed and soaked i n a 4% PFA s o l u t i o n i n 0.1 M phosphate b u f f e r f o r 2-3 hours, and then t r a n s f e r r e d i n t o a 20% sucrose s o l u t i o n i n 0.1 M phosphate b u f f e r (pH 7.4) f o r 24 hours. Each b r a i n was s l i c e d i n 30um c o r o n a l s e c t i o n s on a f r e e z i n g microtome. For v e r i f i c a t i o n of c o r t i c a l i n j e c t i o n s i t e s , 1 out of every 5 s e c t i o n s was c o l l e c t e d from the r o s t r a l p o l e of the f r o n t a l c o r t e x t o the l e v e l of the j u n c t i o n of the corpus callosum. From t h i s p o i n t up t o the l e v e l of the genu of the f a c i a l nerve i n the brainstem, 1 out of every 3 s e c t i o n s was r e t a i n e d f o r v e r i f i c a t i o n of re t r o g r a d e l a b e l i n g . The s e c t i o n s were r i n s e d i n 0.1 M phosphate b u f f e r (pH 7.4), mounted from a 15% phosphate b u f f e r e d sucrose s o l u t i o n onto uncoated g l a s s s l i d e s and l e f t t o dry o v e r n i g h t . The s e c t i o n s were then c o v e r s l i p p e d w i t h f l u o r e s c e n c e immersion o i l ( Z e i s s , West Germany). For m i c r o s c o p i c i n s p e c t i o n of the s e c t i o n s , a L e i t z D i a l u x or Z e i s s f l u o r e s c e n c e microscope equipped w i t h f i l t e r b l o c k s p r o v i d i n g an u l t r a v i o l e t e x c i t a t i o n range t o v i s u a l i z e FG- and TB - l a b e l e d c e l l s and f i l t e r b l o c k s p r o v i d i n g a green e x c i t a t i o n range t o v i s u a l i z e P I- and E B - l a b e l e d c e l l s were used. C e l l s t h a t were l a b e l e d f o l l o w i n g p a i r e d i n j e c t i o n s of PI and FG t y p i c a l l y e x h i b i t e d smooth orange-red and g r a n u l a r g o l d cytoplasms r e s p e c t i v e l y , and d o u b l e - l a b e l e d c e l l s c o u l d be v i s u a l i z e d by a l t e r n a t i n g between e x c i t a t i o n s and emissions 25 f i l t e r s . F o l l o w i n g p a i r e d i n j e c t i o n s of FG and TB a l l c e l l s c o u l d be v i s u a l i z e d w i t h a s i n g l e f i l t e r . In t h i s case, however, FG and TB s i n g l e - l a b e l e d c e l l s showed g r a n u l a r g o l d and smooth blu e cytoplasms r e s p e c t i v e l y , whereas c e l l s c o n t a i n i n g both t r a c e r s ( d o u b l e - l a b e l e d ) emitted a b l e n d of the two c o l o r s . The p o s i t i o n s of r e t r o g r a d e l y l a b e l e d c e l l s i n the b a s a l f o r e b r a i n and mesopontine tegmentum were drawn on s e l e c t e d p l a t e s ( c o r o n a l plane) d e r i v e d from the a t l a s of Paxinos and Watson (1986). The nomenclature t h a t i s used t o d e s c r i b e b r a i n areas i n t h i s study i s t h e r e f o r e i n keeping w i t h the a t l a s of Paxinos and Watson. In 5 animals from the f i r s t group, s e m i - q u a n t i t a t i v e analyses were performed i n an attempt to determine the degree to which the p r o j e c t i o n systems under study are c o l l a t e r a l i z e d . T h i s was done by c o u n t i n g r e t r o g r a d e l y l a b e l e d neurons (FG, and FG + P l - l a b e l e d ) i n thr e e b r a i n s e c t i o n s per l e v e l matching each of the s e l e c t e d p l a t e s . Since the number of c e l l s t h a t were r e t r o g r a d e l y l a b e l e d f o l l o w i n g FG i n j e c t i o n s i n the RTN was s u b s t a n t i a l l y s m a l l e r than l a b e l e d - c o r t i c o p e t a l neurons, percentage e s t i m a t i o n s of d o u b l e - l a b e l e d c e l l s were c a l c u l a t e d r e l a t i v e to the t o t a l number of c e l l s t h a t were s i n g l y l a b e l e d w i t h FG (FG+EI x 100). The number of l a b e l e d c o r t i c o p e t a l neurons was FG not assessed. For photographic purposes, both b l a c k and white ( T r i - X pan, 400 ASA, Kodak) and c o l o r s l i d e s (Ektachrome, 400 ASA, Kodak) f i l m s were used. 2L Immunohistochemical Procedures In f o u r animals of the f i r s t group, a d d i t i o n a l s e t s of s e c t i o n s through the b a s a l f o r e b r a i n and mesopontine tegmentum were r e t a i n e d f o r immunohistochemical i d e n t i f i c a t i o n of r e t r o g r a d e l y l a b e l e d c e l l s . A l l immunohistochemical procedures i n v o l v e d an i n d i r e c t method w i t h f l u o r e s c e i n i s o t h i o c y a n a t e - c o n j u g a t e d (FITC, e x c i t a t i o n range: green) secondary a n t i b o d i e s . A f i r s t s e t of s e c t i o n s f o r each of the f o u r r a t s was processed f o r ChAT immunoreactivity. The p r o t o c o l used f o r ChAT immunohistochemistry was s i m i l a r t o t h a t d e s c r i b e d by Semba e t a l . (1988b). The s e c t i o n s were f i r s t r i n s e d i n 0.05 M b u f f e r e d T r i s (pH 7.4) w i t h 0.9% s a l i n e (TBS) and then incubated w i t h a monoclonal antibody f o r ChAT (1:40, E c k e n s t e i n and Thoenen, 1982) i n a s o l u t i o n c o n t a i n i n g 0.05 M TBS, 0.3% T r i t o n - X and 2% normal goat serum. The s e c t i o n s were i n c u b a t e d a t 4° C f o r 48 hours on a shaker. The s e c t i o n s were then r i n s e d (3x20 minutes) i n 0.05 M TBS and incubated f o r 60 minutes on a shaker at room temperature i n a s o l u t i o n c o n t a i n i n g the secondary antibody (1:20, g o a t - a n t i - r a t ) , 0.05 M TBS, 0.3% T r i t o n - X and 2% normal goat serum. A f i n a l r i n s e (3X20 minutes) i n 0.05 M TBS was performed b e f o r e the s e c t i o n s were mounted from a 15% sucrose s o l u t i o n i n 0.1 M phosphate b u f f e r onto uncoated s l i d e s . The s e c t i o n s were d r i e d o v e r n i g h t and then c o v e r s l i p p e d w i t h f l u o r e s c e n c e immersion o i l . 27 A d d i t i o n a l s e t of s e c t i o n s through the b a s a l f o r e b r a i n from two of the animals t h a t were examined f o r ChAT immunohistochemistry were processed w i t h a monoclonal antibody a g a i n s t s o m a t o s t a t i n (AB 8; 1:20) f o l l o w e d by goat-anti-mouse, secondary antibody (1:40, see V i n c e n t e t a l . , 1985) f o l l o w i n g the procedure d e s c r i b e d f o r ChAT. F i n a l l y , i n t h r e e of the f o u r animals, s e t s of s e c t i o n s through the b a s a l f o r e b r a i n were r e t a i n e d f o r immunohistochemical p r o c e s s i n g w i t h a n t i b o d i e s t o parvalbumin (PV). F o l l o w i n g 3x20 minutes r i n s e s i n 0.1 M phosphate b u f f e r w i t h 0.9% s a l i n e (PBS, pH 7.4), the s e c t i o n s were i n c u b a t e d f o r 48 hours w i t h a r a b b i t anti-mouse muscle parvalbumin p o l y c l o n a l antibody (a g i f t from K.G. Baimbridge) d i l u t e d 1:50 i n a 0.1 M PBS s o l u t i o n c o n t a i n i n g 0.02% sodium a z i d e , 20 u n i t s of h e p a r i n , 1 mM EGTA, 6% goat serum and 0.3% T r i t o n X-100. The i n c u b a t i o n was performed on a shaker a t 4° C. The s e c t i o n s were then r i n s e d (3x20 minutes) i n 0.1 PBS and inc u b a t e d f o r two hours on a shaker a t room temperature w i t h the secondary antibody ( g o a t - a n t i - r a b b i t ) d i l u t e d 1:100 i n a b u f f e r s o l u t i o n prepared as d e s c r i b e d above. F o l l o w i n g t h r e e more r i n s e s (3x20 minutes) i n 0.1 M PBS, the s e c t i o n s were mounted from 15% sucrose, l e f t t o dry o v e r n i g h t and c o v e r s p l i p p e d w i t h f l u o r e s c e n c e immersion o i l . 28 RESULTS Injection Sites C o r t i c a l i n j e c t i o n s with either TB or PI were generally large, each covering an extensive area of the infused gyrus (see Table 1). The c o r t i c a l i n j e c t i o n s were found not to have d i r e c t l y involved the corpus callosum or subcortical regions, except i n two animals which showed p a r t i a l involvement of the corpus callosum with minimal encroachments into s u b c o r t i c a l regions following PI inj e c t i o n s . - In some cases, varying degrees of dye d i f f u s i o n into the corpus callosum or adjacent c o n t r a l a t e r a l cortex could be observed, but t h i s never spread into s u b c o r t i c a l regions such as the hippocampus. Dye d i f f u s i o n was only observed following PI injec t i o n s since the i n j e c t i o n s i t e s following TB infusions were more r e s t r i c t e d as well as more necrotic. Iontophoretic infusions of FG into the RTN resulted i n i n j e c t i o n s i t e s that were small with minimal necrosis. In a l l of the cases, the dense cores of the i n j e c t i o n s i t e s were centered i n the RTN, each f i l l i n g a s i g n i f i c a n t portion of the nucleus along i t s rostro-caudal axis. Although many of these in j e c t i o n s p r i m a r i l y involved the ventral RTN, i n some instances the cores of the in j e c t i o n s were located more c e n t r a l l y i n the RTN. In two cases, the center of the infusion was c l e a r l y r e s t r i c t e d to the r o s t r a l and caudal 29 poles of the RTN respectively. Figure 3 shows a three-dimensional reconstruction of a t y p i c a l RTN i n j e c t i o n . Photomicrographs i l l u s t r a t i n g two i n j e c t i o n s i t e s i n the RTN are shown i n Figure 4. Dye d i f f u s i o n outside the boundaries of the RTN generally involved varying degrees of encroachment into the v e n t r o l a t e r a l and/or ventromedial thalamic nuclei medially. In some cases, dye d i f f u s i o n was also observed i n the i n t e r n a l capsule l a t e r a l l y , the zona incerta v e n t r a l l y , and the anteroventral thalamic nucleus r o s t r a l l y depending on the location of the center of the RTN i n j e c t i o n s i t e . Furthermore, i n j e c t i o n s that were located i n the r o s t r a l pole of the RTN may have resulted i n minor spread of FG into the s t r i a terminalis or medio-dorsal t i p of the substantia innominata. In most animals, FG s p i l l s could be observed at the s i t e of entrance of the micropipette i . e . i n the c o n t r a l a t e r a l c o r t i c a l mantle, but never along the micropipette track i . e . into subcortical regions. D i s t r i b u t i o n of Single-Labeled C e l l s In t h i s study, the pattern of retrograde l a b e l i n g i s described for the hemisphere i p s i l a t e r a l to the i n j e c t i o n s i t e s since retrogradely labeled c e l l s observed i n the c o n t r a l a t e r a l hemisphere were l i k e l y to be the r e s u l t of the spread of the i n j e c t i o n s i t e i n the c o n t r a l a t e r a l cortex. The number of retrogradely single-labeled c e l l s following TB inje c t i o n s i n the cortex tended to be greater than following 30 F i g u r e 3. A r e c o n s t r u c t i o n of a t y p i c a l i n j e c t i o n s i t e i n the r e t i c u l a r t h a l a m i c nucleus (bold) through f i v e c o r o n a l planes ( m o d i f i e d from Paxinos and Watson, 1986). The a n t e r i o r -p o s t e r i o r c o o r d i n a t e s (from bregma) f o r the planes are as f o l l o w s : (A) -0.92mm; (B) -1.3mm; (C) -1.4mm; (D) -1.8mm; (E) -2.12mm. 31 32 F i g u r e 4. P h o t o m i c r o g r a p h s o f f l u o r o - g o l d i n j e c t i o n s i t e s t h r o u g h t h e r o s t r a l (A) and c a u d a l (C) p o l e s of t h e r e t i c u l a r t h a l a m i c n u c l e u s . The e x a c t l o c a t i o n o f t h e i n j e c t i o n s i t e s can be compared w i t h t h e b o u n d a r i e s o f t h e r e t i c u l a r t h a l a m i c n u c l e u s as d e f i n e d w i t h p a r v a l b u m i n i m m u n o s t a i n i n g (B and D; see a l s o d i s c u s s i o n f o r d e t a i l s ) . Note t h a t t h e s e i n j e c t i o n s i n v o l v e d m i n i m a l d i f f u s i o n o u t s i d e t h e b o u n d a r i e s o f t h e r e t i c u l a r t h a l a m i c n u c l e u s . A b b r e v i a t i o n : r t n , r e t i c u l a r t h a l a m i c n u c l e u s . ( m a g n i f i c a t i o n : A + B, 8x; C + D, 93x) . 33 34 35 comparable PI i n f u s i o n s , p o s s i b l y r e f l e c t i n g d i f f e r e n c e s i n the s e n s i t i v i t i e s of the t r a c e r s . Furthermore, T B - l a b e l e d c e l l s g e n e r a l l y tended t o be more d e f i n e d m o r p h o l o g i c a l l y . The d i s t r i b u t i o n of l a b e l e d c o r t i c o p e t a l c e l l s was, however, s i m i l a r f o r the two t r a c e r s ; and c o n s i s t e n t w i t h p r e v i o u s r e p o r t s (see g e n e r a l i n t r o d u c t i o n ) . Numerous l a b e l e d c e l l s were observed i n the t e l e n c e p h a l o n , diencephalon and mesopontine tegmentum. In the b a s a l f o r e b r a i n , l a b e l e d c o r t i c o p e t a l p e r i k a r y a were mainly l o c a t e d i n the r e g i o n s encompassing the h o r i z o n t a l limb of the d i a g o n a l band, ma g n o c e l l u l a r p r e o p t i c area, v e n t r a l p a l l i d u m , s u b s t a n t i a innominata and nucleus b a s a l i s m a g n o c e l l u l a r i s , f o l l o w i n g the nomenclature of Paxinos and Watson (1986). In the mesopontine tegmentum, many l a b e l e d neurons were observed i n the l a t e r o d o r s a l tegmental nucleus, raphe n u c l e i and, to a l e s s e r degree, i n the r o s t r a l p a r t of the pedunculopontine tegmental nucleus. Other r e g i o n s t h a t showed numerous c o r t i c o p e t a l c e l l s t h a t were r e t r o g r a d e l y l a b e l e d i n c l u d e d the s u b s t a n t i a n i g r a and v a r i o u s t h a l a m i c n u c l e i . Of the t o t a l number of s i n g l e - l a b e l e d neurons (FG) t h a t were observed i n the b a s a l f o r e b r a i n and i n the pedunculopontine and l a t e r o d o r s a l tegmental n u c l e i of the mesopontine tegmentum f o l l o w i n g t r a c e r i n j e c t i o n i n t o the RTN, 80% were found i n the b a s a l f o r e b r a i n . The p a t t e r n of r e t r o g r a d e l a b e l i n g i n the b a s a l f o r e b r a i n was e s s e n t i a l l y s i m i l a r r e g a r d l e s s of the d o r s o v e n t r a l l o c a t i o n of the core of the i n j e c t i o n s i t e w i t h i n the RTN. B a s a l f o r e b r a i n c e l l s i n n e r v a t i n g the RTN were observed i n the v e r t i c a l and h o r i z o n t a l limbs of the d i a g o n a l band, ma g n o c e l l u l a r p r e o p t i c area, v e n t r a l p a l l i d u m , s u b s t a n t i a innominata, nucleus b a s a l i s m a g n o c e l l u l a r i s and ansa l e n t i c u l a r i s . The m a j o r i t y of these c e l l s (90%) were l o c a t e d p o s t e r i o r to the d e c u s s a t i o n of the a n t e r i o r commissure (caudal b a s a l f o r e b r a i n ) . Neurons i n the c a u d a l b a s a l f o r e b r a i n were found p r i m a r i l y (>70%) i n the r e g i o n s encompassing the v e n t r a l p a l l i d u m , s u b s t a n t i a innominata and nucleus b a s a l i s m a g n o c e l l u l a r i s and, t o a l e s s e r degree, ansa l e n t i c u l a r i s f o l l o w i n g the nomenclature of Paxinos and Watson (1986). In a d d i t i o n , the d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l e d p e r i k a r y a i n the r e g i o n of the nucleus b a s a l i s m a g n o c e l l u l a r i s i n c l u d e d neurons l o c a t e d i n the medial globus p a l l i d u s as w e l l as neurons embedded i n the i n t e r n a l c a p s u l e (see F i g u r e 5). More r o s t r a l l y , l a b e l e d somata were o c c a s i o n a l l y observed near the bed nucleus of the s t r i a t e r m i n a l i s . In the r o s t r a l b a s a l f o r e b r a i n , almost a l l of the remaining r e t r o g r a d e l y l a b e l e d neurons were found i n the h o r i z o n t a l limb of the d i a g o n a l band and v e n t r a l p a l l i d u m . In a d d i t i o n , l a b e l e d neurons were o c c a s i o n a l l y observed i n the v e r t i c a l limb of the d i a g o n a l band, but r a r e l y i n the medial septum. The b a s a l f o r e b r a i n p r o j e c t i o n system i n n e r v a t i n g the RTN may be t o p o g r a p h i c a l l y o r g a n i z e d s i n c e the p a t t e r n of r e t r o g r a d e l y l a b e l e d c e l l s f o l l o w i n g i n j e c t i o n s c e n t e r e d i n the c a u d a l p o l e of the RTN tended to be r e s t r i c t e d almost e x c l u s i v e l y to the caudal b a s a l f o r e b r a i n , and p r i m a r i l y t o Figure 5. A pair of photomicrographs comparing the d i s t r i b u t i o n of labeled neurons projecting to the r e t i c u l a r nucleus of the thalamus (A) with labeled neurons projecting to the cortex (B) i n the region of the nucleus basalis magnocellularis, medial globus p a l l i d u s and i n t e r n a l capsule. Note the overlap i n the d i s t r i b u t i o n of these two groups of retrogradely labeled neurons. Although i t i s d i f f i c u l t to assess at low magnification, two double-labeled neurons are shown (arrows). Abbreviation: i c , i n t e r n a l capsule, (magnification: 72x). 39 the r e g i o n s encompassing the nucleus b a s a l i s m a g n o c e l l u l a r i s and s u b s t a n t i a innominata. However, i t i s p o s s i b l e t h a t these c a u d a l l y c e n t e r e d i n j e c t i o n s i t e s , which were t y p i c a l l y s m a l l , f a i l e d t o cover adequately the t e r m i n a l f i e l d s of the p r o j e c t i n g neurons of the remaining r o s t r a l r e g i o n s of the b a s a l f o r e b r a i n . The m o r p h o l o g i c a l appearance of r e t r o g r a d e l y l a b e l e d b a s a l f o r e b r a i n neurons f o l l o w i n g i n j e c t i o n i n t o the RTN v a r i e d . The shapes of the RTN p r o j e c t i n g neurons ranged from l a r g e m u l t i p o l a r c e l l s , many of which were observed i n the h o r i z o n t a l limb of the d i a g o n a l band, m a g n o c e l l u l a r p r e o p t i c area and nucleus b a s a l i s m a g n o c e l l u l a r i s (see F i g u r e 6); to s m a l l e r c e l l s w i t h o v a l or f u s i f o r m shapes, which were numerous i n the v e n t r a l p a l l i d u m and s u b s t a n t i a innominata. A l a r g e p r o p o r t i o n of the RTN p r o j e c t i n g neurons t h a t were l a b e l e d throughout the r o s t r o - c a u d a l extent of the b a s a l f o r e b r a i n was admixed w i t h l a b e l e d c o r t i c o p e t a l neurons. T h i s was p a r t i c u l a r l y the case i n the h o r i z o n t a l limb of the d i a g o n a l band, nucleus b a s a l i s m a g n o c e l l u l a r i s and s u b s t a n t i a innominata. In a d d i t i o n , l a b e l e d neurons p r o j e c t i n g t o the RTN tended t o form lo o s e c l u s t e r s i n these r e g i o n s . Other c l u s t e r s of F G - l a b e l e d somata t h a t were c o n s i s t e n t l y observed were found i n the re g i o n s encompassing the v e n t r a l p a l l i d u m , p a r t i c u l a r l y i n an area l o c a t e d immediately v e n t r a l t o the a n t e r i o r commissure; and m a g n o c e l l u l a r p r e o p t i c area. Labeled neurons i n the v e n t r a l p a l l i d u m were g e n e r a l l y not i n t e r m i x e d w i t h r e t r o g r a d e l y l a b e l e d c o r t i c o p e t a l neurons, s i n c e these F i g u r e 6. Photomicrograph of neurons t h a t were r e t r o g r a d e l y l a b e l e d f o l l o w i n g f l u o r o g o l d i n j e c t i o n i n the r e t i c u l a r t h a l a m i c nucleus. These neurons were observed i n the r e g i o n of the nucleus b a s a l i s m a g n o c e l l u l a r i s . Note the m o r p h o l o g i c a l f e a t u r e s t y p i c a l of ma g n o c e l l u l a r neurons i . e . l a r g e and m u l t i p o l a r , ( m a g n i f i c a t i o n : 504x). 41 I 42 FG-labeled neurons tended to be located more d o r s a l l y than labeled c o r t i c o p e t a l neurons. In the magnocellular preoptic area, RTN projecting neurons tended to aggregate i n the medial part where they were also intermingled with c o r t i c o p e t a l neurons. However, some of the retrogradely labeled c e l l s projecting to the RTN that were observed i n the magnocellular preoptic area extended l a t e r a l l y and were generally not admixed with labeled c o r t i c o p e t a l neurons. Numerous single-labeled neurons projecting to the RTN were also observed i n various p r i n c i p a l and intralaminar thalamic n u c l e i . In some cases, single-labeled c e l l s were observed i n the substantia nigra (mainly pars r e t i c u l a t a ) , entopeduncular nucleus and cortex; possibly r e f l e c t i n g the involvement of p r i n c i p a l thalamic n u c l e i , such as those of the ventrobasal complex, i n the i n j e c t i o n s i t e s (see discussion). In the mesopontine tegmentum, neurons that were retrogradely labeled following i n j e c t i o n s into the RTN were found pr i m a r i l y i n the pedunculopontine tegmental nucleus, dorsal raphe, c e n t r a l tegmental area, laterodorsal tegmental nucleus and locus ceruleus. Of the t o t a l number of retrogradely labeled c e l l s (FG) found i n the pedunculopontine and laterodorsal tegmental n u c l e i , 80-90% were observed i n the pedunculopontine tegmental nucleus. With the exception of the FG-labeled neurons found i n the central tegmental area, FG-labeled neurons i n the mesopontine tegmentum were generally adjacent or intermingled with neurons that were retrogradely labeled following c o r t i c a l i n j e c t i o n s . 43 This was p a r t i c u l a r l y the case i n the laterodorsal tegmental nucleus and dorsal raphe. In the region of the pedunculopontine tegmental nucleus, neurons that were labeled following i n j e c t i o n s into the RTN and those that were labeled following c o r t i c a l injections were found primarily i n the r o s t r a l and ce n t r a l portions of the pedunculopontine tegmental nucleus, beginning at a l e v e l just posterior to the caudal pole of the substantia nigra. The morphological appearance of the neurons labeled with FG that were observed i n the mesopontine tegmentum varied from large multipolar neurons, as p a r t i c u l a r l y observed i n the substantia nigra, dorsal raphe, central tegmental area and locus ceruleus; to medium size multipolar c e l l s primarily observed i n the pedunculopontine and laterodorsal tegmental nu c l e i . D i s t r i b u t i o n of Double-Labeled C e l l s Basal forebrain neurons that were double-labeled following PI and FG in j e c t i o n s into both the cortex and RTN were found almost exclu s i v e l y i n the i p s i l a t e r a l caudal basal forebrain i . e . posterior to the l e v e l of the decussation of the anterior commissure. The d i s t r i b u t i o n of double-labeled c e l l s i n the caudal basal forebrain was remarkably similar i n a l l injected animals. Figure 7 shows the representative d i s t r i b u t i o n of double-labeled c e l l s (FG + PI) versus single-labeled c e l l s (FG) through s i x coronal planes of the basal forebrain. In 44 F i g u r e 7. R e p r e s e n t a t i v e d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l e d neurons observed i n the b a s a l f o r e b r a i n t h a t i n n e r v a t e the r e t i c u l a r nucleus of the thalamus ( s i n g l y l a b e l e d ) or t h a t i n n e r v a t e both the r e t i c u l a r t h a l a m i c nucleus and c o r t e x (doubly l a b e l e d ) . The p a t t e r n of d i s t r i b u t i o n i s presented through s i x c o r o n a l planes ( m o d i f i e d from Paxinos and Watson, 1986). The a n t e r o - p o s t e r i o r (from bregma) c o o r d i n a t e s f o r each of the c o r o n a l plane are as f o l l o w s : (A) +0.2mm; (B) -0.3mm; (C) -0.8mm; (D) -1.3mm; (E) -1.8mm; (F) -2.3mm. Symbols: «, approximately f i v e F G - l a b e l e d neurons i n n e r v a t i n g the r e t i c u l a r t h a l a m i c nucleus; *, a neuron l a b e l e d w i t h both FG and PI ( c o r t i c o p e t a l ) . A b b r e v i a t i o n s : ac, a n t e r i o r commissure; vp, v e n t r a l p a l l i d u m ; hdb, h o r i z o n t a l limb of the di a g o n a l band; nbm, nucleus b a s a l i s of Meynart; s i , s u b s t a n t i a innominata; i c , i n t e r n a l c a p s u l e ; a l , ansa l e n t i c u l a r i s . Neurons observed i n the r e g i o n of the hdb a l s o i n c l u d e d neurons found i n the mag n o c e l l u l a r p r e o p t i c area although t h i s i s not shown on the p l a t e s . A l s o not shown i s the d i s t r i b u t i o n of s i n g l y l a b e l e d neurons i n n e r v a t i n g the c o r t e x . 45 46 f i v e animals, the p r o p o r t i o n of d o u b l e - l a b e l e d c e l l s r e l a t i v e to the t o t a l number of F G - l a b e l e d neurons (:EG-'^B-1 x 100) FG counted through the cau d a l b a s a l f o r e b r a i n (see method s e c t i o n f o r d e t a i l s on c e l l counts) was c a l c u l a t e d . A summary of these r e s u l t s i s presented i n Tab l e 2. The p r o p o r t i o n of d o u b l e - l a b e l e d c e l l s r e l a t i v e t o the t o t a l number of s i n g l e -F G - l a b e l e d c e l l s counted through the extent of the c a u d a l b a s a l f o r e b r a i n ranged from 10 to 23% wit h an average of 14%. The m a j o r i t y (at l e a s t 80%) of d o u b l e - l a b e l e d c e l l s were found i n the r e g i o n s encompassing the v e n t r a l p a l l i d u m (caudal p o r t i o n ) , s u b s t a n t i a innominata, nucleus b a s a l i s m a g n o c e l l u l a r i s and ansa l e n t i c u l a r i s . The p r o p o r t i o n of d o u b l e - l a b e l e d c e l l s observed i n these r e g i o n s accounted f o r an average of 13% of the t o t a l number of s i n g l e - l a b e l e d c e l l s (FG) counted through the cau d a l b a s a l f o r e b r a i n ; or an average of 15% of the t o t a l number of s i n g l e - l a b e l e d neurons (FG) counted through the v e n t r a l p a l l i d u m , s u b s t a n t i a innominata, nucleus b a s a l i s m a g n o c e l l u l a r i s and ansa l e n t i c u l a r i s . I t i s estimated t h a t the p r o p o r t i o n of d o u b l e - l a b e l e d c e l l s f o r the r e g i o n of the nucleus b a s a l i s m a g n o c e l l u l a r i s ( i n c l u d i n g neurons found i n the medial globus p a l l i d u s and neurons embedded i n the i n t e r n a l capsule) alone may have been as h i g h as 25%, s i n c e d o u b l e - l a b e l e d c e l l s tended t o be observed l e s s f r e q u e n t l y i n the v e n t r a l p a l l i d u m , and o n l y r a r e l y i n the area of the ansa l e n t i c u l a r i s . S i m i l a r c a l c u l a t i o n s o b t a i n e d f o r the r e g i o n s encompassing the h o r i z o n t a l limb of the d i a g o n a l band and ma g n o c e l l u l a r p r e o p t i c area r e v e a l e d t h a t 47 Table 2. Summary of the r e s u l t s o b t a i n e d i n experiment one. The number of r e t r o g r a d e l y l a b e l e d neurons f o l l o w i n g i n j e c t i o n i n the r e t i c u l a r t h a l a m i c nucleus t h a t were counted through v a r i o u s r e g i o n s of the b a s a l f o r e b r a i n and mesopontine tegmentum.of f i v e animals (see method f o r d e t a i l s on c e l l counts) are presented. The number of neurons t h a t were doubly l a b e l e d i n each of the r e g i o n s examined i s a l s o p r e s e n t e d (values i n b r a c k e t s ) . Note the v a l u e s o b t a i n e d w i t h animal C-7, which r e f l e c t s a p o s s i b l e t o p o g r a p h i c a l o r g a n i z a t i o n as d i s c u s s e d i n more d e t a i l s i n the t e x t . The percentage c a l c u l a t i o n s p r e s e n t e d i n t h i s study were based on the v a l u e s shown i n t h i s t a b l e . A b b r e v i a t i o n s : HDB, h o r i z o n t a l limb of the d i a g o n a l band; MCPO, ma g n o c e l l u l a r p r e o p t i c area; VP, v e n t r a l p a l l i d u m ; SI, s u b s t a n t i a innominata; NBM; nucleus b a s a l i s m a g n o c e l l u l a r i s ; PPT, pedunculopontine tegmental nucleus; LDT, l a t e r o d o r s a l tegmental nucleus; RTN, r e t i c u l a r t h a l a m i c nucleus; x; mean. Table 2. Numbers of Labeled Neurons I n n e r v a t i n g the RTN or RTN + c o r t e x (values i n brac k e t s ) B r a i n Regions Rats HDB\MCPO VP\SI\NBM PPT LDT C-7 0 96 (22) 40 (3) 17 (1) C-13 141 (20) 437 (85) 71 (10) 18 (2) C-14 46 (6) 275 (43) 62 (8) 20 (2) C-16 14 (1) 327 (43) 45 (7) 16 (0) C-21 84 (5) 665 (76) 76 (8) 13 (1) x=57 (6) x=360 (5) x=59 (7) x=17 (2) 49 the p r o p o r t i o n of d o u b l e - l a b e l e d c e l l s accounted f o r an average of 1.5% r e l a t i v e t o the t o t a l number of F G - l a b e l e d c e l l s t h a t were counted through the ca u d a l b a s a l f o r e b r a i n ; or an average of 11% of the t o t a l number of F G - l a b e l e d c e l l s t h a t were counted through the caudal p o r t i o n s of the h o r i z o n t a l limb of the d i a g o n a l band and m a g n o c e l l u l a r p r e o p t i c area alone (see Table 2 ) . In the r e g i o n s encompassing the pedunculopontine and l a t e r o d o r s a l tegmental n u c l e i , d o u b l e - l a b e l e d c e l l s were almost e x c l u s i v e l y found i n the pedunculopontine tegmental nucleus (see Ta b l e 2 ). D o u b l e - l a b e l e d c e l l s i n t h i s r e g i o n accounted f o r an average of 9%-12% of the t o t a l number of s i n g l e - l a b e l e d c e l l s (FG) t h a t were counted across both the pedunculopontine and l a t e r o d o r s a l tegmental n u c l e i or a c r o s s the pedunculopontine tegmental nucleus alone. F i g u r e 8 shows the r e p r e s e n t a t i v e d i s t r i b u t i o n of these neurons through s i x c o r o n a l planes of the mesopontine tegmentum. In F i g u r e 9, examples of d o u b l e - l a b e l e d c e l l s t h a t were observed i n the nucleus b a s a l i s m a g n o c e l l u l a r i s and pedunculopontine tegmental nucleus are shown. Although counts were not o b t a i n e d , numerous d o u b l e - l a b e l e d c e l l s were observed i n the d o r s a l raphe, s u b s t a n t i a n i g r a ( p r i m a r i l y i n the pars r e t i c u l a t a ) and, o c c a s i o n a l l y , i n the r e g i o n of the l o c u s c e r u l e u s . In the diencephalon, double-l a b e l e d c e l l s were a l s o observed i n v a r i o u s i n t r a l a m i n a r t h a l a m i c n u c l e i . However, some of these d o u b l e - l a b e l e d neurons may have r e s u l t e d i n p a r t from the involvement of 50 F i g u r e 8. R e p r e s e n t a t i v e d i s t r i b u t i o n of r e t r o g r a d e l y l a b e l e d neurons observed i n the pedunculopontine and l a t e r o d o r s a l tegmental n u c l e i t h a t i n n e r v a t e the r e t i c u l a r t h a l a m i c nucleus ( s i n g l y l a b e l e d ) or t h a t i n n e r v a t e both the r e t i c u l a r t h a l a m i c nucleus and c o r t e x (doubly l a b e l e d ) . The p a t t e r n of d i s t r i b u t i o n i s presented through s i x c o r o n a l planes ( m o d i f i e d from Paxinos and Watson, 1986) of the mesopontine tegmentum. The a n t e r o - p o s t e r i o r c o o r d i n a t e s (from bregma) f o r each of the planes are as f o l l o w s : (A) -6.8mm; (B) -7.3mm; (C) -7.8mm; (D) -8.3mm; (E) -8.8mm; (F) -9.3mm). Symbols: •, approximately three F G - l a b e l e d neurons i n n e r v a t i n g the r e t i c u l a r t h a l a m i c nucleus; *, a F G - l a b e l e d neuron a l s o l a b e l e d w i t h PI ( c o r t i c o p e t a l ) . A b b r e v i a t i o n s : eg, c e n t r a l gray; dtb, d o r s a l tegmental bundle; mlf, medial l o n g i t u d i n a l f a s c i c u l u s ; ppt, pedunculopontine tegmental nucleus; cp, c e r e b r a l peduncle; ml; medial lemniscus; r s , r u b r o s p i n a l t r a c t ; l d t , l a t e r o d o r s a l tegmental n u c l e u s . Not shown i s the d i s t r i b u t i o n of s i n g l y l a b e l e d c o r t i c o p e t a l neurons. Figure 9. Two pairs of photomicrographs each showing a neuron (arrow) that was doubly labeled following injections i n the RTN and cortex i n d i c a t i n g that t h i s neuron innervated the RTN and cortex through axon c o l l a t e r a l s . These double-labeled neurons were observed i n the nucleus basalis magnocellularis (A + B) and pedunculopontine tegmental nucleus (C + D). Neurons that were labeled following tracer i n j e c t i o n i n the RTN are presented on the far l e f t while those labeled following c o r t i c a l injections are presented on the far ri g h t , (magnification: A + B, 283x; C + D, 126x). 54 f i b e r s o f passage t r a v e l i n g through the RTN. C o n t r o l s t u d i e s are needed to c o n f i r m these r e s u l t s . The d i s t r i b u t i o n o f c e l l s t h a t were d o u b l e - l a b e l e d w i t h FG and TB was s i m i l a r t o t h a t d e s c r i b e d when PI was i n j e c t e d i n the c o r t e x . D o u b l e - l a b e l e d c e l l s w i t h FG and TB r e s u l t e d i n a b l e n d o f the two t r a c e r s t h a t v a r i e d i n c o l o r ( b l u e - l a v e n d e r shades) depending on the i n t e n s i t y of l a b e l i n g f o r each dye. A c c o r d i n g l y , a l though many neurons were u n e q u i v o c a l l y d o u b l e -l a b e l e d w i t h FG and TB (see F i g u r e 1 0 ) , d o u b l e - l a b e l i n g of some neurons was more s u b j e c t t o i n t e r p r e t a t i o n than w i t h PI i n j e c t i o n s (see F i g u r e 1 1 ) . No tw i th s t and ing t h i s p rob lem, the p r o p o r t i o n o f d o u b l e - l a b e l e d c e l l s w i t h FG and TB d i d not appear t o d i f f e r s i g n i f i c a n t l y than t h a t observed f o l l o w i n g PI c o r t i c a l i n j e c t i o n s a l though q u a n t i f i c a t i o n of doub le l a b e l i n g was not at tempted f o r t h i s group of an ima l s . Immunohistochemical Ana ly ses A s u b p o p u l a t i o n of neurons t h a t were r e t r o g r a d e l y l a b e l e d f o l l o w i n g i n j e c t i o n s i n t o the RTN were found t o c o n t a i n c h o l i n e a c e t y l t r a n s f e r a s e i m m u n o r e a c t i v i t y . In the b a s a l f o r e b r a i n , a lmost a l l of the F G - l a b e l e d c e l l s t h a t were a l s o ChAT immunoreact ive were observed i n the r e g i o n o f the nuc leus b a s a l i s m a g n o c e l l u l a r i s . A few of these c e l l s were a l s o observed i n the r e g i o n s encompassing the s u b s t a n t i a innominata and h o r i z o n t a l l imb of the d i a g o n a l band. I t was e s t i m a t e d t h a t up t o 2 0 % o f the F G - l a b e l e d c e l l s t h a t were observed i n F i g u r e 10. C o l o r photomicrograph of a neuron (arrow) t h a t was d o u b l e - l a b e l e d w i t h f l u o r o - g o l d (from the r e t i c u l a r nucleus of the thalamus) and t r u e blue ( c o r t i c o p e t a l ) i n the r e g i o n of the nucleus b a s a l i s m a g n o c e l l u l a r i s . D o u b l e - l a b e l e d neurons r e s u l t i n g from the r e t r o g r a d e t r a n s p o r t of these t r a c e r s through axon c o l l a t e r a l s r e s u l t e d i n a b l e n d of the two c o l o r s which c o u l d be assessed w i t h a s i n g l e f i l t e r b l o c k as i l l u s t r a t e d i n t h i s photomicrograph. S i n g l e l a b e l e d neurons l a b e l e d w i t h t r u e b l u e (deep blue) on the one hand, and f l u o r o - g o l d on the other hand are a l s o shown, ( m a g n i f i c a t i o n : 521x). 56 F i g u r e 11. C o l o r photomicrograph of a neuron (arrow) t h a t was d o u b l e - l a b e l e d w i t h f l u o r o - g o l d (from the r e t i c u l a r t h a l a m i c nucleus) and propidium i o d i d e ( c o r t i c o p e t a l ) i n the r e g i o n of the pedunculopontine tegmental nucleus. Neurons l a b e l e d w i t h f l u o r o - g o l d on the one hand (blue) and propidium i o d i d e (red) on the other are a l s o shown. I t i s thought t h a t neurons t h a t were d o u b l e - l a b e l e d as i l l u s t r a t e d i n t h i s photomicrograph had t r a n s p o r t e d each t r a c e r s through axon c o l l a t e r a l s , ( m a g n i f i c a t i o n : 260x). 59 the r e g i o n of the nucleus b a s a l i s m a g n o c e l l u l a r i s were c h o l i n e r g i c . A l a r g e p r o p o r t i o n (up to 50%) of the FG - l a b e l e d neurons i n t h i s r e g i o n t h a t were ChAT immunoreactive appeared a l s o t o have been l a b e l e d w i t h PI ( t r i p l e - l a b e l e d c e l l s ) . F i g u r e 12 shows photomicrographs of a t r i p l e - l a b e l e d neuron observed i n the nucleus b a s a l i s m a g n o c e l l u l a r i s . However, r e l i a b l e counts were d i f f i c u l t t o o b t a i n s i n c e nucleus b a s a l i s neurons g e n e r a l l y d i d not s t a i n i n t e n s e l y w i t h ChAT a n t i b o d i e s . T h i s problem was f u r t h e r compounded by the f a c t t h a t background s t a i n i n g was h i g h f o l l o w i n g immunohistochemistry. N e v e r t h e l e s s , b a s a l f o r e b r a i n s e c t i o n s processed f o r ChAT immunoreactivity i n two animals were s u f f i c i e n t l y i n t e n s e t o al l o w s e m i - q u a n t i t a t i v e a n a l y s e s . In the mesopontine tegmentum, the v a s t m a j o r i t y of neurons r e t r o g r a d e l y l a b e l e d w i t h FG t h a t were a l s o immunoreactive wit h ChAT a n t i b o d i e s were observed i n the pedunculopontine tegmental n u c l e u s . In t h i s r e g i o n , approximately 40-60% of the F G - l a b e l e d c e l l s a l s o s t a i n e d w i t h ChAT a n t i b o d i e s , p a r t i c u l a r l y i n i t s r o s t r a l p o r t i o n . Furthermore, some of the c e l l s (10-20%) t h a t were d o u b l e - l a b e l e d w i t h FG and ChAT a n t i b o d i e s a l s o appeared t o have been l a b e l e d w i t h PI. However, h i g h background s t a i n i n g prevented r e l i a b l e c e l l counts. T r i p l e - l a b e l e d neurons were a l s o observed i n the l a t e r o d o r s a l tegmental nucleus, but o n l y o c c a s i o n a l l y . The d i s t r i b u t i o n of SOM-positive neurons i n the t e l e n c e p h a l o n was as r e p o r t e d p r e v i o u s l y (Vincent e t a l . , 60 Figure 12. A series of photomicrographs i l l u s t r a t i n g a neuron (arrow) that was retrogradely labeled with fluoro-gold following i n j e c t i o n i n the RTN (A), with propidium iodide following i n j e c t i o n i n the cortex (B), and that was also ChAT immunoreactive (C), i n the region of the nucleus b a s a l i s magnocellularis. I t was therefore concluded that t h i s neuron was cholinergic and that i t innervated both the RTN and cortex through axon c o l l a t e r a l s , (magnification: 96x). 62 1985). For example, many SOM-immunoreactive neurons have been observed i n v a r i o u s c o r t i c a l areas, the s t r i a t u m and the entopeduncular nucleus. In the b a s a l f o r e b r a i n , s c a t t e r e d SOM-positive neurons were observed p r i m a r i l y through the re g i o n s of the h o r i z o n t a l limb of the d i a g o n a l band and v e n t r a l p a l l i d u m . However, none of the b a s a l f o r e b r a i n neurons t h a t were r e t r o g r a d e l y l a b e l e d f o l l o w i n g FG i n j e c t i o n s i n t o the RTN were immunoreactive f o r so m a t o s t a t i n a n t i b o d i e s . In one animal, i n which the RTN i n j e c t i o n s i t e i n c l u d e d t o some degree the v e n t r o l a t e r a l and ventromedial t h a l a m i c n u c l e i , a few F G - l a b e l e d neurons observed i n the entopeduncular nucleus appeared t o be a l s o immunoreactive w i t h SOM a n t i b o d i e s . Numerous F G - l a b e l e d neurons of the b a s a l f o r e b r a i n were found t o be immunoreactive w i t h PV a n t i b o d i e s . The v a s t m a j o r i t y of these d o u b l e d - l a b e l e d neurons (FG + PV) were found i n the r e g i o n of the v e n t r a l p a l l i d u m , and i n p a r t i c u l a r , i n an area j u s t below the a n t e r i o r commissure (see F i g u r e 13). At l e a s t 50% of the FG - l a b e l e d c e l l s found i n t h i s r e g i o n were immunoreactive w i t h PV a n t i b o d i e s . The r e g i o n s encompassing the nucleus b a s a l i s m a g n o c e l l u l a r i s and medial globus p a l l i d u s a l s o c o n t a i n e d F G - l a b e l e d neurons t h a t were PV-immunoreactive (see F i g u r e 12). In these r e g i o n s , the p r o p o r t i o n of double-l a b e l e d neurons (PV + FG l a b e l e d ) was estimated t o range between 20-25%. O c c a s i o n a l l y , P V - p o s i t i v e c e l l s were observed to have been d o u b l e - l a b e l e d w i t h PI ( c o r t i c o p e t a l ) or w i t h both PI and FG ( t r i p l e - l a b e l e d ) i n the re g i o n s of the nucleus Figure 13. Pairs of photomicrographs showing labeled neurons (arrows) projecting to the r e t i c u l a r thalamic nucleus that were also immunoreactive for parvalbumin i n the regions of the nucleus basalis magnocellularis (A + B) and ventral pallidum (C + D). The open arrow shows a neuron labeled only with fluoro-gold (from the r e t i c u l a r thalamic nucleus). Abbreviation: ac, anterior commissure, (magnification: 109x). 65 b a s a l i s m a g n o c e l l u l a r i s and v e n t r a l p a l l i d u m . F i g u r e 14 shows an example of a t r i p l e - l a b e l e d c e l l (FG + PI + PV). However, double-or t r i p l e - l a b e l i n g i n v o l v i n g PI were g e n e r a l l y e q u i v o c a l due to hig h background s t a i n i n g . Figure 14. A series of photomicrographs i l l u s t r a t i n g at least one neuron (arrow) i n the region of the ventral pallidum\substantia innominata that was retrogradely labeled following i n j e c t i o n i n the r e t i c u l a r thalamic nucleus (A) and following i n j e c t i o n s i n the cortex (B). This neuron was also found to be immunoreactive with parvalbumin (C). (magnification: 145x). c 68 DISCUSSION T e c h n i c a l C o n s i d e r a t i o n s The p o s s i b i l i t y t h a t f a l s e - p o s i t i v e l a b e l i n g may undermine the v a l i d i t y of the r e s u l t s must be addressed b e f o r e r e a c h i n g any c o n c l u s i o n s . A p a r t i c u l a r problem w i t h any t r a c t - t r a c i n g study p e r t a i n s to the p o s s i b i l i t y t h a t r e t r o g r a d e l a b e l i n g r e s u l t e d i n p a r t from the involvement of f i b e r s c o u r s i n g through the i n j e c t i o n s i t e . In the case of the c o r t i c a l i n j e c t i o n s , the problem of f i b e r s of passage i s o n l y p e r t i n e n t to the extent t h a t dye d i f f u s i o n had i n v o l v e d s u b c o r t i c a l areas t h a t r e c e i v e d b a s a l f o r e b r a i n a f f e r e n t s such as the hippocampus. Although t h i s cannot be completely r u l e d out, i t i s u n l i k e l y t h a t the r e s u l t s of t h i s study have been a f f e c t e d i n such manner. F i r s t , dye d i f f u s i o n i n t o s u b c o r t i c a l areas was r a r e l y observed. Secondly, the nature of the c o r t i c a l i n j e c t i o n s v a r i e d between animals without a f f e c t i n g s i g n i f i c a n t l y the p r o p o r t i o n or d i s t r i b u t i o n of d o u b l e - l a b e l e d c e l l s (FG + PI\TB). For example, the volume of the i n j e c t e d dye, the l o c a t i o n and number of c o r t i c a l i n j e c t i o n s d i f f e r e d i n many of the animals t h a t were examined. T h i r d l y , the number and d i s t r i b u t i o n of F G - l a b e l e d c e l l s t h a t were doubly l a b e l e d f o l l o w i n g c o r t i c a l i n j e c t i o n s d i d not appear t o d i f f e r i n the two animals t h a t showed p a r t i a l involvement of the corpus c a l l o s u m f o l l o w i n g some of the PI i n j e c t i o n s . Even more c o n v i n c i n g i s the f a c t t h a t the d i s t r i b u t i o n of double-69 l a b e l e d c e l l s d i d not appear to d i f f e r f o l l o w i n g TB i n j e c t i o n s d e s p i t e the f a c t t h a t dye d i f f u s i o n was r e s t r i c t e d to the c o r t i c a l mantle. Thus, i t i s v e r y u n l i k e l y t h a t dye d i f f u s i o n i n t o s u b c o r t i c a l r e g i o n s , even i f undetected, had c o n t r i b u t e d s i g n i f i c a n t l y t o the r e s u l t s of the p r e s e n t study. The p o s s i b i l i t y t h a t f a l s e - p o s i t i v e s may have d i m i n i s h e d the v a l i d i t y of the r e s u l t s due to l a b e l i n g produced by f i b e r s of passage f o l l o w i n g FG i n j e c t i o n s i n t o the RTN i s a l s o u n l i k e l y f o r a number of reasons. F i r s t , a l l RTN i n j e c t i o n s were performed w i t h i o n t o p h o r e s i s v i a p e n e t r a t i o n s through the c o n t r a l a t e r a l hemisphere. P o s s i b l e contamination of the r e s u l t s due t o t r a c e r s p i l l a g e i n the i p s i l a t e r a l c o r t i c a l mantle was t h e r e f o r e avoided. Furthermore, t r a c e r s p i l l a g e was never observed along the m i c r o p i p e t t e t r a c k i . e . i n s u b c o r t i c a l r e g i o n s . However, FG s p i l l s were o f t e n observed at the s i t e of entrance of the m i c r o e l e c t r o d e , i n the c o n t r a l a t e r a l hemisphere. That such s p i l l s may have produced f a l s e - p o s i t i v e s i s u n l i k e l y s i n c e i t has been e s t a b l i s h e d t h a t c o r t i c o p e t a l c h o l i n e r g i c neurons i n both the b a s a l f o r e b r a i n and mesopontine tegmentum are almost e x c l u s i v e l y i p s i l a t e r a l i z e d ( B i g l e t a l . , 1982; Woolf e t a l . , 1984), a f i n d i n g t h a t was c o r r o b o r a t e d i n one c o n t r o l animal to be d i s c u s s e d below. Secondly, i o n t o p h o r e t i c i n f u s i o n of FG produced l i t t l e damage at the i n j e c t i o n s i t e t h e r e f o r e g r e a t l y r e d u c i n g the number of t r a n s e c t e d f i b e r s of passage and consequently, the amount of t r a c e r taken up by these f i b e r s . T h i r d l y , as w i t h c o r t i c a l i n j e c t i o n s , the exact l o c a t i o n of the core of RTN i n j e c t i o n s v a r i e d t o a c e r t a i n e x t e n t between animals without a f f e c t i n g the p a t t e r n of l a b e l i n g . Furthermore, i n a t l e a s t two animals, the i n j e c t i o n s were almost e x c l u s i v e l y l i m i t e d t o the RTN. Although one of these i n j e c t i o n s , which p r i m a r i l y encompassed the cau d a l RTN, d i d not y i e l d r e t r o g r a d e l a b e l i n g i n the r o s t r a l f o r e b r a i n (see r e s u l t s ) , the p a t t e r n and p r o p o r t i o n of d o u b l e - l a b e l e d c e l l s d i d not d i f f e r s i g n i f i c a n t l y i n the cau d a l b a s a l f o r e b r a i n i n comparison t o l e s s r e s t r i c t e d i n j e c t i o n s i t e s . F i n a l l y , t h a t b a s a l f o r e b r a i n f i b e r s may have been i n v o l v e d i n the RTN i n j e c t i o n s i t e i s v e r y u n l i k e l y when c o n s i d e r i n g the course taken by c o r t i c o p e t a l b a s a l f o r e b r a i n f i b e r s . Hence, Saper (1984) has shown t h a t most c o r t i c o p e t a l b a s a l f o r e b r a i n f i b e r s t r a v e l e d through (1) a medial pathway i n v o l v i n g the d i a g o n a l band, f o r n i x , genu of corpus c a l l o s u m and c i n g u l a t e bundle; and (2) a l a t e r a l pathway i n v o l v i n g the s u b s t a n t i a innominata, amygdala, p y r i f o r m c o r t e x and putamen. The p o s s i b i l i t y t h a t dye d i f f u s i o n o u t s i d e the boundaries of the RTN may have confounded the data was f u r t h e r addressed i n s e v e r a l c o n t r o l animals. In one animal, i n which the FG i n j e c t i o n was c e n t e r e d i n the a n t e r o v e n t r a l and anteromedial t h a l a m i c n u c l e i and i n v o l v e d a l s o a major s p i l l of the t r a c e r i n the c o n t r a l a t e r a l c o r t e x , l i t t l e r e t r o g r a d e l a b e l i n g was observed i n the b a s a l f o r e b r a i n . A c c o r d i n g l y , b a s a l f o r e b r a i n c e l l s t h a t were d o u b l e - l a b e l e d f o l l o w i n g such i n j e c t i o n were very r a r e . On the other hand, the p a t t e r n of r e t r o g r a d e l a b e l i n g i n the mesopontine tegmentum f o l l o w i n g FG i n f u s i o n into anterior thalamic nuclei was markedly d i f f e r e n t than following RTN i n j e c t i o n s . Hence, the vast majority of retrogradely labeled c e l l s was found i n the laterodorsal tegmental nucleus r e l a t i v e to the pedunculopontine tegmental nucleus. A small number of these laterodorsal tegmental c e l l s appeared to be have been double-labeled with PI. In a second animal, i n which the core of the FG i n j e c t i o n was centered too rostromedially, involving the s t r i a terminalis, bed nucleus of the s t r i a terminalis, i n t e r n a l capsule, globus p a l l i d u s , substantia innominata and r o s t r a l pole of the RTN, double-labeled c e l l s were observed only occasionally i n either the basal forebrain or i n the pedunculopontine and laterodorsal tegmental nuclei of the mesopontine tegmentum. Furthermore, i t i s possible that the observed double-labeled c e l l s r e f l e c t e d the p a r t i a l involvement of the RTN i n the i n j e c t i o n s i t e rather than being the r e s u l t of f i b e r s t r a v e l i n g through the i n t e r n a l capsule, for example. F i n a l l y , two animals received FG i n j e c t i o n s that were centered i n ventral thalamic nuclei including the ventromedial, v e n t r o l a t e r a l and ventroposterolateral thalamic n u c l e i . The pattern of retrograde la b e l i n g following these injec t i o n s was d i f f e r e n t than following RTN i n j e c t i o n s . FG-labeled c e l l s were found only occasionally i n the basal forebrain. In contrast, numerous FG-labeled neurons were observed i n the RTN, substantia nigra, mammillary bodies, entopeduncular nucleus, dorsal raphe, pedunculopontine and laterodorsal tegmental n u c l e i . Occasional double-labeled c e l l s (FG + PI) were observed i n the pedunculopontine tegmental nucleus, and to a lesser extent, i n the laterodorsal tegmental nucleus. False l a b e l i n g due to d i f f u s i o n of dye into g l i a or e p i t h e l i a l c e l l s rather i s another p o t e n t i a l source of data contamination. However, t h i s i s u n l i k e l y since g l i a and e p i t h e l i a l c e l l s could be r e a d i l y recognized on the basis of several c h a r a c t e r i s t i c s . For example, i n contrast to the majority of retrogradely labeled c e l l s , these c e l l s were small, spherical with no labeled dendrites; they were generally located near the i n j e c t i o n s i t e (which was never included i n the analysis of retrograde labeling) or i n blood vessel walls. Furthermore, i n sections not processed for immunohistochemistry, i t was possible to d i r e c t l y v e r i f y i f a neuron had been labeled by retrograde transport by alternating between emission f i l t e r s , since g l i a and e p i t h e l i a l c e l l s were intensely labeled regardless of the f i l t e r block used. The p o s s i b i l i t y that ChAT immunoreactive neurons (FITC) may have been mistaken for g l i a or e p i t h e l i a l c e l l s i s u n l i k e l y since ChAT immunostaining was c h a r a c t e r i s t i c a l l y not intense. I t i s therefore more probable that the proportion of doubly or t r i p l y - l a b e l e d neurons which had involved ChAT immunostaining was underestimated i n t h i s study as w i l l be discussed below. F i n a l l y , since PV immunostaining was generally intense, only neurons showing morphological c h a r a c t e r i s t i c s of basal forebrain neurons (large, multipolar etc.) .were included i n the analysis. C o l l a t e r a l i z e d Projections Innervating the RTN and Cortex The r e s u l t s of t h i s study have both confirmed and extended knowledge on the organization of basal forebrain and mesopontine tegmental projection systems that innervate the cortex and RTN. By using a t r i p l e - l a b e l i n g approach, the present inves t i g a t i o n has c l e a r l y established that a s i g n i f i c a n t portion (at l e a s t 15%) of either the basal forebrain or mesopontine tegmental input to the RTN i s composed of neurons with axons that bifurcate to innervate the cortex as well. The pattern of d i s t r i b u t i o n of double-labeled c e l l s has revealed that the nucleus basalis magnocellularis of the basal forebrain and the pedunculopontine tegmental nucleus of the mesopontine tegmentum are primary sources of these c o l l a t e r a l i z e d projections. Within these regions, i t was further demonstrated that a large portion of these c o l l a t e r a l i z e d projection systems are cholinergic. The r e l a t i v e contribution of the projection systems that were examined i n r e l a t i o n to the innervation of the RTN or RTN + cortex was assessed by way of semi-quantitative analyses. However, the r e s u l t s of these semi-quantitative analyses, as presented i n Table 2, must be interpreted with caution i . e . within the l i m i t s of the techniques used. Hence, the amount of retrograde l a b e l i n g observed i n t h i s study r e f l e c t e d most probably only a f r a c t i o n of the e n t i r e population of the 74 p r o j e c t i n g neurons under study s i n c e none of the FG i n j e c t i o n s had i n v o l v e d the e n t i r e extent of the RTN, and may have t h e r e f o r e f a i l e d t o cover t e r m i n a l f i e l d s of some p r o j e c t i n g neurons. Given t h a t f a l s e - p o s i t i v e l a b e l i n g was minimal (see above), i t i s t h e r e f o r e b e l i e v e d t h a t the percentage c a l c u l a t i o n s o b t a i n e d i n t h i s study can be i n t e r p r e t e d as rough i n d i c e s p r o v i d i n g the lower l i m i t s of the frequency of s i n g l e , double and t r i p l e l a b e l i n g . T h i s may have been p a r t i c u l a r l y the case f o l l o w i n g immunohistochemistry s i n c e v a r i o u s other f a c t o r s , such as f i x a t i o n p r o t o c o l s , may have been l e s s than o p t i m a l f o r the v i s u a l i z a t i o n of t r i p l y l a b e l e d neurons as w i l l be d i s c u s s e d below. The d i s t r i b u t i o n of l a b e l e d neurons i n the b a s a l f o r e b r a i n and mesopontine tegmentum f o l l o w i n g i n j e c t i o n s i n t o the RTN, a n t e r i o r or v e n t r o b a s a l thalamic n u c l e i as observed i n t h i s study i s i n c l o s e agreement w i t h e a r l i e r r e p o r t s ( H a l l a n g e r e t a l . , 1987; Levey e t a l . , 1987; see a l s o i n t r o d u c t i o n ) . The r e s u l t s of the immunohistochemical analyses have r e v e a l e d t h a t the RTN r e c e i v e s a major i n p u t from b a s a l f o r e b r a i n neurons t h a t c o n t a i n e d PV, and t h a t t h i s i n p u t may be c o l l a t e r a l i z e d t o i n n e r v a t e the c o r t e x as w e l l . Because r e l i a b l e immunohistofluorescence methods f o r gamma-aminobutyric a c i d (GABA) were not a v a i l a b l e , immunostaining f o r PV was chosen as an a l t e r n a t i v e method f o r i d e n t i f y i n g p o t e n t i a l GABAergic neurons s i n c e the d i s t r i b u t i o n of P V - c o n t a i n i n g neurons has been a s s o c i a t e d i n r e c e n t y ears w i t h many of the GABAergic systems of the mammalian b r a i n ( C e l i o and Heizmann, 1981; 75 Gerfen e t a l . , 1985). Hence, the RTN, which i s known to c o n t a i n a major p o p u l a t i o n of GABAergic neurons (Houser e t a l . , 1980), s t a i n e d i n t e n s e l y w i t h PV a n t i b o d i e s (see F i g u r e 4). Furthermore, the l o c a t i o n of F G - l a b e l e d c e l l s t h a t were a l s o immunoreactive w i t h PV a n t i b o d i e s i n the v e n t r a l p a l l i d u m was remarkably s i m i l a r t o t h a t of GABAergic neurons as r e v e a l e d by immunostaining f o r the s y n t h e s i z i n g enzyme of GABA (Young I I I e t a l . , 1984). T h i s i s a l s o c o n s i s t e n t w i t h the o b s e r v a t i o n t h a t w i t h the e x c e p t i o n of the r e g i o n of the nucleus b a s a l i s m a g n o c e l l u l a r i s , the d i s t r i b u t i o n of most PV-c o n t a i n i n g neurons was d i f f e r e n t than t h a t of C h A T - p o s i t i v e neurons as assessed i n the same animals. I t appears t h e r e f o r e q u i t e p l a u s i b l e t h a t the d o u b l e - l a b e l e d (PV + FG) neurons observed i n t h i s study are indeed GABAergic, although PV immunostaining does not c o n s t i t u t e d i r e c t evidence f o r GABA-c o n t a i n i n g neurons. I t would be i n t e r e s t i n g t o determine w i t h other methods i f these neurons are indeed GABAergic; and to v e r i f y the degree to which they may c o l l a t e r a l i z e t o i n n e r v a t e both the RTN and c o r t e x . Furthermore, the presence of a moderate number of PV - c o n t a i n i n g neurons i n the b a s a l f o r e b r a i n as observed i n t h i s study i s , i n i t s e l f , i n t e r e s t i n g and m e r i t s f u r t h e r i n v e s t i g a t i o n p a r t i c u l a r l y i n r e l a t i o n t o t h e i r p o s s i b l e c o n t r i b u t i o n t o c o r t i c a l i n n e r v a t i o n as was suggested i n t h i s study. While s o m a t o s t a t i n d i d not appear t o c o n t r i b u t e t o the b a s a l f o r e b r a i n p r o j e c t i o n system i n n e r v a t i n g the RTN, as mentioned above, the pres e n t study has confirmed t h a t the RTN 76 i s i n n e r v a t e d by c h o l i n e r g i c neurons of the nucleus b a s a l i s m a g n o c e l l u l a r i s and pedunculopontine tegmental nucleus. The r e l a t i v e c o n t r i b u t i o n of these c h o l i n e r g i c i n p u t s to the RTN (up to 20% r e l a t i v e t o the t o t a l number of l a b e l e d neurons p r o j e c t i n g to the RTN) o b t a i n e d i n t h i s study appears, however, lower than p r e v i o u s l y r e p o r t e d by H a l l a n g e r e t a l . (1987, see a l s o i n t r o d u c t i o n ) , but s i m i l a r to t h a t observed by S t e r i a d e e t a l . (1987). However, as mentioned p r e v i o u s l y , i t i s q u i t e p o s s i b l e t h a t the p r o p o r t i o n of b a s a l f o r e b r a i n c h o l i n e r g i c neurons i n n e r v a t i n g the RTN, or RTN and c o r t e x was underestimated i n t h i s study. For example, i t has r e c e n t l y been shown t h a t ChAT immunostaining can be improved w i t h a f i x a t i o n p r o t o c o l t h a t i n c l u d e s p i c r i c a c i d or by adding r a t serum to the secondary antibody ( E c k e n s t e i n e t a l . , 1988). More s e n s i t i v e methods are r e q u i r e d to c l e a r l y e s t a b l i s h e d the r e l a t i v e c o n t r i b u t i o n of c h o l i n e r g i c neurons to the i n n e r v a t i o n of the RTN or of both RTN and c o r t e x . C o n c l u s i o n s S e v e r a l f e a t u r e s of some p r o j e c t i o n s i n n e r v a t i n g the RTN have been d e s c r i b e d . F i r s t , i t was shown t h a t a s u b p o p u l a t i o n of neurons c o n t a i n e d i n the b a s a l f o r e b r a i n and mesopontine tegmentum t h a t i n n e r v a t e the RTN a l s o i n n e r v a t e the c o r t e x through axon c o l l a t e r a l s . I t was f u r t h e r demonstrated t h a t p a r t of t h i s c o l l a t e r a l i z e d p r o j e c t i o n i s c h o l i n e r g i c . I t was a l s o suggested t h a t another s u b p o p u l a t i o n of b a s a l f o r e b r a i n 77 neurons defined by t h e i r immunoreactivity with PV-antibodies also contributed to these c o l l a t e r a l i z e d projection systems. The cholinergic innervation of the RTN derived from both the basal forebrain and mesopontine tegmentum was confirmed i n t h i s study. The present results have extended t h i s knowledge by showing that the RTN also receives a major input from basal forebrain neurons that contains parvalbumin. The neurotransmitter content of t h i s subpopulation of PV-containing neurons as observed i n t h i s study i s uncertain. However, GABA appears a l i k e l y candidate. 78 EXPERIMENT TWO INTRODUCTION The i n t e r p e d u n c u l a r nucleus and the hippocampus are two l i m b i c s t r u c t u r e s t h a t are i n n e r v a t e d by the b a s a l f o r e b r a i n . Retrograde t r a c t - t r a c i n g s t u d i e s combined w i t h ChAT immunohistochemistry have r e v e a l e d t h a t p a r t of t h i s i n p u t i s d e r i v e d from c h o l i n e r g i c neurons of the r o s t r a l b a s a l f o r e b r a i n (Woolf and Butcher, 1985; Woolf e t a l . , 1984; Amaral and Kurz, 1985; Saper, 1984). A more d e t a i l e d a n a l y s i s of these s t u d i e s f u r t h e r r e v e a l s t h a t the v a s t m a j o r i t y of the r o s t r a l b a s a l f o r e b r a i n c h o l i n e r g i c neurons i n n e r v a t i n g the i n t e r p e d u n c u l a r nucleus on the one hand, and the hippocampus on the other hand, are d i s t r i b u t e d i n areas t h a t appear t o o v e r l a p s i g n i f i c a n t l y . Hence, c h o l i n e r g i c neurons a s s o c i a t e d w i t h the medial s e p t a l nucleus and the v e r t i c a l and h o r i z o n t a l limbs of the d i a g o n a l band were d e s c r i b e d i n these s t u d i e s as p r o v i d i n g a major i n p u t to the i n t e r p e d u n c u l a r nucleus and hippocampus r e s p e c t i v e l y . In c o n t r a s t t o the hippocampus, the i n t e r p e d u n c u l a r nucleus has a l s o been shown t o r e c e i v e a minor c h o l i n e r g i c i n p u t from more caud a l r e g i o n s of the b a s a l f o r e b r a i n i n c l u d i n g the r e g i o n s encompassing the s u b s t a n t i a innominata and nucleus b a s a l i s m a g n o c e l l u l a r i s (Woolf and Butcher, 1985). Since o v e r l a p i n the o r i g i n s of b a s a l f o r e b r a i n p r o j e c t i o n s to the i n t e r p e d u n c u l a r nucleus and hippocampus appears w e l l e s t a b l i s h e d , the q u e s t i o n a r i s e s as t o the e x i s t e n c e of b a s a l f o r e b r a i n neurons t h a t c o l l a t e r a l i z e t o i n n e r v a t e both of these 79 s t r u c t u r e s s i m u l t a n e o u s l y . I t i s i n t e r e s t i n g t o note t h a t the i n t e r p e d u n c u l a r nucleus and hippocampus have been shown to be a n a t o m i c a l l y l i n k e d v i a r e c i p r o c a l connections (Fass and H a m i l l , 1987) and t h a t some of these connections may be c o l l a t e r a l i z e d (Montone e t a l . , 1987). A c c o r d i n g l y , the f o l l o w i n g study sought t o determine the degree t o which s i n g l e b a s a l f o r e b r a i n neurons have axons t h a t branch t o i n n e r v a t e both the i n t e r p e d u n c u l a r nucleus and hippocampus u s i n g a r e t r o g r a d e f l u o r e s c e n c e double-l a b e l i n g procedure. A schematic r e p r e s e n t a t i o n of t h i s h y p o t h e t i c a l c o l l a t e r a l i z e d p r o j e c t i o n system i s shown i n F i g u r e 15. Such a c o l l a t e r a l i z e d p r o j e c t i o n would p r o v i d e y e t another anatomical l i n k a g e between the i n t e r p e d u n c u l a r nucleus and hippocampus. 80 F i g u r e 15. A schematic r e p r e s e n t a t i o n of a h y p o t h e t i c a l p r o j e c t i o n system a r i s i n g i n the b a s a l f o r e b r a i n t h a t c o l l a t e r a l i z e d ( d o t t e d l i n e ) t o i n n e r v a t e both the i n t e r p e d u n c u l a r nucleus and hippocampus. The course of the f i b e r s as drawn are not r e p r e s e n t a t i v e of a c t u a l t r a j e c t o r i e s . A b b r e v i a t i o n s : b f , b a s a l f o r e b r a i n ; i p n , i n t e r p e d u n c u l a r nucleus; h, hippocampus. (modi f i e d s a g g i t a l p l a t e from Paxinos and Watson, 1986). 81 82 METHODS The experimental procedure and m a t e r i a l s used i n t h i s experiment were as d e s c r i b e d i n Experiment one. For t h i s reason, o n l y d e t a i l s p e r t i n e n t t o the p r e s e n t experiment and not d e s c r i b e d e a r l i e r are presented here. Three male Wistar r a t s (250-300g) were used. Each animal r e c e i v e d a s i n g l e i o n t o p h o r e t i c i n f u s i o n of FG (same parameters as experiment one) i n t o the i n t e r p e d u n c u l a r nucleus f o l l o w e d by fou r i n j e c t i o n s of PI (0. 3 u . l \ i n j e c t i o n ) i n t o the hippocampus. The i n t e r p e d u n c u l a r nucleus was approached a t an angle from the hemisphere c o n t r a l a t e r a l t o the hippocampal i n j e c t i o n s . The s t e r e o t a x i c c o o r d i n a t e s used f o r the i n t e r p e d u n c u l a r nucleus were as f o l l o w s : 6.72mm p o s t e r i o r t o bregma, 1mm l a t e r a l t o the m i d l i n e and 8.1mm v e n t r a l t o dura, w i t h an angle of 7° o f f the v e r t i c a l p l a n e . The c o o r d i n a t e s used f o r each of hippocampal i n j e c t i o n were as f o l l o w s ( i n o r d e r ) : 2.12; 2.56; 3.60 and 4.52mm p o s t e r i o r t o bregma; 1; 1.4; 2; and 2.2mm l a t e r a l t o the m i d l i n e ; 3; 3.1; 2.6 and 2.6mm v e n t r a l t o dura. 83 RESULTS I n j e c t i o n s i t e s Two of the FG i n f u s i o n s r e s u l t e d i n i n j e c t i o n s i t e s t h a t were l a r g e l y c o n f i n e d to the i n t e r p e d u n c u l a r nucleus whereas; d i f f u s i o n i n t o the v e n t r a l tegmental area was observed i n the t h i r d animal. A photomicrograph i l l u s t r a t i n g an i n j e c t i o n s i t e c e n t e r e d i n the i n t e r p e d u n c u l a r nucleus i s shown i n F i g u r e 16. T r a c e r s p i l l a g e was observed at the s i t e of entrance of the m i c r o e l e c t r o d e , and except f o r one animal, i n which the i n j e c t i o n s i t e was r e s t r i c t e d to the i n t e r p e d u n c u l a r nucleus, no t r a c e r s p i l l a g e was observed i n s u b c o r t i c a l areas. I n j e c t i o n s of PI i n t o the hippocampus produced l a r g e i n j e c t i o n s i t e s t h a t covered e x t e n s i v e l y the CAI, CA2, CA3 and CA4 f i e l d s of Ammon's horn as w e l l as the dentate gyrus. Although the i n j e c t i o n s were aimed at the r i g h t hippocampal formation; d i f f u s i o n had encompassed t o some degree the c o n t r a l a t e r a l hippocampal f o r m a t i o n e s p e c i a l l y f o r the hippocampal i n j e c t i o n s t h a t were c l o s e to the m i d l i n e (see methods f o r d e t a i l s on c o o r d i n a t e s ) . In a d d i t i o n , a l l PI i n j e c t i o n s produced v a r y i n g degree of t r a c e r s p i l l a g e i n the c o r t i c a l mantle immediately d o r s a l to the hippocampal i n j e c t i o n s i t e s . F i g u r e 16. The r e p r e s e n t a t i v e FG i n j e c t i o n s i t e through the i n t e r p e d u n c u l a r nucleus i s i l l u s t r a t e d w i t h a photomicrograph which corresponds to the shaded area of the c o r o n a l p l a t e . Note the presence of l a b e l e d neurons i n the r e g i o n of the v e n t r a l tegmental area, ( m a g n i f i c a t i o n : 70x). o c. 86 P a t t e r n of Retrograde L a b e l i n g F o l l o w i n g i n j e c t i o n s i n t o the i n t e r p e d u n c u l a r nucleus, numerous l a b e l e d somata were observed i n the r o s t r a l b a s a l f o r e b r a i n . The v a s t m a j o r i t y of these neurons were observed i n the h o r i z o n t a l limb of the d i a g o n a l band. O c c a s i o n a l l y , l a b e l e d neurons were found i n the medial septum and v e r t i c a l limb of the d i a g o n a l band. At more caudal l e v e l s of the b a s a l f o r e b r a i n , l a b e l e d c e l l s found i n the h o r i z o n t a l limb of the d i a g o n a l band and extended l a t e r a l l y t o i n c l u d e the m a g n o c e l l u l a r p r e o p t i c area. In a d d i t i o n , many l a b e l e d somata were observed i n the v e n t r a l p a l l i d u m , but these decreased i n number i n more c a u d a l r e g i o n s such as the s u b s t a n t i a innominata and nucleus b a s a l i s m a g n o c e l l u l a r i s . Although the i n t e r p e d u n c u l a r nucleus i s a m i d l i n e s t r u c t u r e , the core of the i n t e r p e d u n c u l a r nucleus i n j e c t i o n was c e n t e r e d on the s i d e i p s i l a t e r a l t o the hippocampal i n j e c t i o n s , a c c o r d i n g l y b a s a l f o r e b r a i n neurons were mainly found i p s i l a t e r a l t o the t r a c e r i n j e c t i o n s i t e s . However, a few l a b e l e d neurons were a l s o found on the c o n t r a l a t e r a l s i d e . The p a t t e r n of r e t r o g r a d e l a b e l i n g i n the b a s a l f o r e b r a i n was not s i g n i f i c a n t l y d i f f e r e n t i n the animal i n which the v e n t r a l tegmental area was p a r t i a l l y i n v o l v e d i n the i n j e c t i o n s i t e . However, i n c o n t r a s t t o i n j e c t i o n s i t e s c o n f i n e d w i t h i n the l i m i t s of the i n t e r p e d u n c u l a r n u c l e u s , a few l a b e l e d somata were observed i n the l a t e r a l s e p t a l and m edial p r e o p t i c areas. On the other hand, i n the two animals i n which the i n j e c t i o n s were lim i t e d to the interpeduncular nucleus, labeled neurons i n the ventral tegmental area could be observed. F i n a l l y , a l l FG i n j e c t i o n s resulted i n massive retrograde lab e l i n g i n the medial habenular nucleus. Neurons projecting to the hippocampus that were retrogradely labeled were found b i l a t e r a l l y i n the medial septum and i n the v e r t i c a l and horizontal limbs of the diagonal band. Very few labeled c e l l s were observed i n the caudal portion of the basal forebrain. Double-labeled c e l l s were r a r e l y (less than 2%) observed i n the basal forebrain. A comparison of the d i s t r i b u t i o n of labeled c e l l s following injections i n the hippocampus or interpeduncular nucleus revealed that neurons innervating each of these structures generally formed separate populations. F i r s t , the horizontal limb of the diagonal band was the only area that provided substantial afferents to both the hippocampus and interpeduncular nucleus. Within t h i s region, labeled neurons innervating the interpeduncular nucleus tended to be located dorsal to those labeled following injections into the hippocampus. The r e s u l t s of t h i s experiment are i l l u s t r a t e d i n Figure 17 which shows the representative d i s t r i b u t i o n of the retrogradely labeled neurons that were observed through three coronal planes of the basal forebrain. F i g u r e 17. The r e p r e s e n t a t i v e d i s t r i b u t i o n of neurons t h a t were r e t r o g r a d e l y l a b e l e d f o l l o w i n g i n j e c t i o n s i n the in t e r p e d u n c u l a r nucleus and hippocampus through t h r e e c o r o n a l planes of the b a s a l f o r e b r a i n . The a n t e r o - p o s t e r i o r (from bregma) c o o r d i n a t e s f o r each of the plane are as f o l l o w : +0.2mm; -0.3mm and -0.8mm. Symbols:. F G - l a b e l e d neurons i n n e r v a t i n g the i n t e r p e d u n c u l a r nucleus; . P i - l a b e l e d neurons i n n e r v a t i n g the hippocampus. 89 DISCUSSION The major fi n d i n g of the present experiment was the demonstration that the interpeduncular nucleus and the hippocampus are independently innervated by basal forebrain neurons. Although negative r e s u l t s do not necessarily mean that a c o l l a t e r a l i z e d projection, as hypothesized i n t h i s study, does not e x i s t , the p o s s i b i l i t y of undetected c o l l a t e r a l s i s however un l i k e l y . F i r s t , the retrograde fluorescence double-labeling procedure appears s u f f i c i e n t l y s ensitive for detecting c o l l a t e r a l i z e d projections based on the r e s u l t s of Experiment one. Secondly, both the interpeduncular nucleus and hippocampus were extensively f i l l e d by each of the respective tracers. In addition, dye d i f f u s i o n and tracer s p i l l a g e were s i g n i f i c a n t i n the c o r t i c a l mantle. If anything, these factors should have enhanced the l i k e l i h o o d of detecting c o l l a t e r a l i z a t i o n . Thus, i t would appear reasonable to conclude that very few basal forebrain neurons have axons that innervate both the hippocampus and interpeduncular nucleus. The pattern of retrograde labeling obtained i n t h i s experiment generally confirmed the r e s u l t s of previous investigations (Woolf and Butcher, 1985; see introduction). Unreported i n previous studies was the demonstration that neurons innervating the interpeduncular nucleus are found b i l a t e r a l l y i n the basal forebrain. A more systematic analysis i s however required before any firm conclusions can be advanced. The r e s u l t s of t h i s study have further established that basal forebrain neurons innervating the hippocampus and interpeduncular nucleus are segregated within the regions of or i g i n s thereby corroborating the conclusion that these neurons do not provide c o l l a t e r a l s to the interpeduncular nucleus and hippocampus. 92 GENERAL DISCUSSION Anatomical Considerations On the basis of the results of the present studies alone, i t i s d i f f i c u l t to reach an o v e r a l l conclusion as to the degree to which basal forebrain projection systems are c o l l a t e r a l i z e d . Hence, i t was shown that cholinergic and possibly GABAergic neurons o r i g i n a t i n g i n the basal forebrain have axons that branch to innervate the RTN and cortex, but not the interpeduncular nucleus and hippocampus. The r e s u l t s obtained i n experiment one provide additional evidence for the existence of basal forebrain neurons with c o l l a t e r a l s (see general introduction). Since both p o s i t i v e and negative evidence for the existence of such neurons have been reported as reviewed e a r l i e r , and as obtained i n t h i s study, no generalization can be made on the preponderance of c o l l a t e r a l i z e d projections a r i s i n g from the basal forebrain. I t can however be suggested that the degree of c o l l a t e r a l i z a t i o n constitutes an anatomical c r i t e r i a for d i s t i n g u i s h i n g subpopulations of basal forebrain neurons. By contrast, the demonstration of a c o l l a t e r a l i z e d cholinergic projection a r i s i n g i n the mesopontine tegmentum i n experimemt one i s i n agreement with the proposition of Woolf and Butcher (1985) that these neurons tend to c o l l a t e r a l i z e extensively. To that e f f e c t , i t i s i n t e r e s t i n g to note that, although not 93 s y s t e m a t i c a l l y analyzed, d o u b l e - l a b e l e d c e l l s were a l s o observed i n the mesopontine tegmentum of c o n t r o l animals t h a t had r e c e i v e d i n j e c t i o n s i n a n t e r i o r or v e n t r a l t h a l a m i c n u c l e i . F u n c t i o n a l C o n s i d e r a t i o n s The demonstration t h a t the RTN and c o r t e x are i n n e r v a t e d by s i n g l e neurons o r i g i n a t i n g i n the b a s a l f o r e b r a i n and mesopontine tegmentum may have s e v e r a l i n t e r e s t i n g f u n c t i o n a l i m p l i c a t i o n s . A l a r g e body of e l e c t r o p h y s i o l o g i c a l and p h a r m a c o l o g i c a l data have e s t a b l i s h e d t h a t the b a s a l f o r e b r a i n and mesopontine tegmental c h o l i n e r g i c systems are important s u b s t r a t e s f o r e x t r a t h a l a m i c modulation of c o r t i c a l f u n c t i o n s such as a r o u s a l and s l e e p ( Borst e t a l . , 1987; Baghdoyan e t a l . , 1987; Stewart e t a l . , 1984)). Ascending c h o l i n e r g i c p r o j e c t i o n s o r i g i n a t i n g i n the mesopontine tegmentum are known to modulate th a l a m i c a c t i v i t i e s i n c l u d i n g RTN t r a n s m i s s i o n (Kayama e t a l . , 1986) and may thereby i n f l u e n c e c o r t i c a l a c t i v i t y through t h i s a d d i t i o n a l i n d i r e c t t halamic r o u t e . R e c e n t l y , i t was suggested t h a t b a s a l f o r e b r a i n c h o l i n e r g i c neurons may a l s o e x e r t an i n f l u e n c e on c o r t i c a l a c t i v i t y through an i n d i r e c t route i . e v i a the RTN (Hallanger e t a l . , 1987). I t i s thought t h a t the RTN p l a y s a c r i t i c a l r o l e i n mediating c h o l i n e r g i c i n f l u e n c e s on t h a l a m i c t r a n s m i s s i o n and c o r t i c a l a c t i v i t y (see Jones, 1985). Of p a r t i c u l a r i n t e r e s t are the a n a t o m i c a l , e l e c t r o p h y s i o l o g i c a l and p h a r m a c o l o g i c a l 94 s t u d i e s which converge i n e s t a b l i s h i n g a r o l e f o r the RTN i n g a t i n g t h a l a m o c o r t i c a l and c o r t i c o t h a l a m i c t r a n s m i s s i o n s (see Jones, 1985). I t has a l s o been e s t a b l i s h e d t h a t c h o l i n e r g i c i n p u t t o the RTN leads t o i n h i b i t i o n of the spontaneous a c t i v i t y of the GABA neurons i n the RTN, as r e v e a l e d w i t h i o n t o p h o r e t i c a p p l i c a t i o n of a c e t y l c h o l i n e (McCormick and P r i n c e , 1986). Since the GABAergic c e l l s of the RTN, which are known to i n n e r v a t e the d o r s a l thalamus, are b e l i e v e d t o be i n h i b i t o r y (see Houser e t a l . , 1980), i t has been proposed t h a t c h o l i n e r g i c i n p u t v i a the RTN leads to d i s i n h i b i t i o n of r e l a y neurons i n the d o r s a l thalamus (see Jones, 1985). Such a d i s i n h i b i t i o n would r e s u l t i n the f a c i l i t a t i o n of sensory t r a n s m i s s i o n which would then r e s u l t i n modulation of c o r t i c a l a c t i v i t y t h a t c o u l d be f u n c t i o n a l l y r e l a t e d to s t a t e s of a r o u s a l . Thus, i t would appear t h a t the RTN i s an important c e n t e r f o r i n t e g r a t i n g the i n f o r m a t i o n conveyed by ascending c h o l i n e r g i c systems w i t h the i n f o r m a t i o n r e l a y e d through the d o r s a l thalamus. The r e s u l t s o b t a i n e d i n t h i s study f u r t h e r suggests t h a t p a r t of the i n f o r m a t i o n t r a n s m i t t e d t o the neocortex from b a s a l f o r e b r a i n and mesopontine tegmentum c h o l i n e r g i c neurons i s a l s o s i m u l t a n e o u s l y conveyed t o the RTN. Thus, i t can be hyp o t h e s i z e d the b a s a l f o r e b r a i n and mesopontine tegmentum c h o l i n e r g i c systems can r e a d i l y i n f l u e n c e , through axon c o l l a t e r a l s , c o r t i c o t h a l a m i c and t h a l a m o c o r t i c a l t r a n s m i s s i o n i n the d o r s a l thalamus v i a the RTN. Taken a ste p f u r t h e r , i t can be hypothesized t h a t such c o l l a t e r a l i z e d p r o j e c t i o n s , by e n a b l i n g b a s a l f o r e b r a i n and 95 mesopontine tegmental cholinergic systems to concomitantly influence neocortical and RTN a c t i v i t i e s , constitute a powerful anatomical substrate capable of exerting a t i g h t control over c o r t i c a l transmission through a series of thalamic feedback loops. The nature of the functional role of the c o l l a t e r a l i z e d projections i d e n t i f i e d remains to be established. The functional implications of a RTN input from PV-containing neurons i n the basal forebrain remains to be established. Aside from the p o s s i b i l i t y that GABA may be the neurotransmitter involved i n these projections and may constitute another important neurochemical pathway for the in d i r e c t modulation of c o r t i c a l a c t i v i t y , the presence of PV i n these neurons may y i e l d important clues as to the physi o l o g i c a l properties of these basal forebrain neurons. Hence, since parvalbumin i s a calcium binding protein, i t has been suggested that parvalbumin may be a marker of a se l e c t i v e neuronal population with calcium dependent action potentials (Celio and Heizmann, 1981). Interestingly, i t has been further suggested that the influence of PV on i n t r a c e l l u l a r concentrations of calcium ions i n neurons such as those containing GABA may contribute i n enhancing metabolic and e l e c t r i c a l a c t i v i t i e s of these neurons (Celio, 1986). 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