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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 B.A.,  Jourdain  Concordia  A THESIS SUBMITTED  University,  1986  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES GRADUATE PROGRAM IN  We  accept to  this  NEUROSCIENCE  thesis  the required  as c o n f o r m i n g standard  THE UNIVERSITY OF B R I T I S H COLUMBIA July  1988  ® Anne J o u r d a i n ,  1988  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department  this or  thesis by  for scholarly  his  publication of this thesis  or  her  Neuroscience  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  the  I agree  requirements  for  may  representatives.  It  be is  advanced  that the Library shall make it  I further agree that permission  purposes  an  granted  for extensive  by the head  understood  that  for financial gain shall not be allowed without  permission.  Department of  of  of  my  copying  or  my written  Abstract  Many 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 p r o j e c t i o n systems tend to o v e r l a p i n t h e i r o r i g i n s .  This  r a i s e s the p o s s i b i l i t y t h a t these p r o j e c t i o n systems are 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 d i v e r g e n t areas. one,  In experiment  the degree to which b a s a l f o r e b r a i n and mesopontine  tegmental neurons t h a t i n n e r v a t e the r e t i c u l a r nucleus  thalamic  have axons t h a t c o l l a t e r a l i z e to i n n e r v a t e the  as w e l l was  examined w i t h a r e t r o g r a d e  fluorescence  method combined w i t h immunohistochemistry.  cortex  labeling  A significant  p o r t i o n of the l a b e l e d neurons 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 and pedunculopontine tegmental  nucleus  p r o j e c t i n g to the r e t i c u l a r t h a l a m i c nucleus were observed to be a l s o l a b e l e d (double-labeled) tracer injections.  following i n t r a c o r t i c a l  Many of these d o u b l e - l a b e l e d  neurons  displayed choline acetyltransferase choline acetyltransferase immunoreactivity.  I t was  a l s o shown t h a t numerous b a s a l  f o r e b r a i n neurons t h a t i n n e r v a t e d the r e t i c u l a r nucleus  contained  the c a l c i u m - b i n d i n g  thalamic  p r o t e i n , parvalbumin.  These neurons tended t o be l o c a t e d more r o s t r a l l y than the ChAT immunoreactive neurons; p r i m a r i l y i n the r e g i o n of ventral pallidum.  There was  the  some i n d i c a t i o n t h a t parvalbumin-  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 t h a t i n n e r v a t e r e t i c u l a r t h a l a m i c nucleus i n n e r v a t e the c o r t e x .  the  a l s o have axons t h a t branch to  F i n a l l y , none of the b a s a l f o r e b r a i n  neurons i n n e r v a t i n g the r e t i c u l a r thalamic nucleus was to c o n t a i n  found  somatostatin.  In experiment two,  the degree t o which b a s a l f o r e b r a i n  neurons have axons t h a t c o l l a t e r a l i z e to i n n e r v a t e interpeduncular  nucleus  and hippocampus was  r e t r o g r a d e f l u o r e s c e n c e l a b e l i n g methods. p r o j e c t i n g t o both of these only o c c a s i o n a l l y .  the  examined w i t h Labeled  neurons  l i m b i c s t r u c t u r e s were observed  Comparison of the 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 neurons i n n e r v a t i n g each of these s t r u c t u r e s r e v e a l e d t h a t w i t h i n the r e g i o n of o r i g i n , 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, neurons i n n e r v a t i n g the nucleus  interpeduncular  tended t o be l o c a t e d d o r s a l l y t o those  i n n e r v a t i n g the  hippocampus. The  r e s u l t s of these experiments are d i s c u s s e d i n r e l a t i o n  t o t h e i r anatomical  and f u n c t i o n a l i m p l i c a t i o n s toward a  g r e a t e r understanding  of the b a s a l f o r e b r a i n and mesopontine  c h o l i n e r g i c and n o n - c h o l i n e r g i c p r o j e c t i o n systems.  iv Table  of Contents  GENERAL INTRODUCTION  1  EXPERIMENT ONE  12  Introduction  12  Methods  16  Animals  16  Stereotaxic  S u r g i c a l Procedures  Fluorescent  Tracer  Histology  19  and  Microscopic  Examination  23  Immunohistochemical Procedures  26  Results  28  Injection  Sites  28  Distribution  of Single-Labeled  Cells  29  Distribution  of Double-Labeled C e l l s  43  Immunohistochemical Analyses Discussion Technical  68 Considerations  Collateralized the  Projections  RTN a n d C o r t e x  Conclusions  EXPERIMENT TWO  54  6 Innervating 73 76  78  Introduction  78  Methods  82  V  Results  83  Injection Pattern  Sites  of Retrograde Labeling  83 86  Discussion  90  GENERAL DISCUSSION  92  Anatomical Considerations  92  Functional  93  REFERENCES  Considerations  96  vi List  Table  1.  of  Tables  Stereotaxic Coordinates  of  Cortical  Injections  Table  2.  22  Summary o f t h e D i s t r i b u t i o n  L a b e l e d Neurons Tegmentum  o f S i n g l y and  i n t h e B a s a l F o r e b r a i n and  Doubly  Mesopontine 48  vii List  Figure Basal  1.  Schematic  Forebrain  Collateralizing Nucleus  Figure  and  2.  of  Figures  Illustration  and M e s o p o n t i n e to Innervate  of  Hypothetical  Tegmental P r o j e c t i o n s  the R e t i c u l a r Thalamic  Cortex  15  Schematic  Illustration  of the I n j e c t i o n  Protocols  Figure  3.  Injection  Figure in  4.  18  Three-Dimensional Reconstruction Site  of a T y p i c a l  i n the R e t i c u l a r Thalamic Nucleus  Photomicrographs  Illustrating  31  Injection Sites  the R e t i c u l a r Thalamic Nucleus  Figure Labeled  5.  Photomicrographs  N e u r o n s i n The  Innervate  Illustrating  33  the D i s t r i b u t i o n  Nucleus B a s a l i s M a g n o c e l l u l a r i s  the R e t i c u l a r Thalamic Nucleus  that  and  Cortex  Figure  38  6.  Photomicrographs  Characteristics Labeled Nucleus  of  of Basal  Following  Illustrating  the  F o r e b r a i n Neurons  Morphological  Retrogradely  I n j e c t i o n i n the R e t i c u l a r Thalamic 41  F i g u r e 7. Through  Representative Distribution  o f L a b e l e d Neurons  S i x Coronal Planes of the B a s a l F o r e b r a i n  Injection  i n the R e t i c u l a r  Thalamic Nucleus  Following  and  Cortex  Figure Through  45  8.  Representative Distribution  of L a b e l e d Neurons  S i x C o r o n a l Planes of the Mesopontine  Following  Injection  Tegmentum  i n the R e t i c u l a r Thalamic Nucleus  and  Cortex  Figure  51  9.  Photomicrographs  of Double-Labeled  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 Pedunculopontine Tegmental  Figure  10.  Color  and  Neurons  i n the  Nucleus  Photomicrograph  53  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  Figure  11  Color  Photomicrograph  56  of a Double-Labeled  Neuron i n the P e d u n c u l o p o n t i n e Tegmental  Nucleus  Figure  a Single Labeled  12  Neuron t h a t  Photomicrographs  Illustrating  Innervate the R e t i c u l a r  C o r t e x t h a t was  also ChAT-Positive  Thalamic Nucleus  58  and 61  ix Figure  13.  Photomicrographs  Innervate the R e t i c u l a r  Illustrating  L a b e l e d Neurons  T h a l a m i c N u c l e u s t h a t were  also  Immunoreactive w i t h Parvalbumin  Figure  14.  Photomicrographs  Immunoreactive Neuron t h a t Thalamic Nucleus  Figure  15.  Interpeduncular Nucleus  Site  16.  Illustrating  a Parvalbumin-  Innervate both the R e t i c u l a r 66  Illustration  Basal Forebrain Projection  Figure  64  and C o r t e x  Schematic  that  of a Hypothetical  Innervate both the  and t h e Hippocampus  Photomicrograph  Illustrating  a Typical  81  Injection  i n the Interpeduncular Nucleus  Figure Through  17.  Representative Distribution  Three  Injections Hippocampus  that  85  o f L a b e l e d Neurons  Coronal Planes of the Basal Forebrain  i n the Interpeduncular Nucleus  Following  and • 89  X  Acknowledgements D r . 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 h i m 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 lab. I am i n d e b t e d t o D r . S.R. V i n c e n t and D r . K.B. B a i m b r i d g e who h a v e g e n e r o u s l y a l l o w e d me t o u s e 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 h a v e g i v e n me p r e c i o u s advices. To K a z u e , I w i l l a l w a y s 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 n e u r o a n a t o m y . I wish her w e l l i n a f u t u r e endeavors. C h u i - S e , P a u l , M a u r e e n and L o r i h a v e b e e n 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 K i n s m e n ' s L a b , t h a n k y o u a l l , and a s p e c i a l m e r c i t o Peter f o r h i s contagious enthusiasm. S i c e 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 these n ' a u r a i t jamais vu j o u r . M e r c i du f o n d d u 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 reve, pour a v o i r g a g n e . . . p a r - d e s s u s t o u t . A nous deux.  1 GENERAL INTRODUCTION  Two  relatively  generating  r e c e n t d e v e l o p m e n t s have b e e n p i v o t a l i n  the impetus f o r a d e t a i l e d  analysis  a n a t o m i c a l 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 cholinergic markers  neurons.  that  neurons.  The  first  of  of the  central  i s the development  allow 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  I m m u n o h i s t o c h e m i c a l methods f o r  acetyltransferase acetylcholine  (ChAT), the s y n t h e t i c  (Cozzari  and Hartman,  E c k e n s t e i n and T h o e n e n ,  of  reliable  cholinergic  choline  enzyme f o r  1980;  L e v e y e t a l . , 1981;  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 acetylcholinesterase acetylcholine currently central  ( A C h E ) , t h e d e g r a d a t i v e enzyme f o r  ( S a t o h e t a l . , 1983;  Mesulam e t a l . , 1 9 8 3 ) , a r e  t h e most r e l i a b l e methods f o r t h e v i s u a l i z a t i o n o f  cholinergic  neurons  (see F i b i g e r ,  g e n e r a t e d by t h e d e v e l o p m e n t by t h e l a r g e the  of  and F i b i g e r ,  that  1985a;  Hirata,  1986;  Vincent  and R e i n e r ,  followed  mapping  e t a l . , 1983; 1987;  progress  Wainer  studies  ( K i m u r a e t a l . , 1981;  1985b; S a t o h e t a l . , 1983;  Houser  The  o f t h e s e methods i s i l l u s t r a t e d  number o f r e c e n t c h o l i n e r g i c  mammalian b r a i n  1982).  in  Satoh  I c h i k a w a and  Mesulam e t a l . , 1983;  e t a l . , 1984;  McGeer e t a l . ,  1984). The  second development  implicating including  acetylcholine  learning  pertains i n a wide  and memory  t o t h e body variety  of evidence  of behaviours  ( B a r t u s e t a l . , 1987).  The  2 functional  significance  o f c h o l i n e r g i c mechanisms,  particularly  illustrated  by  pathogenesis  of Alzheimer's  their  proposed  disease  McGeer e t a l . , 1 9 8 4 ) , i l l u s t r a t e s understanding  of the  as  involvement  in  the  ( C o y l e e t a l . , 1983;  the need f o r a  anatomy o f c h o l i n e r g i c  comprehensive  systems i n the  mammalian b r a i n . Although on  there are  the d i s t r i b u t i o n s  still  and  unresolved  questions,  p r o j e c t i o n s of major  n u c l e a r g r o u p s i n t h e mammalian b r a i n has level  of consensus.  b a s a l f o r e b r a i n on  m e s o p o n t i n e tegmentum on of c h o l i n e r g i c been a c h i e v e d The  hand, and  the other  those  over  the  c o n t a i n s an  last  to the  subthalamic  (Fibiger,  1982), c o i n c i d i n g w i t h  l e v e l of the  end  nucleus  of the globus  cat b r a i n (Vincent  t h e b a s a l f o r e b r a i n encompasses v a r i o u s  limbs  of c h o l i n e r g i c  the v e r t i c a l  o f t h e d i a g o n a l band r o s t r a l l y ;  cholinergic  and  neurons regions,  and h o r i z o n t a l  more c a u d a l l y ,  n e u r o n s have b e e n a s s o c i a t e d w i t h  magnocellular  the  pallidus  through  septum and  tubercle  caudally  r o s t r a l pole of  1987).  the medial  distribution  cholinergic  Reiner,  including  has  basal  of the o l f a c t o r y  the  as d e s c r i b e d i n t h e The  i n the  populations  or  p o p u l a t i o n of  rostrally  respectively,  i n the  consensus  telencephalon,  important  the caudal  general  decade.  level  septum and  a  contained  hand, a r e two  neurons t h a t extends from the  medial  reached  neurons about which r e a s o n a b l e  v e n t r a l p o r t i o n of the  forebrain,  cholinergic  C h o l i n e r g i c complexes c o n t a i n e d t h e one  knowledge  the  p r e o p t i c area, v e n t r a l pallidum, substantia  3 innominata, nucleus b a s a l i s magnocellularis  and  the  nucleus  the  Vincent  et a l . ,  ansa l e n t i c u l a r i s  1986). and  An  important  o r g a n i z a t i o n of  forebrain pertains (Satoh  1987).  i n u n d e r s t a n d i n g the  c h o l i n e r g i c neurons w i t h i n to t h e i r  proposed columnar  W a i n e r e t a l . , 1984)  e m p i r i c a l support  reconstructions al.,  concept  e t a l . , 1983;  received  (Wainer e t a l . , 1984;  using  with  rostro-caudal  extent  the  term  c h o l i n e r g i c column of  and  "cholinergic basal  account f o r t h i s al.,  1987;  principle the  of  the  encompasses in  the  a continuum  the  basal  (Satoh  e t a l . , 1986).  as  of b a s a l  i s the  with  region  proximity  with  regions  region of  the  Rye  the  i t aspect  f o r e b r a i n c h o l i n e r g i c neurons  often  non-cholinergic  W a i n e r e t a l . , 1984;  et  that  Another important  f o r e b r a i n neurons.  s i n c e they are  to  the  realization  the  evidence demonstrating the  of b a s a l  such,  Schwaber  neurochemical 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 n e u r o n a l subpopulation given  through  forebrain"  e t a l . , 1983;  Consistent  et  that  c o m p l e x " have b e e n p r o p o s e d  (Schwaber e t a l . , 1 9 8 7 ) .  heterogeneity  (Schwaber  f o r e b r a i n , and  t r a d i t i o n a l boundaries of  to the  recently  of c h o l i n e r g i c neurons extends beyond  distribution  pertains  basal  of columnar o r g a n i z a t i o n ,  distribution  limits  of the  organization  Vincent  arrangement  implies  d i s t r i b u t e d along  nuclear  basal  three-dimensional  the  "rostral  the  a d i g i t a l microscopy system  neurons are  distribution  w h i c h has  Such a columnar o r g a n i z a t i o n  cholinergic  of  intermingled  neurons  e t a l . , 1984).  h o r i z o n t a l limb  of  the  or  within  i n close  (Schwaber e t a l . , For  diagonal  a  example, band,  in  1987; the  GABAergic  4 neurons are al.,  intermingled  1986).  cortically obtained The  w i t h c h o l i n e r g i c neurons  Physiological projecting  (Reiner et rostral  (Lehmann e t  amygdala  (Woolf e t  (Carlsen  o l f a c t o r y bulb  et  a l . , 1980;  t h a l a m i c nucleus of  mesencephalon  Nagai et  structures,  including  a l . , 1984),  the  Kurz,  In  Butcher,  1985;  1985), and  the the  addition,  diencephalon  habenulo-interpeduncular  (Woolf and  been  forebrain  n e u r o n s h a v e b e e n shown t o the  of  also  a l . , 1982)  a l . , 1986).  reticular  the  et  et  a l . , 1985).  basal  A m a r a l and  a l . , 1985;  (Zaborszky et  and  Rye  a l . , 1984;  cholinergic  1987)  the  telencephalic  forebrain  al.,  n e u r o n s has  Aston-Jones et  c h o l i n e r g i c column o f  the  hippocampus  forebrain  a l . , 1987;  a wide range of  cortex  evidence f o r heterogeneity  basal  innervates  (Brashear  basal  innervate  the  (Hallanger  system of  Contestabile  et  the et a l . ,  1987). As  k n o w l e d g e on  forebrain  the  cholinergic  d i s t r i b u t i o n and  neurons p r o g r e s s e s ,  increasingly d i f f i c u l t  to c l a s s i f y  subgroups of  forebrain  of  their  ch4)  the  basal  respective  projections  nomenclature of  classification  projection 1987).  For  Mesulam e t  scheme d o e s n o t  extensive overlap  i n the  systems of example,  the  on  forebrain  basis  a l . (1983). reflect  basal  of  the  the  (chl-  this the  cholinergic  (Schwaber e t a l . ,  rostro-caudal  neocortex  the  Hence,  been demonstrated t h a t  the  origins  sufficiently well  forebrain  basal  nuclear  p r o p o s e d by  o r i g i n s o f many o f  i t has  innervate  the  of  i t i s becoming  cholinergic  s u c h as  neurons d i s t r i b u t e d throughout the basal  organization  cholinergic  extent of  (Saper,1984)  and  the  5 amygdala  (Woolf  localization  and B u t c h e r , 1 9 8 5 ) .  of c h o l i n e r g i c  neurons  Furthermore, innervating  the neocortex  c o i n c i d e s w i t h that of amygdalopetal c h o l i n e r g i c (Carlsen et a l . ,  p o p u l a t i o n of c h o l i n e r g i c  the mesencephalic  neurons  nucleus, caudally, (Woolf  Satoh e t a l . , mesopontine  and  1983).  et  al.,  neurons.  i s similar  column"  Hence, t h e s e  1983;  1983;  Vincent et a l . ,  neurons  of the locus  Armstrong 1986).  i n t h e c a t ( J o n e s and B e a u d e t ,  Reiner,  1987).  limited Butcher,  ascending cholinergic  nigra,  ceruleus,  et a l . ,  1983;  Furthermore,  Mesulam  mesopontine  intermingled  1987;  V i n c e n t and  projections arising  from  the  c o l u m n 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  regions of the cortex 1986;  which  s u c h 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  caudal cholinergic  basal  "the  1983),  have a l s o b e e n shown t o be  with non-cholinergic c e l l s  of  cholinergic  (see Satoh e t a l . ,  to the a n t e r i o r p a r t  (Satoh e t a l . ,  cholinergic  The  to that of  from the c a u d a l p o l e of the s u b s t a n t i a  rostrally, caudally  neurons  1983;  the o r g a n i z a t i o n  a r r a n g e d i n a l o n g i t u d i n a l column,  caudal cholinergic extends  Mesulam e t a l . ,  I n many r e s p e c t s ,  cholinergic  are also  tegmental  the pedunculopontine tegmental nucleus,  and B u t c h e r , 1986;  forebrain cholinergic neurons  i s found  and p o n t i n e tegmentum where i t i s  predominantly a s s o c i a t e d with the l a t e r o d o r s a l  rostrally  neurons  1985).  A second major in  the  (Vincent et a l . ,  S a t o h and F i b i g e r ,  1986),  the  1983; thalamus  Woolf  and  6 ( S o f r o n i e w e t a l . , 1985; interpeduncular superior  nucleus  colliculus  forebrain Semba e t  S a t o h and (Woolf and  ( B e n i n a t o and  (Woolf and  Butcher;  the  Butcher;  the  Spencer,  1986;  numbers o f  J o n e s and  detail  the  c h o l i n e r g i c systems; f o c u s i n g  neuropeptide c o - l o c a l i z a t i o n (Vincent topographical  organization  ( I c h i k a w a and  Hirata,  and  the  Beaudet,  basal 1987;  (Saper,  1986)  and  organization  on  of  such questions  as  e t a l . , 1983),  1984), c o u r s e t r a j e c t o r y  axonal c o l l a t e r a l i z a t i o n  (Bigl  a l . , 1982). As  alluded  various  basal  number o f  i s considerable  i n the  o r i g i n s of  i n d i c a t i v e of  individual innervate  overlap  independent p r o j e c t i o n s .  such o v e r l a p be  t o above, when c o m p a r i n g t h e  divergent  Most s t u d i e s collateralized  i n the  I t has  column of  the  r a t and  double-labeling  systems  that to  ( F i b i g e r , 1982). degree  o r i g i n a t i n g i n the basal  primate brain,  of rostral  f o r e b r a i n have f o c u s e d  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 both the  in  that  have axons t h a t b r a n c h  t h a t have e x a m i n e d t h e  projections  a  been s u g g e s t e d  axonal c o l l a t e r a l i z a t i o n  regions  o r i g i n s of  cholinergic projection  c h o l i n e r g i c n e u r o n s may  cholinergic  d i s t r i b u t i o n of  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  may  1986)  a n a t o m i c a l i n v e s t i g a t i o n s have  sought to a n a l y z e i n g r e a t e r  et  1985),  a l . , 1988a).  Increasing  central  F i b i g e r ; 1986);  studies  the  using  on  neocortex. a  the In  retrograde  technique w i t h f l u o r e s c e n t probes  (Saper,  7  1984; al.,  L u s k i n and 1985)  or  procedure  Price,  a l . , 1982)  proportion  (0-5%) o f  project  the  divergent the  (Lamour e t  (Aston-Jones et  areas. way  et  an  a l . , 1985).  a l . , 1984;  Price  Stern,  forebrain  antidromic  projecting  basal  1983;  Levine  (15-30%) o f  projecting  basal  sporadically forebrain  to  tracers  that  other  subpopulations of  innervate  neurons i n the  i n the  paired  cholinergic,  divergent  respectively.  h a v e axons  small  rat brain  that been  p e r c e n t a g e of  basal  have been o b s e r v e d  i n j e c t i o n s of d i f f e r e n t  o l f a c t o r y b u l b and  cortical  basal  s u b c o r t i c a l a r e a s has A  have  cortically  t o be  brain  investigated.  branch f o l l o w i n g  innervate  n e u r o n s may  (Lamour  a l . , 1986)  cat  cholinergic  collateralize  neurons b e l i e v e d to  area  axons  and  proportion  forebrain  these  double-labeling  a significant  possibility  are  Aston-Jones et a l . ,  shown t h a t  The  innervate  of  a given c o r t i c a l  retrograde  collateralize  that  forebrain  Adams e t  r a t and  small  activation  a l . , 1986;  areas i n the  a  neurons  branch to  (Boylan et  have a x o n s t h a t  only  with these r e s u l t s  a l . (1983) and  forebrain  et  anterograde t r a c t - t r a c i n g  within  using  t e c h n i q u e s , McKinney e t collaborators  Walker  c h o l i n e r g i c were o b s e r v e d t o have  and  In c o n t r a s t ,  that  However, i n s e v e r a l  individual cortically  branched e x t e n s i v e l y  1985).  have r e v e a l e d  Consistent of  1983;  AChE p h a r m a c o h i s t o c h e m i s t r y  a l . , 1 9 8 4 ) , and  n e u r o n s t h o u g h t t o be that  Stern,  n e o c o r t e x have a x o n s t h a t  cortical  studies,  and  cholinergic basal  d a t a o b t a i n e d by  method  Price  c o m b i n e d w i t h an  (Bigl et  to  1982;  p i r i f o r m cortex  to  fluorescent (Luskin  and  8 Price, al.,  1 9 8 2 ) , a m y g d a l a and  1 9 8 5 ) , o l f a c t o r y b u l b and  1 9 8 7 ) , as w e l l primate b r a i n significant to  a l l o c o r t e x or  as  i n the  send c o l l a t e r a l s  h i p p o c a m p u s and  basal  to the  p e r i b r a c h i a l area,  forebrain reticular  and  the  and  other thalamic n u c l e i or  but  demonstrated with retrograde (Parent  et  a l . , 1987).  projections  hippocampus  ( K o l i a t s o s e t a l . , 1987).  number o f  arising  not  cortex  the  reticular  o t h e r hand identity in  the  (Steriade  of  projections  the  basal  from the the  and  et  Further  amygdala c o u l d  basal  forebrain  there  projections  basal  one  head of  the  hand, and  in  was  For  not  also  collateralization  from b a s a l  to the  f o r e b r a i n n e u r o n s was  revealed  by  retrograde  i n the  been  example, h a l f  is a collateralized  In c o n t r a s t ,  assessed  branched  from the  a m y g d a l a and  of  activated  fimbria  of  projection  hippocampus  primate b r a i n ,  a m y g d a l a and  f o u n d t o be  the  chemical  f o r e b r a i n has  activated  to the  degree of  branched  magnocellularis  However, t h e  evidence for  a l s o be  a l . , 1986).  as  the  as  fluorescence  f o r e b r a i n n e u r o n s t h a t were a n t i d r o m i c a l l y  (Dutar e t  (2%),  primate b r a i n  cat brain,  p h y s i o l o g i c a l approaches.  rat, suggesting that  from the  the  on  thalamus  p e r i b r a c h i a l area  n e o c o r t e x and  a l . , 1987).  studies.  the  found  m e d i o d o r s a l t h a l a m i c n u c l e i on  o r i g i n a t i n g i n the  obtained using  nucleus of  nucleus b a s a l i s  these c o l l a t e r a l i z e d  latter  a  double-labeling  ( F i s h e r e t a l . , 1985)  thalamic  In c o n t r a s t ,  n e u r o n s have b e e n  of  et  (Okoyama e t a l . , the  S i m i l a r l y , i n the  from the  (Carlsen  neocortex of  to the  have b e e n shown t o t e r m i n a t e i n t h e caudate nucleus  neocortex  hippocampus  relatively  double-labeling  the  low  fluorescence  9 ( K o l i a t s o s e t a l . , 1987). the  Finally,  r a t d i a g o n a l band have b e e n shown by  labeling  a n a l y s i s combined w i t h  innervate nucleus  simultaneously  (Stratford  Evidence cholinergic  combining  and  t h e m e d i a n r a p h e and  from  n e u r o n s o f t h e m e s o p o n t i n e tegmentum i s  less  In a retrograde t r i p l e - l a b e l i n g  evidence  for collateralized  thalamic  area  and  thalamic  geniculate nucleus  nucleus  of the  nucleus  ( 1 2 % ) , and  tegmental rat.  were d o u b l e  fluorescent nuclei,  and  (Cornwall  and  (10%),  hypothalamic area  (18%)  neurons c o n t a i n e d  nucleus  and  study,  neurons i n the  reticular  collateralized  Phillipson,  1987;  area  or  caudate-  laterodorsal  the  tegmental  a high percentage  and  lateral  i n both  laterodorsal  labeled following injections  strongly suggesting  significantly  obtained  s u b s t a n t i a n i g r a and  In another  t r a c e r s i n the  thus  lateral  p o s t e r i o r thalamic  of presumed c h o l i n e r g i c nucleus  (1985)  p r o j e c t i o n s to the a n t e r i o r  ( 5 % ) , from c h o l i n e r g i c  pedunculopontine  Butcher  study  ChAT  s e p t a l - d i a g o n a l b a n d complex  s u b t h a l a m u s and  putamen  interpeduncular  projections arising  f l u o r e s c e n t retrograde probes with  mediodorsal  double-  W i r t s h a f t e r , 1987).  i m m u n o h i s t o c h e m i s t r y , W o o l f and  (8%),  retrograde  of  AChE h i s t o c h e m i s t r y t o  for collateralized  controversial.  is  n o n - c h o l i n e r g i c neurons  (50%)  tegmental  of  mediodorsal  thalamic  t h a t t h i s p r o j e c t i o n system i n both  the  r a t and  cat brain  S t e r i a d e e t a l . , 1987).  10 It  has  been c o n c l u d e d t h a t  cholinergic generally  neurons contained  non-collateralized  the  projections  i n the  basal  (Carlsen  Butcher,  1985). the  forebrain,  t h i s conclusion  small  rostral  number o f  collateralized sensitivities used. size  of  the  injection site  fields  activation and  the  the  i t i s possible  the  the  antidromic  (1985) t h e  For  d i d not  that  fail  also  study of  with  This  antidromic  neurons i s  often  at points  of  Aston-Jones et a l .  cells  total  forebrain projection  s y s t e m s as  possible  thereby  the  of b a s a l  and  the  activation  differences  limitations  the  used f o r antidromic  to p o t e n t i a l species  (see  the  projections  a c t i v a t i o n can  cat brain  been  branched p r o j e c t i o n s .  example, i n t h e  the  detect  cover optimally  of  of  have  i n some s t u d i e s  number o f  A n o t h e r f a c t o r w h i c h seems t o be  i n the  to  sampling population  l e s s t h a n 10%  basal  r e l a t e d to  pertains  studies  the  t r a c t - t r a c i n g methods  collateralized  sampling population  accounted f o r  and  projection  case i n p h y s i o l o g i c a l studies  since  bifurcation.  studied.  of  mesopontine  systems t h a t  ability  is directly  i n a underestimation of  a l s o be  small  the  l i m i t a t i o n s of  example,  resulting can  projections  are  i s perhaps premature i n view of  In a d d i t i o n ,  and  the  from  whereas  (Woolf  c h o l i n e r g i c column of  For  terminal  case of  subcortical projection  examined t o d a t e .  the  extensively  However, i n t h e  systems of  forebrain  e t a l . , 1985)  those a r i s i n g from c h o l i n e r g i c neurons of tegmentum t e n d t o c o l l a t e r a l i z e  derived  above).  species  of  importance  i n the  organization  illustrated  Such  from  technical  d i f f e r e n c e s may  be  at  the  11 basis  of  the  discrepancies  reviewed e a r l i e r ,  and  between t h e  further  various  emphasize the  studies  need  for  additional data before firm conclusions  can  be  N o t w i t h s t a n d i n g the  cat  brain  results  d a t a have b e e n o b t a i n e d by  support the  contained  conclusion  i n the  projections  to  basal  the  projection  column t h a t the  and  extended to other b a s a l  inconsistency  relationships  of  the  way  studies  included  that  to  of  system of  the  rostral  candidate  for  showed a x o n a l  of  available.  cholinergic  collateralization or  functional  examined. studies  basal  retrograde  i d e n t i f y the  i n view  data c u r r e n t l y  systems o r i g i n a t i n g i n the  effort  s u b c o r t i c a l areas,  axonal c o l l a t e r a l i z a t i o n  also  forebrain  the  following  tegmentum by  neurons  conclusion  s i g n i f i c a n t anatomical overlap,  s h o u l d be  Accordingly,  that  of  appears a l i k e l y  basis  degree of  However, s u c h a  systems i n n e r v a t i n g  paucity  the  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  Hence, e a c h p r o j e c t i o n  on  and  different laboratories  t h a t most c h o l i n e r g i c  neocortex.  c a n n o t a t p r e s e n t be  the  i n the  of McKinney e t a l . (1983), i t would appear  sufficient to  observations  reached.  sought to determine  for several  f o r e b r a i n or  double-labeling  immunohistochemical neurotransmitter  collateralization.  the  projection mesopontine methods.  analyses  content of  in any  These  an neurons  12 EXPERIMENT 1  INTRODUCTION  On  the  basis  cholinergic the  recent anatomical observations,  n u c l e a r groups c o n t r i b u t i n g  t h a l a m u s , and  nucleus of  of  i n p a r t i c u l a r to  (RTN), a r e  the  innervation  reticular  a t t r a c t i v e c a n d i d a t e s f o r the  of  thalamic examination  collateralization. Numerous s t u d i e s  that  have c o m b i n e d t r a c t - t r a c i n g methods  w i t h ChAT i m m u n o h i s t o c h e m i s t r y o r revealed various the  that  the  thalamic nuclei  caudal cholinergic  i n the  and  1986;  1983).  Recently,  cholinergic particularly  W o o l f and  Butcher,  i n the  i n the  well  cholinergic  m e s o p o n t i n e tegmentum Steriade  realization that  et  that  input  a l . , 1987). i n the  i s derived  afferents  RTN  cat  n e u r o n s have b e e n shown t o  sole  basal brain,  cat  brain, and have b e e n  in addition  derived  from  to  the  the  Hallanger et a l . ,  particular interest RTN  appears  r e c i p i e n t of forebrain basal  and Satoh  forebrain,  a l . , 1987; Of  from  Mesulam e t a l . ,  basal  the  to  a l . , 1985;  r a t and  r a t thalamus, the  from the  1987), whereas i n the  to  (Levey e t  i t i s a p p a r e n t l y the  cholinergic  input  nucleus b a s a l i s magnocellularis,  a major i n p u t  have  pedunculopontine  1986;  however, i n b o t h t h e  shown t o p r o v i d e  1987;  mammalian b r a i n  (Sofroniew et  neurons c o n t a i n e d  established  cholinergic  column, i . e . t h e  tegmental n u c l e i  Fibiger,  AChE h i s t o c h e m i s t r y  p r i n c i p a l source of  laterodorsal  in  the  to  the  the  unique  a major  (Hallanger et a l . ,  forebrain  t e r m i n a t e i n the  is  cholinergic  mediodorsal  13 thalamic  n u c l e u s as w e l l  interesting and  to note that  collaborators  1987), the  (Steriade  e t a l . , 1987).  according  to the  (Hallanger  proportion  studies  e t a l . , 1987;  of b a s a l  approximately  labeled c e l l s signature  of  25-50% o f  observed the  the  f o r e b r a i n and  total  of  also  Hallanger  Levey e t a l . ,  tegmental c h o l i n e r g i c neurons i n n e r v a t i n g for  It is  mesopontine  the  r a t RTN  number o f  i n these regions.  The  accounts  retrogradely  chemical  remaining neurons i n n e r v a t i n g  the  RTN  is  unknown. The  localization  of  the  basal  f o r e b r a i n and  tegmental c h o l i n e r g i c neurons i n n e r v a t i n g c o i n c i d e w i t h the n e u r o n s as  localization  described  i n the  Lehmann e t a l . , 1980,  of  RTN  appears  to  cholinergic corticopetal  rat brain  S a p e r and  the  mesopontine  (Vincent  Loewy, 1982;  et a l . ,  1983;  Mesulam e t a l . ,  1983). On the  the  basis  the  significant  cholinergic projections  following  to the  overlap RTN  and  i n the  origins  cortex,  of  the  s t u d y e x a m i n e d w h e t h e r axons o f i n d i v i d u a l  cholinergic one  of  neurons o r i g i n a t i n g i n the  hand, and  i n the  send c o l l a t e r a l s rat brain. hypothetical  that  Figure  basal  f o r e b r a i n on  m e s o p o n t i n e tegmentum on innervate  both the  cortex  following  and  RTN  study a l s o  included  the  neurons.  these  of  the  the  several  attempt to  c o n t e n t of  of  hand,  study.  i m m u n o h i s t o c h e m i c a l e x p e r i m e n t s i n an neurotransmitter  other  1 shows a s c h e m a t i c r e p r e s e n t a t i o n  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  Furthermore, the  the  the  characterize  14 Figure  1.  A schematic  projections  arising  and m e s o p o n t i n e  r e p r e s e n t a t i o n of  i n the basal  lines).  illustrated  ppt,  Note  do n o t  Abbreviations:  cx,  saggital  that  thalamic  the t r a j e c t o r i e s  cortex;  plate  branch  and  cortex  nucleus;  of  the  fibers  to  as  trajectories.  rtn, reticular  tegmental  hand,  that  nucleus  correspond to actual  pedunculopontine  (modified  f o r e b r a i n on t h e one  tegmentum on t h e o t h e r h a n d ,  innervate both the r e t i c u l a r (dotted  hypothetical  thalamic bf,  basal  f r o m P a x i n o s and W a t s o n ,  nucleus; forebrain. 1986).  15  METHODS  The  retrograde fluorescence d o u b l e - l a b e l i n g procedure  involves,  a s d e s c r i b e d by Van d e r Kooy e t a l . ( 1 9 7 8 ) ,  fluorochromes  which emit  characteristics.  Each t e r m i n a l f i e l d  systems under study (see  fluorescence with d i f f e r e n t  F i g u r e 2) .  fluoresce  a t both  wavelengths  (double-labeled) are thought  transported  the t r a c e r s v i a divergent axonal  Fluoro-gold  (FG), propidium  iodide  were t h e f l u o r e s c e n t d y e s c h o s e n d y e s a r e known t o be r e a d i l y resistant little  compatible al.,  excitation  collaterals.  ( P I ) and t r u e b l u e experiment.  (TB)  These  transported retrogradely, are illumination,  t o d i f f u s e out of the c e l l  w i t h immunohistochemical  1984; Schmued  tracers  t o have r e t r o g r a d e l y  for this  t o f a d i n g by f l u o r e s c e n t  tendency  spectral  of the p r o j e c t i o n  i s i n j e c t e d w i t h one o f t h e two  Neurons t h a t  two  and F a l l o n ,  bodies  procedures  exhibit and a r e  (Skirboll et  1986).  Animals  Male W i s t a r  rats  (U.B.C A n i m a l  Breeding  250-300g were u s e d .  Upon a r r i v a l ,  t h e a n i m a l s were a l l o w e d t o  accommodate  new e n v i r o n m e n t  to their  f o r at least  one d a y  to surgery.  after  s u r g e r y , i n g r o u p s o f two o r f o u r and m a i n t a i n e d light\dark cycle with free  were h o u s e d , b o t h  weighing  prior  hour  The a n i m a l s  Center)  b e f o r e and o n a 12  a c c e s s t o f o o d and w a t e r .  F i g u r e 2.  A schematic r e p r e s e n t a t i o n of the  protocols  (see t e x t f o r d e t a i l s ) .  propidium  i o d i d e ; FG,  thalamic  injection  Abbreviations:  PI,  f l u o r o - g o l d ; cx, c o r t e x ; r t n ; r e t i c u l a r  nucleus.  (modified c o r o n a l p l a t e from Paxinos and Watson, 1986).  28  19  S t e r e o t a x i c S u r g i c a l Procedures  Each animal was a n e s t h e t i z e d w i t h sodium p e n t o b a r b i t a l (Nembutal;  50mg/kg, i . p . ) and p l a c e d i n a s t e r e o t a x i c  instrument (David Kopf Instruments; T i j i n g a , CA, USA). Supplemental doses of a n e s t h e t i c were administered when required. A l l animals r e c e i v e d a s i n g l e 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. prepared.  A 4% s o l u t i o n of FG d i s s o l v e d i n 0.9% s a l i n e was The i n t r a c e r e b r a l i n f u s i o n of the t r a c e r was  performed by i o n t o p h o r e s i s (ByTech, Vancouver, B.C., Can.). The parameters used f o r the i o n t o p h o r e t i c i n f u s i o n of FG were determined p r i o r t o the experiment.  A s i n g l e i n f u s i o n w i t h an  anodal DC c u r r e n t of 2.6-2.8 u.A a d m i n i s t e r e d a t 6 second i n t e r v a l s f o r a p e r i o d of 10 minutes was chosen.  These  e j e c t i o n parameters g e n e r a l l y r e s u l t e d i n a s m a l l i n j e c t i o n of FG w i t h s a t i s f a c t o r y retrograde t r a n s p o r t y e t produced  little  n e c r o s i s and minimal d i f f u s i o n of the t r a c e r from the s i t e of i n j e c t i o n o r along the m i c r o p i p e t t e t r a c k .  Glass c a p i l l a r y  t u b i n g (outer diameter: 1.5mm) was p u l l e d on the same day of the s u r g e r y and broken a t the t i p t o a diameter of 20uxn-30ura. The m i c r o p i p e t t e s were then f i l l e d by c a p i l l a r y a c t i o n w i t h the FG s o l u t i o n , b a c k f i l l e d w i t h 0.9% s a l i n e and maintained i n a humid environment u n t i l surgery.  20  The  RTN o f t h e r i g h t h e m i s p h e r e was a p p r o a c h e d a t a n a n g l e  from the l e f t  hemisphere  parameters used i n t h i s o f P a x i n o s a n d Watson positioned incisor  so that  2).  e x p e r i m e n t were d e r i v e d  (1986).  the surface  o f t h e s k u l l was f l a t ; t h e  t o the midline  1.4mm p o s t e r i o r t o bregma,  plane.  were l e f t  i n place  10-15 m i n u t e s . D u r i n g t h e same s u r g i c a l s e s s i o n  o r , o n some o c c a s i o n s , o n  d a y f o l l o w i n g t h e FG i n f u s i o n , 15 a n i m a l s  received multiple  t o t h e FG i n f u s i o n . distilled  ( g r o u p 1)  i n f u s i o n s o f P I ( C a i B i o c h e m ; L a J o l l a , CA,  USA; Sigma; S t - L o u i s ,  MO, USA) i n t o t h e n e o c o r t e x  w a t e r b y s o n i c a t i o n was p r e p a r e d .  0 . 2 - 0 . 4 u l / i n j e c t i o n o f PI over a p e r i o d Following  the i n f u s i o n , the syringe  minutes t o allow  A one u l s y r i n g e  was l e f t  the tracer t o d i f f u s e .  received  a volume o f  o f 5-10 m i n u t e s . i n place  Multiple  i n j e c t i o n s were p e r f o r m e d t o m a x i m i z e r e t r o g r a d e basal  ipsilateral  A 10% s o l u t i o n o f P I d i s s o l v e d i n  ( H a m i l t o n Co., Reno, NV, USA) was u s e d t o i n j e c t  (See  with  Following the  i n f u s i o n of the t r a c e r , the micropipettes  the  The  a n d 6.4mm v e n t r a l t o d u r a ,  angle o f 24° o f f the v e r t i c a l  the  from t h e a t l a s  T h e h e a d s o f t h e a n i m a l s were  u s e d were a s f o l l o w :  1.0mm l a t e r a l  for  The s t e r e o t a x i c  b a r was s e t 3.3mm b e l o w t h e i n t e r a u r a l l i n e .  coordinates  an  (see Figure  f o r e b r a i n a n d m e s o p o n t i n e tegmentum.  f o r 5-10  cortical labelingi n  Each  animal  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  Table 1 f o r coordinates).  fields  To c o n t r o l f o r p o s s i b l e  labeling  of transected  f i b e r s o f p a s s a g e , many o f t h e a n i m a l s  received  d i f f e r e n t combinations o f c o r t i c a l  infusions.  Table  1.  List  the c o r t i c a l plane;  L-M,  o f 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 c h o s e n f o r  injections.  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  latero-medial plane;  ( B a s e d on P a x i n o s and Watson,  D-V,  1986).  dorso-ventral  plane.  22  Table  Cortical  1.  target  Stereotaxic coordinates cortical injections  A-P ( f r o m bregma)  ( i n nun)  for  L-M (from  D-V dura)  +3.7  -3.4  -2.8  frontal  +3.7  -1.6  -0.8  cingulate\ infralimbic  +3.2  -0.6  -2.6  frontal\ cingulate  +2.7  -1.  -1.3  frontal\ parietal  +2.7  -4.0  -1.2  granular insular\ agranular insular  +1.7  -5.0  -3.4  frontal  -2.3  -1.4  -0.6  retrosplenial  -3.8  -1.4  -0.4  retrosplenial  -4.3  -1.6  -0.4  agranular  insular  23  However, a l l a n i m a l s the f r o n t a l  and m e d i a l  A second  same p r o c e d u r e  MO,  USA) was u s e d TB d i s s o l v e d  The to  prefrontal  group of animals  the  of  received injections  f o r the c o r t i c a l  i n j e c t i o n procedure those  used  adjacent (EB;  into  cortex.  (n=4) was p r e p a r e d  as above e x c e p t  in distilled  of the t r a c e r  t h a t TB  according to  (Sigma; S t - L o u i s ,  injections.  A 4%  solution  w a t e r by s o n i c a t i o n was  prepared.  and i n f u s i o n p a r a m e t e r s were  identical  f o r t h e i n j e c t i o n o f PI w i t h  s e c t i o n s were r i n s e d w i t h  Sigma; S t - L o u i s , MO,  the exception that  0.0001% e t h i d i u m  bromide  USA) w h i c h p r o v i d e s a r e d  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 .  This  c o u n t e r s t a i n i n g method a l l o w e d  f o r a more p r e c i s e  cytoarchitectonic  o f TB r e t r o g r a d e l y l a b e l e d  localization  cells. Finally,  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  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  Following allowed  FG  injections  of the t r a c e r s ,  Examination  the animals  t o s u r v i v e f o r a minimum o f 4 d a y s .  This  s u r v i v a l p e r i o d h a s b e e n shown t o be s u f f i c i e n t  were  post-surgery  f o r adequate  FG and P I r e t r o g r a d e t r a n s p o r t a n d l a b e l i n g  of c e l l  bodies  (Skirboll  1986).  A t t h e end  of  e t a l . , 1984; Schmued  and F a l l o n ,  t h e 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 , e a c h a n i m a l was  anesthetized with transcardially  chloral  hydrate  (400mg/kg,  p e r f u s e d w i t h an i n i t i a l  deeply  i . p . ) and  then  50-100ml b o l u s o f 0.9%  24  saline  followed  b y 400ml o f 4% p a r a f o r m a l d e l y d e  phosphate b u f f e r  (pH 7.4) a d m i n i s t e r e d  (PFA) i n 0.1 M  through a p e r i s t a l t i c  pump a t a s l o w r a t e o v e r a 20-30 m i n u t e p e r i o d .  The b r a i n s  were removed a n d s o a k e d 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 20%  f o r 2-3 h o u r s , a n d t h e n t r a n s f e r r e d i n t o a  s u c r o s e s o l u t i o n i n 0.1 M p h o s p h a t e b u f f e r  hours.  E a c h b r a i n was s l i c e d  f r e e z i n g microtome. sites,  For v e r i f i c a t i o n  1 out of every  rostral  pole  i n 30um c o r o n a l  of the f r o n t a l cortex  l e v e l o f t h e genu o f t h e f a c i a l every  3 s e c t i o n s was r e t a i n e d  retrograde  labeling.  phosphate b u f f e r buffered  or  to the level  From t h i s p o i n t nerve  i n the brainstem,  f o rv e r i f i c a t i o n of  s l i d e s and l e f t  inspection of the sections,  an u l t r a v i o l e t  excitation  range t o v i s u a l i z e P I - and E B - l a b e l e d  gold  Dialux  blocks  e x c i t a t i o n r a n g e t o v i s u a l i z e FG- a n d  and f i l t e r  blocks  providing  a green  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  a n d FG t y p i c a l l y  a Leitz  microscope equipped with f i l t e r  cells  PI  with  i m m e r s i o n o i l ( Z e i s s , West Germany).  TB-labeled  used.  1 out  The s e c t i o n s were r i n s e d i n 0.1 M  fluorescence  providing  up t o t h e  The s e c t i o n s were t h e n c o v e r s l i p p e d  microscopic  Zeiss  of the  (pH 7 . 4 ) , mounted f r o m a 15% p h o s p h a t e  to dry overnight.  For  on a  of c o r t i c a l i n j e c t i o n  sucrose s o l u t i o n onto uncoated g l a s s  fluorescence  sections  5 s e c t i o n s was c o l l e c t e d f r o m t h e  j u n c t i o n o f the corpus callosum.  of  (pH 7.4) f o r 24  exhibited  cells  were  injections of  smooth o r a n g e - r e d a n d g r a n u l a r  c y t o p l a s m s 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  cells  could  be v i s u a l i z e d b y a l t e r n a t i n g b e t w e e n e x c i t a t i o n s a n d e m i s s i o n s  25  filters. could  Following  paired  i n j e c t i o n s o f FG and TB a l l c e l l s  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 .  however, FG and TB s i n g l e - l a b e l e d c e l l s and  In t h i s case,  showed g r a n u l a r  smooth b l u e c y t o p l a s m s r e s p e c t i v e l y , w h e r e a s  containing the  both t r a c e r s  gold  cells  (double-labeled) emitted a blend of  two c o l o r s . The  positions  forebrain  of retrogradely  labeled  (coronal  plane) derived  Watson  (1986).  The n o m e n c l a t u r e t h a t  group, s e m i - q u a n t i t a t i v e  i s used t o  I n 5 a n i m a l s from t h e  brain  plates.  sections  This  neurons  was done by c o u n t i n g  (FG, and FG + P l - l a b e l e d )  of c e l l s  t h a t were  labeled  following  smaller  than l a b e l e d - c o r t i c o p e t a l neurons,  number  (FG+EI x 1 0 0 ) .  in  retrogradely  FG i n j e c t i o n s i n t h e RTN was  of double-labeled  to the t o t a l  systems under  per l e v e l matching each of the s e l e c t e d  S i n c e t h e number  estimations  first  a n a l y s e s were p e r f o r m e d i n an a t t e m p t  study are c o l l a t e r a l i z e d . labeled  describe  i n keeping with the  to determine the degree t o which the p r o j e c t i o n  retrogradely  selected  f r o m t h e a t l a s o f P a x i n o s and  areas i n t h i s study i s therefore  a t l a s o f P a x i n o s and Watson.  three  i n the basal  and m e s o p o n t i n e tegmentum were drawn on  plates  brain  cells  of c e l l s  The number  substantially  percentage  c e l l s were c a l c u l a t e d r e l a t i v e t h a t were s i n g l y l a b e l e d w i t h  of labeled  c o r t i c o p e t a l neurons  FG not  assessed. For  pan,  photographic purposes, both black  400 ASA, Kodak) and c o l o r  Kodak) f i l m s were u s e d .  and w h i t e  (Tri-X  s l i d e s ( E k t a c h r o m e , 400  ASA,  FG was  2L  Immunohistochemical In four  Procedures  animals of the f i r s t  sections through the basal  group,  f o r e b r a i n and mesopontine  were r e t a i n e d f o r i m m u n o h i s t o c h e m i c a l retrogradely  labeled cells.  procedures involved  A l l immunohistochemical  (FITC, e x c i t a t i o n range:  set of sections  f o r each of the four  f o r ChAT i m m u n o r e a c t i v i t y .  ChAT i m m u n o h i s t o c h e m i s t r y was Semba e t a l . ( 1 9 8 8 b ) . M buffered  Tris  rats  similar to that described  a n t i b o d y f o r ChAT  (1:40,  and T h o e n e n , 1982) i n a s o l u t i o n c o n t a i n i n g  were t h e n r i n s e d 60 m i n u t e s  and 2% n o r m a l g o a t serum.  (3x20 m i n u t e s )  The  sections The  sections  i n a solution  and 2% n o r m a l  g o a t serum.  A final  rinse  i n 0.05 M TBS was p e r f o r m e d b e f o r e t h e s e c t i o n s  were mounted f r o m a 15% s u c r o s e s o l u t i o n i n 0.1 M  oil.  M  t h e s e c o n d a r y a n t i b o d y (1:20, g o a t - a n t i - r a t ) , 0.05  (3X20 m i n u t e s )  onto uncoated s l i d e s .  overnight  0.05  i n 0.05 M TBS and i n c u b a t e d  on a s h a k e r a t room t e m p e r a t u r e  M TBS, 0.3% T r i t o n - X  buffer  by  r i n s e d i n 0.05  were i n c u b a t e d a t 4 ° C f o r 48 h o u r s on a s h a k e r .  containing  was  (pH 7.4) w i t h 0.9% s a l i n e (TBS) a n d t h e n  TBS, 0.3% T r i t o n - X  for  green)  The p r o t o c o l u s e d f o r  The s e c t i o n s were f i r s t  incubated w i t h a monoclonal Eckenstein  i d e n t i f i c a t i o n of  antibodies.  A first processed  tegmentum  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  isothiocyanate-conjugated secondary  a d d i t i o n a l sets of  phosphate  The s e c t i o n s were d r i e d  and t h e n 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  27  Additional  s e t of sections  through the basal  forebrain  f r o m two o f t h e a n i m a l s t h a t were e x a m i n e d f o r ChAT immunohistochemistry against  were p r o c e s s e d w i t h a m o n o c l o n a l  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  s e c o n d a r y a n t i b o d y (1:40, the  procedure described Finally,  Following  with  0.9% s a l i n e  for  in  3x20 m i n u t e s  anti-mouse  antibody (a g i f t  f r o m K.G.  EGTA,  i n 0.1 M p h o s p h a t e  muscle  parvalbumin  Baimbridge)  0.02% s o d i u m  diluted  azide,  (3x20 m i n u t e s )  s o l u t i o n p r e p a r e d as d e s c r i b e d  mounted f r o m  (3x20 m i n u t e s )  above.  1:100  coversplipped with fluorescence  t o d r y o v e r n i g h t and immersion  The  oil.  with in a  Following three  i n 0.1 M PBS, t h e s e c t i o n s  15% s u c r o s e , l e f t  X-  i n 0.1 PBS and  f o r two h o u r s on a s h a k e r a t room t e m p e r a t u r e  more r i n s e s  1:50  20  on a s h a k e r a t 4 ° C.  secondary antibody ( g o a t - a n t i - r a b b i t ) d i l u t e d  buffer  buffer  6% g o a t serum and 0.3% T r i t o n  The i n c u b a t i o n was p e r f o r m e d  s e c t i o n s were t h e n r i n s e d  the  rinses  to parvalbumin  (PBS, pH 7 . 4 ) , t h e s e c t i o n s were i n c u b a t e d  u n i t s o f h e p a r i n , 1 mM  incubated  animals, sets of sections  processing with antibodies  a 0.1 M PBS s o l u t i o n c o n t a i n i n g  100.  following  f o r ChAT.  48 h o u r s w i t h a r a b b i t  polyclonal  1985)  f o r e b r a i n were r e t a i n e d f o r  immunohistochemical (PV).  goat-anti-mouse,  see V i n c e n t e t a l . ,  i n three of the four  through the b a s a l  antibody  were  28  RESULTS  Injection  Sites  C o r t i c a l i n j e c t i o n s w i t h e i t h e r TB or PI were g e n e r a l l y l a r g e , each c o v e r i n g an e x t e n s i v e area o f the i n f u s e d 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 t o have  d i r e c t l y i n v o l v e d the corpus c a l l o s u m or s u b c o r t i c a l r e g i o n s , except i n two animals which showed p a r t i a l involvement corpus c a l l o s u m w i t h minimal  encroachments i n t o  r e g i o n s f o l l o w i n g PI i n j e c t i o n s . degrees  o f the  subcortical  In some cases, v a r y i n g  of dye d i f f u s i o n i n t o the corpus callosum or adjacent  c o n t r a l a t e r a l c o r t e x c o u l d be observed,  but t h i s never  i n t o s u b c o r t i c a l r e g i o n s such as the hippocampus. d i f f u s i o n was o n l y observed  spread  Dye  f o l l o w i n g PI i n j e c t i o n s s i n c e the  i n j e c t i o n s i t e s f o l l o w i n g TB i n f u s i o n s were more r e s t r i c t e d as w e l l as more n e c r o t i c . I o n t o p h o r e t i c i n f u s i o n s of FG i n t o the RTN 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 s m a l l w i t h 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 c e n t e r e d i n the RTN, each f i l l i n g a s i g n i f i c a n t p o r t i o n o f the nucleus along i t s r o s t r o - c a u d a l a x i s .  Although many of these  i n j e c t i o n s p r i m a r i l y i n v o l v e d the v e n t r a l RTN, i n some i n s t a n c e s the c o r e s of the i n j e c t i o n s were l o c a t e d more c e n t r a l l y i n the RTN.  In two cases, the c e n t e r of the  i n f u s i o n was c l e a r l y r e s t r i c t e d t o the r o s t r a l and c a u d a l  29  p o l e s of the RTN dimensional  respectively.  F i g u r e 3 shows a t h r e e -  r e c o n s t r u c t i o n of a t y p i c a l RTN  Photomicrographs i l l u s t r a t i n g two are shown i n F i g u r e 4 . of the RTN  Dye  i n j e c t i o n s i t e s i n the  g e n e r a l l y i n v o l v e d v a r y i n g degrees of encroachment thalamic n u c l e i  In some cases, dye d i f f u s i o n was  the i n t e r n a l c a p s u l e  a l s o observed i n  l a t e r a l l y , the zona i n c e r t a v e n t r a l l y ,  and the a n t e r o v e n t r a l thalamic nucleus the l o c a t i o n of the c e n t e r of the RTN  r o s t r a l l y depending on injection  site.  Furthermore, i n j e c t i o n s t h a t were l o c a t e d i n the r o s t r a l of the RTN  RTN  d i f f u s i o n o u t s i d e the boundaries  i n t o the v e n t r o l a t e r a l and/or ventromedial medially.  injection.  may  pole  have r e s u l t e d i n minor spread of FG i n t o the  s t r i a t e r m i n a l i s or medio-dorsal t i p of the s u b s t a n t i a innominata.  In most animals,  the s i t e of entrance  FG s p i l l s c o u l d be observed at  of the m i c r o p i p e t t e  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 s u b c o r t i c a l regions.  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  In t h i s study,  Cells  the p a t t e r n of r e t r o g r a d e  labeling i s  d e s c r i b e d f o r the hemisphere i p s i l a t e r a l t o the  injection  s i t e s s i n c e 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 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 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 c o r t e x .  the The  number of r e t r o g r a d e l y s i n g l e - l a b e l e d c e l l s f o l l o w i n g TB i n j e c t i o n s i n the c o r t e x tended to be g r e a t e r than f o l l o w i n g  30  Figure  3.  reticular  A reconstruction of a typical thalamic nucleus  ( m o d i f i e d from posterior follows: -2.12mm.  Paxinos  (bold) through  a n d Watson, 1 9 8 6 ) .  c o o r d i n a t e s ( f r o m bregma)  injection five  site  i n the  coronal planes  The a n t e r i o r -  f o r the planes are as  (A) -0.92mm; (B) -1.3mm; (C) -1.4mm; (D) -1.8mm; (E)  31  32  Figure 4. through  Photomicrographs of f l u o r o - g o l d i n j e c t i o n  the r o s t r a l  thalamic nucleus.  (A) a n d c a u d a l The e x a c t  (C) p o l e s o f t h e  location  reticular  of the i n j e c t i o n  c a n be c o m p a r e d w i t h t h e b o u n d a r i e s  of the r e t i c u l a r  nucleus  immunostaining  as d e f i n e d w i t h p a r v a l b u m i n  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 ) . involved minimal reticular thalamic  diffusion  thalamic nucleus.  Note t h a t these  outside the boundaries  sites  thalamic  (B a n d  C + D,  93x).  D;  injections of the  Abbreviation: r t n , reticular  nucleus.  ( m a g n i f i c a t i o n : A + B, 8x;  sites  33  34  35 comparable PI i n f u s i o n s , the  sensitivities  cells The  generally  possibly  reflecting  of the t r a c e r s .  Furthermore,  t e n d e d t o be more d e f i n e d  d i s t r i b u t i o n of labeled  differences i n TB-labeled  morphologically.  c o r t i c o p e t a l c e l l s was, however,  similar  f o r t h e two t r a c e r s ;  reports  (see g e n e r a l  and c o n s i s t e n t  introduction).  with  Numerous  previous  labeled  cells  were o b s e r v e d i n t h e t e l e n c e p h a l o n , d i e n c e p h a l o n a n d m e s o p o n t i n e tegmentum.  In the basal  corticopetal perikarya  forebrain,  were m a i n l y l o c a t e d  encompassing the h o r i z o n t a l magnocellular preoptic  i n the regions  limb of the diagonal  area, v e n t r a l pallidum,  i n n o m i n a t a and n u c l e u s b a s a l i s m a g n o c e l l u l a r i s , n o m e n c l a t u r e o f P a x i n o s a n d Watson In  t h e m e s o p o n t i n e tegmentum, many l a b e l e d  t o a l e s s e r degree,  i n the r o s t r a l  pedunculopontine tegmental nucleus. numerous c o r t i c o p e t a l c e l l s included Of  the substantia  the t o t a l  were o b s e r v e d  number  that  nigra  80%  were f o u n d  forebrain  retrograde similar the  i n the basal  regardless  n e u r o n s were raphe  nuclei  of the  labeled  thalamic neurons  showed  nuclei. (FG)  that  tegmental n u c l e i of the  tracer  i n j e c t i o n i n t o t h e RTN, The p a t t e r n  of  f o r e b r a i n was e s s e n t i a l l y  of the dorsoventral  injection site within  the  and i n t h e  forebrain.  l a b e l i n g i n the basal  following  Other regions t h a t  and v a r i o u s  following  substantia  were r e t r o g r a d e l y  p e d u n c u l o p o n t i n e and l a t e r o d o r s a l m e s o p o n t i n e tegmentum  part  of s i n g l e - l a b e l e d  i n the basal  band,  (1986).  observed i n the l a t e r o d o r s a l tegmental nucleus, and,  labeled  t h e RTN.  l o c a t i o n of the core of  Basal forebrain  cells  innervating horizontal  the  RTN  limbs of  area, v e n t r a l  (90%)  anterior  the  and  substantia  were l o c a t e d  caudal basal  basal  ventral  retrogradely  (1986).  labeled  In  perikarya  included  g l o b u s p a l l i d u s as w e l l  as  (see  Figure  5).  o b s e r v e d near the  terminalis.  In  the  bed  rostral basal  d i a g o n a l b a n d and  vertical  labeled limb of  d i a g o n a l band, but  the  the  to  a  distribution of  the  of  of  nucleus  i n the  medial  internal  somata were  the  stria  almost a l l of  n e u r o n s were f o u n d ventral  n e u r o n s were o c c a s i o n a l l y the  the  nomenclature  labeled  forebrain,  horizontal addition,  the  nucleus of  labeled  the  the  neurons l o c a t e d  remaining retrogradely limb of  and,  n e u r o n s embedded i n t h e  occasionally  these  Neurons i n  (>70%) i n  region  More r o s t r a l l y ,  of  substantia  following  i n the  basalis  d e c u s s a t i o n of  forebrain).  addition,  basalis magnocellularis  capsule  the  nucleus b a s a l i s magnocellularis  Watson  preoptic  majority  pallidum,  l e s s e r degree, ansa l e n t i c u l a r i s P a x i n o s and  to  The  were f o u n d p r i m a r i l y  r e g i o n s encompassing the i n n o m i n a t a and  posterior  (caudal  forebrain  and  innominata, nucleus  ansa l e n t i c u l a r i s .  commissure  vertical  d i a g o n a l band, m a g n o c e l l u l a r  pallidum,  magnocellularis cells  were o b s e r v e d i n t h e  in  the  the  pallidum.  observed  r a r e l y i n the  in  In  the  medial  septum. The may  be  basal  topographically  retrogradely the  forebrain  labeled  c a u d a l p o l e of  exclusively  to  the  projection organized  cells  the  RTN  system i n n e r v a t i n g  since  following  pattern  restricted  forebrain,  and  RTN  of  injections centered  t e n d e d t o be  caudal basal  the  the  in  almost  primarily  to  F i g u r e 5.  A p a i r of photomicrographs comparing  the  d i s t r i b u t i o n of l a b e l e d neurons p r o j e c t i n g to the nucleus  reticular  of the thalamus (A) w i t h l a b e l e d neurons p r o j e c t i n g to  the c o r t e x  (B) 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 , medial globus p a l l i d u s and  internal  Note the o v e r l a p i n the d i s t r i b u t i o n of these two r e t r o g r a d e l y l a b e l e d neurons.  (magnification:  double-labeled  Abbreviation: i c , i n t e r n a l 72x).  groups of  Although i t i s d i f f i c u l t  assess at low m a g n i f i c a t i o n , two shown (arrows).  capsule.  to  neurons are  capsule,  39  the  regions  and  substantia  caudally failed  encompassing the nucleus b a s a l i s innominata.  centered  However,  injection  sites,  i t i s possible that  w h i c h were t y p i c a l l y  t o cover adequately the terminal  fields  p r o j e c t i n g neurons of the remaining r o s t r a l basal  regions  of the  injection  labeled  i n t o t h e RTN  cells,  horizontal  limb  numerous  of the diagonal  band, m a g n o c e l l u l a r  c e l l s with oval or fusiform i n the v e n t r a l pallidum  A large proportion labeled  diagonal  In addition,  of FG-labeled  anterior  extent  i n an a r e a  commissure;  l a b e l e d c o r t i c o p e t a l neurons.  This  of the  and  substantia  l a b e l e d neurons p r o j e c t i n g t o the Other  somata t h a t were c o n s i s t e n t l y o b s e r v e d encompassing the v e n t r a l  located  pallidum,  immediately v e n t r a l t o the  and m a g n o c e l l u l a r p r e o p t i c  neurons i n the v e n t r a l p a l l i d u m with retrogradely  were  of the basal  c l u s t e r s i n these regions.  were f o u n d i n t h e r e g i o n s particularly  innominata.  the case i n the h o r i z o n t a l limb  tended t o form loose  clusters  s h a p e s , w h i c h were  band, n u c l e u s b a s a l i s m a g n o c e l l u l a r i s  innominata.  6); to  o f t h e RTN p r o j e c t i n g n e u r o n s t h a t  admixed w i t h  was p a r t i c u l a r l y  preoptic  (see Figure  and s u b s t a n t i a  throughout the rostro-caudal  f o r e b r a i n was  large  many o f w h i c h were o b s e r v e d i n t h e  and n u c l e u s b a s a l i s m a g n o c e l l u l a r i s  smaller  basal  varied.  s h a p e s o f t h e RTN p r o j e c t i n g n e u r o n s r a n g e d f r o m  multipolar  RTN  small,  of 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  f o r e b r a i n neurons f o l l o w i n g  area  these  forebrain.  The  The  magnocellularis  were g e n e r a l l y  area. not  Labeled  intermixed  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  6.  Figure labeled  thalamic of  P h o t o m i c r o g r a p h o f n e u r o n s t h a t were  following fluoro gold nucleus.  injection  large  features  and m u l t i p o l a r ,  (magnification:  reticular  T h e s e n e u r o n s were o b s e r v e d i n t h e r e g i o n  the nucleus b a s a l i s magnocellularis.  morphological  i n the  retrogradely  504x).  typical  Note t h e  of magnocellular  neurons i . e .  41  I  42  F G - l a b e l e d neurons tended t o be l o c a t e d more d o r s a l l y than l a b e l e d c o r t i c o p e t a l neurons.  In the m a g n o c e l l u l a r  preoptic  area, RTN p r o j e c t i n g neurons tended t o aggregate i n the medial p a r t where they were a l s o i n t e r m i n g l e d neurons.  with c o r t i c o p e t a l  However, some of the r e t r o g r a d e l y  labeled  cells  p r o j e c t i n g t o the RTN t h a t were observed i n the m a g n o c e l l u l a r preoptic  area extended l a t e r a l l y and were g e n e r a l l y not  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. Numerous s i n g l e - l a b e l e d neurons p r o j e c t i n g t o the RTN were a l s o observed i n v a r i o u s nuclei. the  p r i n c i p a l and i n t r a l a m i n a r  thalamic  In some cases, s i n g l e - l a b e l e d c e l l s were observed i n  substantia  nigra  nucleus and c o r t e x ;  (mainly pars r e t i c u l a t a ) ,  entopeduncular  p o s s i b l y r e f l e c t i n g the involvement of  p r i n c i p a l t h a l a m i c n u c l e i , such as those of the v e n t r o b a s a l complex, i n the i n j e c t i o n s i t e s  (see d i s c u s s i o n ) .  In the mesopontine tegmentum, neurons t h a t were retrogradely  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 p r i m a r i l y i n the pedunculopontine tegmental nucleus, d o r s a l raphe, c e n t r a l tegmental area, l a t e r o d o r s a l tegmental nucleus and l o c u s c e r u l e u s . retrogradely and  labeled c e l l s  Of the t o t a l number of (FG) found i n the pedunculopontine  l a t e r o d o r s a l tegmental n u c l e i , 80-90% were observed i n the  pedunculopontine tegmental n u c l e u s . With the e x c e p t i o n of the F G - l a b e l e d neurons found i n the c e n t r a l tegmental area, F G - l a b e l e d neurons i n the mesopontine tegmentum were g e n e r a l l y t h a t were r e t r o g r a d e l y  adjacent or i n t e r m i n g l e d  w i t h neurons  labeled following c o r t i c a l injections.  43  T h i s was p a r t i c u l a r l y the case i n the l a t e r o d o r s a l tegmental nucleus and d o r s a l raphe.  In the r e g i o n  of the  pedunculopontine tegmental n u c l e u s , neurons t h a t were  labeled  following  labeled  i n j e c t i o n s i n t o the RTN and those t h a t were  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 were found p r i m a r i l y i n the r o s t r a l and c e n t r a l p o r t i o n s  of the pedunculopontine tegmental  nucleus, b e g i n n i n g a t a l e v e l j u s t p o s t e r i o r t o the c a u d a l p o l e of the s u b s t a n t i a The  nigra.  m o r p h o l o g i c a l appearance of the neurons l a b e l e d w i t h  FG t h a t were observed i n the mesopontine tegmentum v a r i e d large multipolar substantia  from  neurons, as p a r t i c u l a r l y observed i n the  n i g r a , d o r s a l raphe, c e n t r a l tegmental area and  locus c e r u l e u s ;  t o medium s i z e m u l t i p o l a r  cells  primarily  observed i n the pedunculopontine and l a t e r o d o r s a l tegmental nuclei.  D i s t r i b u t i o n of Double-Labeled C e l l s  B a s a l f o r e b r a i n neurons t h a t were d o u b l e - l a b e l e d PI and FG i n j e c t i o n s i n t o both the c o r t e x  following  and RTN were found  almost e x c l u s i v e l y i n the i p s i l a t e r a l c a u d a l b a s a l  forebrain  i . e . p o s t e r i o r t o the l e v e l of the d e c u s s a t i o n of the a n t e r i o r commissure.  The 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 i n the  caudal b a s a l  f o r e b r a i n was remarkably s i m i l a r i n a l l i n j e c t e d  animals.  Figure  7 shows the r e p r e s e n t a t i v e  double-labeled c e l l s (FG)  d i s t r i b u t i o n of  (FG + PI) versus s i n g l e - l a b e l e d  through s i x c o r o n a l  planes of the b a s a l  cells  forebrain.  In  44  Figure  7.  Representative  neurons observed  distribution  i n the basal  reticular  nucleus  innervate  both the r e t i c u l a r  (doubly  labeled).  forebrain that innervate the  o f t h e thalamus  (singly  thalamic  labeled)  nucleus  The p a t t e r n o f d i s t r i b u t i o n  through s i x c o r o n a l planes 1986).  of retrogradely labeled  (modified  The a n t e r o - p o s t e r i o r  each o f the c o r o n a l plane  (from  or that  and c o r t e x i s presented  f r o m P a x i n o s a n d Watson,  bregma) c o o r d i n a t e s f o r  a r e 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: the FG  «, a p p r o x i m a t e l y  reticular  thalamic  five  nucleus;  and PI ( c o r t i c o p e t a l ) .  FG-labeled  band; nbm, n u c l e u s  innominata;  i c ,  Neurons o b s e r v e d  Abbreviations:  i n t e r n a l capsule;  of singly  anterior  hdb, h o r i z o n t a l l i m b  al,  ansa  i n t h e r e g i o n o f t h e hdb a l s o  n o t shown o n t h e p l a t e s .  distribution  ac,  both  of the  b a s a l i s o f Meynart; s i , s u b s t a n t i a  neurons found i n t h e m a g n o c e l l u l a r is  innervating  *, a n e u r o n l a b e l e d w i t h  commissure; v p , v e n t r a l p a l l i d u m ; diagonal  neurons  lenticularis. included  p r e o p t i c area  although  this  A l s o n o t shown i s t h e  l a b e l e d neurons i n n e r v a t i n g t h e c o r t e x .  45  46 five to  animals,  the t o t a l  the proportion of double-labeled number o f F G - l a b e l e d  neurons  cells  relative  ( -'^ - x 100) :EG  B  1  FG counted through the caudal for  details  these  on c e l l  results  FG-labeled  c o u n t s ) was c a l c u l a t e d .  i s presented  double-labeled  cells  cells  b a s a l f o r e b r a i n ( s e e method s e c t i o n  i n Table  relative  2.  The p r o p o r t i o n o f  to the t o t a l  in  majority  (at least  the regions  portion),  nucleus  and ansa l e n t i c u l a r i s . cells  observed  counted through the caudal  15% o f t h e t o t a l  region of the nucleus  frequently  for  i n the medial  pallidus alone  cells  i n the v e n t r a l pallidum,  the regions  innominata,  cells  Itis f o r the  (including  and neurons  may have b e e n a s h i g h  t e n d e d t o be o b s e r v e d and o n l y  rarely  less  i n the  Similar c a l c u l a t i o n s obtained  encompassing the h o r i z o n t a l limb  band a n d m a g n o c e l l u l a r  (FG)  and ansa l e n t i c u l a r i s .  globus  o f the ansa l e n t i c u l a r i s .  diagonal  accounted f o r  substantia  basalis magnocellularis  25%, s i n c e d o u b l e - l a b e l e d  area  regions  b a s a l f o r e b r a i n ; o r an a v e r a g e  embedded i n t h e i n t e r n a l c a p s u l e ) as  basalis  that the proportion of double-labeled  neurons found  (caudal  number o f s i n g l e - l a b e l e d n e u r o n s  basalis magnocellularis  found  number o f s i n g l e - l a b e l e d c e l l s  counted through the v e n t r a l pallidum,  estimated  c e l l s were  The p r o p o r t i o n o f  i n these  an a v e r a g e o f 13% o f t h e t o t a l  nucleus  an a v e r a g e o f 14%.  80%) o f d o u b l e - l a b e l e d  s u b s t a n t i a innominata,  double-labeled  of  of the caudal  encompassing t h e v e n t r a l p a l l i d u m  magnocellularis  (FG)  number o f s i n g l e -  counted through the extent  b a s a l f o r e b r a i n r a n g e d f r o m 10 t o 23% w i t h The  A summary o f  p r e o p t i c area  of the  revealed  that  47 Table The in  2.  Summary o f t h e r e s u l t s  obtained  i n experiment  number o f r e t r o g r a d e l y l a b e l e d n e u r o n s f o l l o w i n g i n j e c t i o n the r e t i c u l a r  various  thalamic nucleus  t h a t were c o u n t e d  regions of the basal forebrain  tegmentum.of f i v e  animals  ( s e e method f o r d e t a i l s  are presented.  labeled  i n each o f t h e r e g i o n s examined i s a l s o  (values i n b r a c k e t s ) . which r e f l e c t s  discussed  table.  doubly  presented  Note t h e v a l u e s o b t a i n e d w i t h a n i m a l  i n the text.  i n this  The  C-  percentage  s t u d y were b a s e d  Abbreviations:  HDB,  t h e d i a g o n a l band; MCPO, m a g n o c e l l u l a r  p r e o p t i c area; NBM;  b a s a l i s m a g n o c e l l u l a r i s ; PPT, p e d u n c u l o p o n t i n e LDT, l a t e r o d o r s a l x; mean.  tegmental  on t h e v a l u e s  horizontal  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;  thalamic nucleus;  cell  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  calculations presented  nucleus;  on  The number o f n e u r o n s t h a t were  i n more d e t a i l s  shown i n t h i s  through  and m e s o p o n t i n e  counts)  7,  one.  limb of VP,  nucleus  tegmental  n u c l e u s ; RTN,  reticular  Table  2.  Numbers o f L a b e l e d N e u r o n s I n n e r v a t i n g t h e RTN o r RTN + c o r t e x ( v a l u e s i n b r a c k e t s ) Brain  Rats C-7 C-13  HDB\MCPO 0  VP\SI\NBM 96 (22)  Regions PPT  LDT  40 (3)  17 (1)  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  proportion of double-labeled c e l l s  average cells  o f 1.5% r e l a t i v e  to the t o t a l  accounted number  f o r an  of FG-labeled  t h a t were c o u n t e d t h r o u g h t h e c a u d a l b a s a l  an a v e r a g e  o f 11% o f t h e t o t a l  were c o u n t e d  number  forebrain; or  of FG-labeled c e l l s  through the caudal p o r t i o n s of the h o r i z o n t a l  l i m b o f t h e d i a g o n a l band a n d m a g n o c e l l u l a r p r e o p t i c alone  area  (see Table 2 ) .  In  t h e r e g i o n s encompassing t h e pedunculopontine  laterodorsal  tegmental  almost e x c l u s i v e l y nucleus  nuclei,  Double-labeled c e l l s  f o r an average  single-labeled  cells  pedunculopontine  double-labeled c e l l s  found i n the pedunculopontine  (see Table 2 ) .  accounted  (FG) t h a t were c o u n t e d  and l a t e r o d o r s a l  pedunculopontine  tegmental  the  representative distribution  tegmental  were  tegmental region  number o f  across both the n u c l e i or across  nucleus alone.  F i g u r e 8 shows  of these neurons through s i x  c o r o n a l p l a n e s of the mesopontine  tegmentum.  F i g u r e 9, e x a m p l e s o f d o u b l e - l a b e l e d c e l l s  observed  and  i n this  o f 9%-12% o f t h e t o t a l  the  In  that  that  were  i n t h e n u c l e u s b a s a l i s m a g n o c e l l u l a r i s and  pedunculopontine  t e g m e n t a l n u c l e u s a r e shown.  A l t h o u g h c o u n t s were n o t o b t a i n e d , numerous d o u b l e - l a b e l e d cells  were o b s e r v e d  (primarily  i n the d o r s a l  i n the pars r e t i c u l a t a )  region of the locus ceruleus. labeled cells  substantia  nigra  and, o c c a s i o n a l l y ,  i n the  In the diencephalon, double-  were a l s o o b s e r v e d  thalamic n u c l e i . n e u r o n s may  raphe,  i n various intralaminar  However, some o f t h e s e d o u b l e - l a b e l e d  have r e s u l t e d  i n part  from  the involvement of  50  Figure  8.  Representative d i s t r i b u t i o n  of retrogradely labeled  neurons observed  i nthe pedunculopontine  tegmental  that innervate ther e t i c u l a r  nuclei  (singly  labeled) o r t h a t innervate both  nucleus  and c o r t e x  (doubly  labeled).  thalamic  the r e t i c u l a r  i s presented  from  a n d Watson, 1986) o f t h e m e s o p o n t i n e  The  antero-posterior coordinates  planes  thalamic  s i x coronal planes  (modified  tegmentum.  ( f r o m bregma) f o r e a c h o f t h e  a r e a s 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: •, a p p r o x i m a t e l y  t h r e e F G - l a b e l e d neurons i n n e r v a t i n g t h e r e t i c u l a r nucleus;  *, a F G - l a b e l e d n e u r o n a l s o  (corticopetal). tegmental  mlf, medial tegmental  lemniscus;  tegmental  longitudinal  nucleus;  labeled corticopetal  gray;  fasciculus;ppt,  l d t , laterodorsal  N o t shown i s t h e d i s t r i b u t i o n neurons.  dtb, dorsal  cp, c e r e b r a l peduncle; ml;  r s , rubrospinal tract;  nucleus.  thalamic  l a b e l e d with PI  Abbreviations: eg, c e n t r a l  bundle;  pedunculopontine medial  nucleus  The p a t t e r n o f  distribution Paxinos  through  and l a t e r o d o r s a l  of singly  Figure  9.  Two p a i r s of photomicrographs each showing a neuron  (arrow) t h a t was doubly l a b e l e d f o l l o w i n g RTN and c o r t e x and c o r t e x  i n j e c t i o n s i n the  i n d i c a t i n g t h a t t h i s neuron i n n e r v a t e d  through axon c o l l a t e r a l s .  the RTN  These d o u b l e - l a b e l e d  neurons were observed i n the nucleus b a s a l i s  magnocellularis  (A + B) and pedunculopontine tegmental nucleus (C + D). Neurons t h a t were l a b e l e d 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 the RTN are p r e s e n t e d on the f a r l e f t w h i l e those 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 are p r e s e n t e d on the f a r r i g h t , (magnification:  A + B, 283x; C + D, 126x).  54  fibers  of  passage t r a v e l i n g  through  are needed t o c o n f i r m these The d i s t r i b u t i o n o f and TB was  similar  the cortex. blend of shades)  d e s c r i b e d when PI  that varied in color  (see  Figure  some n e u r o n s was more s u b j e c t (see  p r o p o r t i o n of appear  Figure  A subpopulation  choline  although  not attempted f o r  Immunohistochemical  following  of  injections  almost  labeling  f o r each dye.  this  group  the  not  observed f o l l o w i n g  q u a n t i f i c a t i o n of this  PI  problem,  w i t h FG and TB d i d  neurons  of  PI  double  animals.  into  all  of  t h a t were r e t r o g r a d e l y  contain  immunoreactivity.  the  In  the F G - l a b e l e d c e l l s  observed i n the regions  encompassing  and h o r i z o n t a l l i m b o f  labeled  t h e RTN were f o u n d t o  A few o f  2 0 % of  of  i n t e r p r e t a t i o n than with  basalis magnocellularis.  up t o  double-  10), double-labeling  than that  a  (blue-lavender  the d i a g o n a l  these c e l l s  the  were  the s u b s t a n t i a band.  the F G - l a b e l e d c e l l s  It  basal  t h a t were  ChAT i m m u n o r e a c t i v e were o b s e r v e d i n t h e r e g i o n o f  that  in  Analyses  acetyltransferase  forebrain,  of  Notwithstanding  significantly  injections  l a b e l i n g was  11).  to  double-labeled c e l l s  to d i f f e r  cortical  injected  a l t h o u g h many n e u r o n s were u n e q u i v o c a l l y  l a b e l e d w i t h FG and TB  injections  was  w i t h FG and TB r e s u l t e d i n  d e p e n d i n g on t h e i n t e n s i t y  Accordingly,  studies  t h a t were d o u b l e - l a b e l e d w i t h FG  Double-labeled c e l l s  t h e two t r a c e r s  Control  results.  cells  to that  t h e RTN.  was  also nucleus  also innominata  estimated  t h a t were o b s e r v e d  in  Figure  10.  Color  photomicrograph of  double-labeled with fluoro-gold t h a l a m u s ) and  the  nucleus b a s a l i s magnocellularis. from the  blue  (from the  the  resulting  true  which could  be  illustrated  in this  (magnification:  resulted  i n the  of  these  the  (deep b l u e ) on  o t h e r hand a r e  521x).  also  Single the  one  shown,  was  nucleus  region  Double-labeled  i n a b l e n d of  photomicrograph.  blue  (arrow) t h a t  reticular  assessed with a s i n g l e f i l t e r  labeled with true on  (corticopetal)  retrograde transport  t h r o u g h axon c o l l a t e r a l s  fluoro-gold  a neuron  of  of  neurons  tracers the  block  two as  labeled hand,  colors  neurons and  56  Figure  11.  Color photomicrograph  double-labeled with fluoro-gold n u c l e u s ) and p r o p i d i u m i o d i d e the  of a neuron  (from the r e t i c u l a r  (corticopetal)  pedunculopontine tegmental nucleus.  fluoro-gold  on t h e one  on t h e o t h e r a r e a l s o  hand shown.  (magnification:  260x).  that  i n the region  ( b l u e ) and p r o p i d i u m i o d i d e I t i s thought t h a t neurons  t h r o u g h axon  of  with (red) that  photomicrograph  collaterals,  was  thalamic  Neurons l a b e l e d  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 t r a n s p o r t e d each t r a c e r s  (arrow)  had  59 the  region of the nucleus  cholinergic.  basalis magnocellularis  A large proportion  neurons i n t h i s  (up t o 50%) o f t h e  PI ( t r i p l e - l a b e l e d  reliable  basalis magnocellularis.  c o u n t s were d i f f i c u l t  to obtain  neurons g e n e r a l l y d i d not s t a i n antibodies.  T h i s p r o b l e m was  processed  f o r ChAT  sufficiently  labeled with  basal  forebrain sections  i n two a n i m a l s were analyses.  the vast majority  FG t h a t were a l s o  of neurons  immunoreactive  ChAT a n t i b o d i e s were o b s e r v e d i n t h e p e d u n c u l o p o n t i n e  tegmental nucleus. the  ChAT  semi-quantitative  I n t h e m e s o p o n t i n e tegmentum, retrogradely  basalis  following  Nevertheless,  to allow  However,  f u r t h e r compounded by t h e f a c t  immunoreactivity  intense  neuron  since nucleus  i n t e n s e l y with  t h a t b a c k g r o u n d s t a i n i n g was h i g h immunohistochemistry.  appeared  cells).  12 shows p h o t o m i c r o g r a p h s o f a t r i p l e - l a b e l e d  observed i n the nucleus  with  FG-labeled  r e g i o n t h a t were ChAT i m m u n o r e a c t i v e  a l s o t o have been l a b e l e d w i t h Figure  were  FG-labeled  particularly cells  In t h i s  cells  region,  approximately  also stained with  i n i t srostral portion.  ChAT  FG and ChAT  background s t a i n i n g prevented  reliable  cell  T r i p l e - l a b e l e d n e u r o n s were a l s o o b s e r v e d i n t h e  laterodorsal  The  with  some o f t h e  a l s o appeared t o have been l a b e l e d w i t h P I .  However, h i g h counts.  antibodies,  Furthermore,  (10-20%) t h a t were d o u b l e - l a b e l e d  antibodies  40-60% o f  tegmental nucleus,  distribution  telencephalon  was  but only o c c a s i o n a l l y .  of SOM-positive neurons i n the  as r e p o r t e d  previously  (Vincent e t a l . ,  60  Figure  12.  A s e r i e s of photomicrographs i l l u s t r a t i n g a neuron  (arrow) t h a t was  retrogradely labeled with fluoro-gold  f o l l o w i n g i n j e c t i o n i n the RTN  (A), w i t h propidium i o d i d e  f o l l o w i n g i n j e c t i o n i n the c o r t e x immunoreactive  I t was  c h o l i n e r g i c and  a l s o ChAT  t h e r e f o r e concluded t h a t t h i s neuron  that i t innervated  through axon c o l l a t e r a l s , (magnification:  t h a t was  (C), i n the r e g i o n of the nucleus b a s a l i s  magnocellularis. was  (B), and  96x).  both the RTN  and  cortex  62  1985).  F o r e x a m p l e , many SOM-immunoreactive n e u r o n s have b e e n  observed  i n various c o r t i c a l  entopeduncular SOM-positive  nucleus.  a r e a s , t h e s t r i a t u m and t h e  In the basal forebrain, scattered  n e u r o n s were o b s e r v e d  regions of the h o r i z o n t a l ventral pallidum.  primarily  l i m b o f t h e d i a g o n a l band and  However, none o f t h e b a s a l  n e u r o n 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 t o t h e RTN were i m m u n o r e a c t i v e I n one a n i m a l ,  nucleus  site  and v e n t r o m e d i a l  appeared  injections  f o r somatostatin antibodies.  a few F G - l a b e l e d n e u r o n s o b s e r v e d  entopeduncular  forebrain  f o l l o w i n g FG  i n w h i c h t h e RTN i n j e c t i o n  some d e g r e e t h e v e n t r o l a t e r a l nuclei,  through the  included to thalamic  i n the  t o be a l s o i m m u n o r e a c t i v e  with  SOM a n t i b o d i e s . Numerous F G - l a b e l e d n e u r o n s o f t h e b a s a l f o r e b r a i n found  t o be i m m u n o r e a c t i v e w i t h PV a n t i b o d i e s .  m a j o r i t y o f these d o u b l e d - l a b e l e d neurons in  The v a s t  (FG + PV) were  found  t h e r e g i o n o f t h e 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 a r e a At  were  least  just  below t h e a n t e r i o r  commissure  50% o f t h e F G - l a b e l e d c e l l s  i m m u n o r e a c t i v e w i t h PV a n t i b o d i e s . the nucleus  found  i n this  The r e g i o n s  b a s a l i s m a g n o c e l l u l a r i s and m e d i a l  a l s o c o n t a i n e d F G - l a b e l e d n e u r o n s t h a t were (see F i g u r e 1 2 ) . labeled  (see F i g u r e 13).  neurons  b e t w e e n 20-25%.  In these  encompassing globus  pallidus  PV-immunoreactive  regions, the p r o p o r t i o n of double-  (PV + FG l a b e l e d ) was e s t i m a t e d  t o range  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  t o have b e e n 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 ) both  r e g i o n were  P I and FG ( t r i p l e - l a b e l e d )  observed  or with  i n the regions of the nucleus  Figure  13.  P a i r s of photomicrographs showing l a b e l e d  (arrows) p r o j e c t i n g t o the r e t i c u l a r  neurons  thalamic nucleus t h a t  were a l s o immunoreactive f o r parvalbumin i n the r e g i o n s 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 (C + D).  (A + B) and v e n t r a l  The open arrow shows a neuron l a b e l e d o n l y  fluoro-gold Abbreviation:  (from the r e t i c u l a r ac, a n t e r i o r  (magnification:  109x).  thalamic  commissure,  nucleus).  pallidum with  65  basalis magnocellularis an  and v e n t r a l  example o f a t r i p l e - l a b e l e d c e l l  double-or  triple-labeling involving  e q u i v o c a l due t o h i g h b a c k g r o u n d  pallidum.  Figure  (FG + P I + P V ) . P I were g e n e r a l l y  staining.  14  shows  However,  F i g u r e 14. one neuron  A s e r i e s of photomicrographs i l l u s t r a t i n g  at l e a s t  (arrow) 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 \ s u b s t a n t i a innominata t h a t was  retrogradely 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 t h a l a m i c nucleus (A) and f o l l o w i n g i n j e c t i o n s i n the c o r t e x  (B).  T h i s neuron was  found t o be immunoreactive w i t h parvalbumin ( C ) . (magnification:  145x).  also  c  68 DISCUSSION  Technical  The  Considerations  p o s s i b i l i t y that  the  v a l i d i t y of  any  conclusions.  study pertains resulted  the  false-positive  r e s u l t s must be  A particular  to  i n part  the  injection site.  injections,  the  to  the  extent  areas that  dye  received  hippocampus. unlikely  in  s u c h manner.  that  the  dye,  (FG the  forebrain  the  For  and  number o f  number and  d i s t r i b u t i o n of  the  animals that  following two  afferents  cortical pertinent  subcortical s u c h as  the  completely ruled  the  following  more c o n v i n c i n g  i s the  volume o f  cortical  double-labeled the  injected  cortical injections  differed  were e x a m i n e d .  Thirdly,  FG-labeled c e l l s  some o f  fact that  areas  affecting  d i s t r i b u t i o n of  did  the the  PI  it  affected  that  not  were  the doubly  appear t o  showed p a r t i a l i n v o l v e m e n t o f  corpus callosum  out,  subcortical  nature of  cortical injections  animals that  coursing  s t u d y have been  example, the  the  in  involved  diffusion into  p r o p o r t i o n or  the  labeling  passage i s o n l y  this  Secondly, the  i n many o f  labeled  fibers  between a n i m a l s w i t h o u t  + PI\TB). location  dye  of  reaching  tract-tracing  retrograde  case of  f i b e r s of  results  First,  varied  significantly cells  the  d i f f u s i o n had  basal  r a r e l y observed.  injections  In  A l t h o u g h t h i s c a n n o t be  is  was  problem of  that  p r o b l e m w i t h any  involvement of  through the  undermine  addressed before  p o s s i b i l i t y that  from the  l a b e l i n g may  injections.  d i s t r i b u t i o n of  differ  the Even double-  69 labeled  cells  despite  the  did  not  fact that  c o r t i c a l mantle. into  significantly  the of  to  v a l i d i t y of  the  the  due  m a n t l e was  at  tracer  never observed  the  contralateral  corticopetal and  avoided.  a l o n g the  (Bigl et  was  little  r e d u c i n g the  Thirdly,  as  the  First,  the  contributed  have  diminished  RTN  is  a l l RTN  injections  i t has  a l . , 1982;  amount o f  spillage  i.e.  in  may  in observed  the  have  produced  been e s t a b l i s h e d basal  forebrain  Woolf e t control  a l . , 1984), animal to  infusion  of  f i b e r s of t a k e n up  with c o r t i c a l injections,  the  passage by  exact  these  a  be FG  injection site therefore  tracer  that  exclusively  i n one  transected  the  the  were o f t e n  neurons i n b o t h the  the  through  ipsilateral cortical  spills  almost  fibers  also  c o n t a m i n a t i o n of  Secondly, iontophoretic damage a t  diffusion  study.  microelectrode,  since  corroborated  number o f  consequently,  into  That such s p i l l s  is unlikely  d i s c u s s e d below. produced  dye  Furthermore, t r a c e r  the  the  l a b e l i n g p r o d u c e d by  i n the  However, FG  cholinergic  that  that  micropipette track  m e s o p o n t i n e tegmentum a r e  finding  present  Possible  hemisphere.  ipsilateralized  to  reasons.  entrance of  false-positives  the  injections  spillage  regions.  s i t e of  injections  iontophoresis v i a penetrations  therefore  subcortical  FG  hemisphere.  to  of  r e s u l t s due  were p e r f o r m e d w i t h  results  TB  r e s t r i c t e d to  f a l s e - p o s i t i v e s may  a number o f  contralateral  d i f f u s i o n was  results  passage f o l l o w i n g for  following  r e g i o n s , e v e n i f u n d e t e c t e d , had  p o s s i b i l i t y that  unlikely  was  dye  differ  Thus, i t i s v e r y u n l i k e l y  subcortical  The  appear to  greatly  and fibers.  location  of  the  core  o f RTN  i n j e c t i o n s v a r i e d to a c e r t a i n extent  animals without a f f e c t i n g the p a t t e r n Furthermore, i n a t l e a s t  not  yield  did  t o t h e RTN.  and p r o p o r t i o n  significantly  f o r e b r a i n f i b e r s may site  i s very  f o r e b r a i n (see  injection  basal  sites.  have b e e n i n v o l v e d  traveled  that  the course  Hence,  Saper  forebrain  fibers  (1) a m e d i a l pathway  i n v o l v i n g the  diagonal  band, f o r n i x , genu o f c o r p u s c a l l o s u m  and c i n g u l a t e  bundle;  and  through  forebrain i n  i n t h e RTN  forebrain fibers.  (1984) h a s shown t h a t most c o r t i c o p e t a l b a s a l  cells  Finally,  u n l i k e l y when c o n s i d e r i n g  t a k e n by c o r t i c o p e t a l b a s a l  RTN, d i d  of double-labeled  i n the caudal  comparison t o l e s s r e s t r i c t e d  injection  the caudal  l a b e l i n g i n the r o s t r a l  the p a t t e r n  not d i f f e r  basal  A l t h o u g h one o f t h e s e  w h i c h p r i m a r i l y encompassed  retrograde  results),  of l a b e l i n g .  two a n i m a l s , t h e 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 injections,  between  (2) a l a t e r a l pathway  amygdala, p y r i f o r m The p o s s i b i l i t y may  cortex  i n v o l v i n g the substantia and  t h e RTN  in  s e v e r a l c o n t r o l animals.  i n j e c t i o n was thalamic  centered  I n one a n i m a l ,  i n the a n t e r o v e n t r a l  little  forebrain.  t h a t were d o u b l e - l a b e l e d rare.  labeling  was  On t h e o t h e r  the boundaries  further  addressed  i n w h i c h t h e FG and  n u c l e i and i n v o l v e d a l s o a m a j o r s p i l l  observed i n the basal  very  outside  have confounded t h e d a t a  i n the c o n t r a l a t e r a l cortex,  cells  putamen.  t h a t dye d i f f u s i o n  of  innominata,  retrograde  Accordingly,  anteromedial of the t r a c e r l a b e l i n g was basal  forebrain  f o l l o w i n g such i n j e c t i o n  hand, t h e p a t t e r n  i n t h e m e s o p o n t i n e tegmentum  of  were  retrograde  f o l l o w i n g FG i n f u s i o n  i n t o a n t e r i o r t h a l a m i c n u c l e i was following  RTN  retrogradely  injections.  markedly d i f f e r e n t than  Hence, the v a s t m a j o r i t y of  l a b e l e d 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 s m a l l number of these l a t e r o d o r s a l tegmental c e l l s  appeared t o be have been d o u b l e - l a b e l e d with  PI.  In a second animal, i n which the core of the FG was  c e n t e r e d too r o s t r o m e d i a l l y ,  t e r m i n a l i s , bed  nucleus of the  i n v o l v i n g the  p o l e of the RTN, occasionally  i n e i t h e r the b a s a l  innominata and  rostral  f o r e b r a i n or i n  the  l a t e r o d o r s a l tegmental n u c l e i of  mesopontine tegmentum.  Furthermore, i t i s p o s s i b l e  observed d o u b l e - l a b e l e d c e l l s r e f l e c t e d the involvement of the RTN  i n the  F i n a l l y , two  animals r e c e i v e d  nuclei.  The  pattern  i n j e c t i o n s was  than being  i n t e r n a l capsule, FG  injections  forebrain.  In c o n t r a s t ,  observed i n the RTN,  thalamic  of r e t r o g r a d e l a b e l i n g f o l l o w i n g  l a b e l e d c e l l s were found o n l y o c c a s i o n a l l y  these  injections. i n the  that  the  ventroposterolateral  d i f f e r e n t than f o l l o w i n g RTN  the  partial  were c e n t e r e d i n v e n t r a l t h a l a m i c n u c l e i i n c l u d i n g v e n t r o m e d i a l , v e n t r o l a t e r a l and  the  that  i n j e c t i o n s i t e rather  r e s u l t of f i b e r s t r a v e l i n g through the  f o r example.  internal  d o u b l e - l a b e l e d c e l l s were observed o n l y  pedunculopontine and  the  stria  s t r i a terminalis,  c a p s u l e , globus p a l l i d u s , s u b s t a n t i a  injection  FG-  basal  numerous F G - l a b e l e d neurons were  substantia  n i g r a , mammillary b o d i e s ,  entopeduncular n u c l e u s , d o r s a l raphe, pedunculopontine l a t e r o d o r s a l tegmental n u c l e i .  and  Occasional double-labeled  cells  (FG + PI) were observed i n the pedunculopontine  tegmental nucleus,  and  to a l e s s e r extent,  i n the l a t e r o d o r s a l  F a l s e 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  tegmental  nucleus. or  e p i t h e l i a l c e l l s r a t h e r 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 s i n c e g l i a  e p i t h e l i a l c e l l s c o u l d be r e a d i l y recognized  and  on the b a s i s of  s e v e r a l c h a r a c t e r i s t i c s . For example, i n c o n t r a s t t o majority  the  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 , these c e l l s were  s m a l l , s p h e r i c a l w i t h no  labeled dendrites;  g e n e r a l l y l o c a t e d near the i n j e c t i o n s i t e i n c l u d e d i n the a n a l y s i s of r e t r o g r a d e vessel walls.  (which was  never  l a b e l i n g ) or i n blood  Furthermore, i n s e c t i o n s not processed f o r  immunohistochemistry, i t was neuron had  they were  p o s s i b l e to d i r e c t l y v e r i f y i f a  been l a b e l e d by r e t r o g r a d e  between e m i s s i o n f i l t e r s ,  t r a n s p o r t by a l t e r n a t i n g  s i n c e g l i a and  e p i t h e l i a l c e l l s were  i n t e n s e l y l a b e l e d r e g a r d l e s s of the f i l t e r b l o c k used.  The  p o s s i b i l i t y t h a t ChAT immunoreactive neurons (FITC) may  have  been mistaken f o r 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 s i n c e ChAT immunostaining was  c h a r a c t e r i s t i c a l l y not i n t e n s e .  It is  t h e r e f o r e more probable t h a t the p r o p o r t i o n of doubly or t r i p l y - l a b e l e d neurons which had was  i n v o l v e d ChAT immunostaining  underestimated i n t h i s study as w i l l be d i s c u s s e d  F i n a l l y , s i n c e PV  immunostaining was  below.  generally intense,  neurons showing 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  only  basal  f o r e b r a i n neurons ( l a r g e , m u l t i p o l a r e t c . ) .were i n c l u d e d i n the a n a l y s 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 s I n n e r v a t i n g the RTN  The  and  r e s u l t s of t h i s study have both confirmed  Cortex  and  extended  knowledge on 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 and mesopontine tegmental p r o j e c t i o n systems t h a t i n n e r v a t e c o r t e x and  the  RTN.  By u s i n g a t r i p l e - l a b e l i n g approach, the  present  i n v e s t i g a t i o n has c l e a r l y e s t a b l i s h e d t h a t a s i g n i f i c a n t p o r t i o n (at l e a s t 15%)  of e i t h e r the b a s a l f o r e b r a i n or  mesopontine tegmental i n p u t to the RTN  i s composed of neurons  with axons t h a t b i f u r c a t e to i n n e r v a t e the c o r t e x as w e l l . The  p a t t e r n of 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  r e v e a l e d t h a t the nucleus  cells  has  b a s a l i s m a g n o c e l l u l a r i s of the b a s a l  f o r e b r a i n and the pedunculopontine tegmental nucleus mesopontine tegmentum are primary sources collateralized projections.  of  of  the  these  W i t h i n these r e g i o n s , i t was  f u r t h e r demonstrated t h a t a l a r g e p o r t i o n 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 systems are c h o l i n e r g i c . The  r e l a t i v e c o n t r i b u t i o n of the p r o j e c t i o n systems t h a t  were examined i n r e l a t i o n to the i n n e r v a t i o n of the RTN + c o r t e x was  assessed  by way  However, the r e s u l t s of these presented  i n Table  of s e m i - q u a n t i t a t i v e  probably  RTN  analyses.  semi-quantitative analyses,  as  2, must be i n t e r p r e t e d with c a u t i o n i . e .  w i t h i n the l i m i t s of the techniques of r e t r o g r a d e  or  used.  Hence, the amount  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  o n l y a f r a c t i o n of the e n t i r e p o p u l a t i o n of  the  74  projecting had  n e u r o n s u n d e r s t u d y s i n c e none o f  involved  therefore  the  e n t i r e extent  failed  obtained  particularly various  the  other  ventrobasal  study i s i n c l o s e al.,  1987; of  the  receives  RTN  the  innervate  be  and  cortex  of  the  an  as  frequency  of  have b e e n since have  v i s u a l i z a t i o n of  labeled  triply  forebrain  i n j e c t i o n s i n t o the  n u c l e i as  see  basal  observed  reports  in  RTN, this  (Hallanger  also introduction).  from b a s a l  this  i n p u t may  as w e l l .  Because  forebrain be  a v a i l a b l e , immunostaining  that  neurons  collateralized  reliable  f o r PV  was  acid  chosen  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 G A B A e r g i c  neurons s i n c e  the  been a s s o c i a t e d systems of  the  d i s t r i b u t i o n of PV-containing  i n recent  y e a r s w i t h many o f  mammalian b r a i n  ( C e l i o and  et  The  have r e v e a l e d  i m m u n o h i s t o f l u o r e s c e n c e methods f o r g a m m a - a m i n o b u t y r i c (GABA) were n o t  (see  f i x a t i o n p r o t o c o l s , may  thalamic  that  minimal  interpreted  l a b e l e d neurons i n the  a major i n p u t  the  some p r o j e c t i n g  T h i s may  agreement w i t h e a r l i e r  PV,  have  percentage  immunohistochemical analyses  that contained  may  injections  below.  L e v e y e t a l . , 1987;  results  to  f o r the  FG  immunohistochemistry  m e s o p o n t i n e tegmentum f o l l o w i n g  a n t e r i o r or  of  the  limits  labeling.  discussed  d i s t r i b u t i o n of  that  lower  case f o l l o w i n g  be  and  fields  study can  f a c t o r s , s u c h as  n e u r o n s as w i l l  and  the  triple  been l e s s t h a n o p t i m a l  The  believed  in this  rough i n d i c e s p r o v i d i n g d o u b l e and  RTN,  f a l s e - p o s i t i v e l a b e l i n g was  above), i t i s t h e r e f o r e  single,  the  to cover terminal  neurons. Given t h a t  calculations  of  the  the  neurons  has  GABAergic  Heizmann,  1981;  as  75 Gerfen et a l . ,  1985).  c o n t a i n a major  Hence, t h e RTN,  p o p u l a t i o n of GABAergic  al.,  1980),  4).  Furthermore, the l o c a t i o n  also  i m m u n o r e a c t i v e w i t h PV  was  stained  (Young  i s known t o  neurons  (Houser e t  i n t e n s e l y w i t h PV a n t i b o d i e s  remarkably s i m i l a r  r e v e a l e d by  which  (see F i g u r e  of FG-labeled c e l l s  antibodies  to that  i n the v e n t r a l  of GABAergic  neurons  observation  1984).  This  containing  than that of  n e u r o n s was  different  as a s s e s s e d i n t h e same a n i m a l s .  quite plausible  that  observed i n t h i s  the d o u b l e - l a b e l e d  neurons.  I t would  be  o t h e r methods i f t h e s e n e u r o n s  and c o r t e x .  direct  their  possible  suggested While basal  e v i d e n c e f o r GABAto determine with  i n the  to  innervate  basal interesting  in relation  i n n e r v a t i o n as  to  was  study.  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  above,  and  to  study i s , i n i t s e l f ,  investigation particularly  forebrain projection  mentioned  a l t h o u g h PV  collateralize  contribution to c o r t i c a l  i n this  neurons  Furthermore, the presence of a  as o b s e r v e d i n t h i s further  therefore  a r e i n d e e d GABAergic;  m o d e r a t e number o f P V - c o n t a i n i n g n e u r o n s  and m e r i t s  PV-  ChAT-positive  (PV + FG)  interesting  t h e d e g r e e t o w h i c h t h e y may  b o t h t h e RTN  o f most  I t appears  study are indeed GABAergic,  immunostaining does not c o n s t i t u t e  forebrain  as  that w i t h the exception of the r e g i o n of the the d i s t r i b u t i o n  verify  pallidum  i s a l s o c o n s i s t e n t with the  nucleus b a s a l i s magnocellularis,  containing  were  i m m u n o s t a i n i n g f o r t h e s y n t h e s i z i n g enzyme o f GABA  III et al.,  neurons  that  system i n n e r v a t i n g  the present  t o the  t h e RTN,  as  s t u d y has c o n f i r m e d t h a t t h e  RTN  76  is  innervated  by  magnocellularis relative (up  to  c h o l i n e r g i c neurons of and  projecting  relative to the  (1987, see  also  to the  RTN)  total  obtained  e t a l . (1987).  cholinergic  b e e n shown t h a t  that  the For  appears,  Hallanger  includes  RTN,  of  the  RTN  by  forebrain  o r RTN  can  picric  secondary antibody  of b a s a l  and  be  a c i d o r by  (Eckenstein  required  and  was  recently  adding  a rat  e t a l . , 1988).  to c l e a r l y  o r o f b o t h RTN  cortex  improved w i t h  established  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  innervation  et a l .  observed  e x a m p l e , i t has  ChAT i m m u n o s t a i n i n g that  by  RTN  neurons  s i m i l a r to that  proportion  study.  More s e n s i t i v e methods a r e relative  reported  to the  labeled  study  The  However, as m e n t i o n e d p r e v i o u s l y , i t the  in this  fixation protocol serum t o t h e  in this  neurons i n n e r v a t i n g  underestimated  number o f  i n t r o d u c t i o n ) , but  quite possible  basalis  pedunculopontine tegmental nucleus.  however, l o w e r t h a n p r e v i o u s l y  is  nucleus  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 inputs  20%  Steriade  the  the  the  cortex.  Conclusions  Several  features  have b e e n d e s c r i b e d . of neurons c o n t a i n e d tegmentum t h a t  of  some p r o j e c t i o n s  First, i n the  innervate  part  of  this  also  suggested that  basal  the  t h r o u g h axon c o l l a t e r a l s . collateralized another  i t was  RTN  I t was  innervating  shown t h a t  f o r e b r a i n and also innervate  a  the  subpopulation mesopontine the  cortex  f u r t h e r demonstrated  projection is cholinergic. subpopulation  RTN  of b a s a l  that It  was  forebrain  77  neurons d e f i n e d by t h e i r immunoreactivity  with  PV-antibodies  a l s o c o n t r i b u t e d t o 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 systems. The  c h o l i n e r g i c i n n e r v a t i o n of the RTN  d e r i v e d from both  the b a s a l f o r e b r a i n and mesopontine tegmentum was t h i s study.  The  present  by showing t h a t the RTN  r e s u l t s have extended t h i s knowledge a l s o 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 s parvalbumin. neurotransmitter  content  confirmed i n  of t h i s subpopulation  The of  PV-  c o n t a i n i n g neurons as observed i n t h i s study i s u n c e r t a i n . However, GABA appears a l i k e l y  candidate.  78 EXPERIMENT  TWO  INTRODUCTION  The limbic  interpeduncular  nucleus  a n d t h e h i p p o c a m p u s a r e two  structures that are innervated  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  i m m u n o h i s t o c h e m i s t r y have r e v e a l e d derived  ChAT  that part of t h i s  from c h o l i n e r g i c neurons o f t h e r o s t r a l  (Woolf a n d B u t c h e r , 1985;  by t h e b a s a l f o r e b r a i n .  Saper,  further  1984).  input i s  basal forebrain  1985; W o o l f e t a l . , 1984; A m a r a l and K u r z , A more d e t a i l e d a n a l y s i s o f t h e s e  reveals that the vast majority  of the r o s t r a l  studies basal  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 t h e i n t e r p e d u n c u l a r nucleus  on t h e one hand, and t h e h i p p o c a m p u s on t h e o t h e r  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  Hence, c h o l i n e r g i c n e u r o n s a s s o c i a t e d w i t h nucleus  and t h e v e r t i c a l  band were d e s c r i b e d  i n these  to the interpeduncular contrast  significantly.  the medial  and h o r i z o n t a l l i m b s  s t u d i e s as p r o v i d i n g a major  nucleus  nucleus  Since  (Woolf and B u t c h e r ,  overlap  has  f r o m more  basalis  1985).  i n the o r i g i n s of basal forebrain projections  to the interpeduncular established,  In  of the basal f o r e b r a i n i n c l u d i n g the regions  e n c o m p a s s i n g t h e s u b s t a n t i a i n n o m i n a t a and n u c l e u s magnocellularis  input  and h i p p o c a m p u s r e s p e c t i v e l y .  t o t h e hippocampus, t h e i n t e r p e d u n c u l a r  regions  septal  of the diagonal  a l s o b e e n shown t o r e c e i v e a m i n o r c h o l i n e r g i c i n p u t caudal  hand,  nucleus  the question  and hippocampus appears  a r i s e s as t o t h e e x i s t e n c e  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  to innervate  well  of basal  both of  these  79 structures  simultaneously.  interpeduncular anatomically 1987)  nucleus  I t i s interesting  and h i p p o c a m p u s h a v e b e e n shown t o be  linked v i a reciprocal  a n d t h a t some o f t h e s e  (Montone e t a l . , 1 9 8 7 ) .  connections  connections  Accordingly,  h a v e axons t h a t b r a n c h t o i n n e r v a t e and h i p p o c a m p u s u s i n g  l a b e l i n g procedure.  Such a c o l l a t e r a l i z e d  anatomical  collateralized  the f o l l o w i n g study  sought  f o r e b r a i n neurons  both the interpeduncular  a retrograde  fluorescence  double-  p r o j e c t i o n s y s t e m i s shown i n F i g u r e p r o j e c t i o n would p r o v i d e  l i n k a g e between t h e i n t e r p e d u n c u l a r  hippocampus.  and H a m i l l ,  A schematic r e p r e s e n t a t i o n of t h i s  hypothetical collateralized 15.  (Fass  may be  t o determine the degree t o which s i n g l e b a s a l  nucleus  t o note t h a t the  y e t another  nucleus  and  80 Figure  15.  A schematic  projection  system  collateralized interpeduncular fibers  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  arising  (dotted  i n the b a s a l f o r e b r a i n t h a t  line)  n u c l e u s and  to innervate hippocampus.  both The  the course of  as drawn a r e n o t r e p r e s e n t a t i v e o f a c t u a l  Abbreviations: n u c l e u s ; h, (modified  bf, basal  forebrain; ipn,  trajectories.  interpeduncular  hippocampus.  saggital  p l a t e from  Paxinos  and Watson,  the  1986).  81  82 METHODS  The  e x p e r i m e n t a l p r o c e d u r e and m a t e r i a l s used  e x p e r i m e n t were a s d e s c r i b e d reason, only d e t a i l s described earlier  pertinent  received a single  four The  into  midline  t o the hippocampal  6.72mm p o s t e r i o r  a n d 8.1mm v e n t r a l  v e r t i c a l plane.  4.52mm p o s t e r i o r  animal  parameters  t h e hippocampus.  a t an a n g l e from t h e injections.  The  nucleus  t o bregma, 1mm l a t e r a l  t o the  t o d u r a , w i t h an a n g l e o f 7 ° o f f t h e  The c o o r d i n a t e s used  i n j e c t i o n were a s f o l l o w s  midline;  into  c o o r d i n a t e s used f o r t h e i n t e r p e d u n c u l a r  were a s f o l l o w s :  and n o t  the i n t e r p e d u n c u l a r n u c l e u s f o l l o w e d by  n u c l e u s was a p p r o a c h e d  hemisphere c o n t r a l a t e r a l  Each  i n f u s i o n o f FG (same  o f P I (0. 3 u . l \ i n j e c t i o n )  interpeduncular  stereotaxic  For this  t o the present experiment  (250-300g) were u s e d .  iontophoretic  one)  injections  one.  are presented here.  Three male W i s t a r r a t s  as e x p e r i m e n t  i nExperiment  i n this  ( i n order):  f o r each o f hippocampal 2.12; 2.56; 3.60 a n d  t o bregma; 1; 1.4; 2; a n d 2.2mm l a t e r a l  3; 3.1; 2.6 a n d 2.6mm v e n t r a l  t o dura.  to the  83  RESULTS  Injection  Two  sites  of  the  were l a r g e l y diffusion third  Tracer  infusions  confined to  into  animal.  centered  FG  the  resulted  the  ventral  t e g m e n t a l a r e a was  interpeduncular nucleus  spillage  was  observed at  microelectrode,  and  except for  tracer of  PI  s i t e was  spillage into  the  restricted  was  h o r n as aimed a t  well the  as  the  right  encompassed t o  (see  addition,  spillage  midline a l l PI  i n the  hippocampal  animal,  the  CA2,  some d e g r e e t h e  close In  to  s i t e of  observed i n  the  large  CA3  and  injection  entrance of i n which  areas.  injection CA4  sites  that  Ammon's were  had  hippocampal  injections  details  no  Injections  diffusion  contralateral  methods f o r  sites.  16.  the  injections  on  that  were  coordinates).  produced v a r y i n g degree of  c o r t i c a l mantle immediately d o r s a l  injection  site  the  f i e l d s of  Although the  hippocampal  injections  an  the  interpeduncular nucleus,  hippocampal formation;  for  the  one  dentate gyrus.  formation e s p e c i a l l y to  the  hippocampus p r o d u c e d CAI,  that  i s shown i n F i g u r e  observed i n s u b c o r t i c a l  c o v e r e d e x t e n s i v e l y the  sites  i n t e r p e d u n c u l a r n u c l e u s whereas;  A photomicrograph i l l u s t r a t i n g  i n the  injection  in injection  to  the  tracer  Figure  16.  The  interpeduncular  r e p r e s e n t a t i v e FG  injection  nucleus i s i l l u s t r a t e d  which c o r r e s p o n d s t o the shaded  tegmental area,  (magnification:  70x).  with a  through  the  photomicrograph  area of the c o r o n a l  Note t h e p r e s e n c e o f l a b e l e d neurons ventral  site  plate.  i n the r e g i o n of  the  o c.  86  Pattern  of Retrograde  Following  Labeling  i n j e c t i o n s i n t o the  interpeduncular  numerous l a b e l e d somata were o b s e r v e d i n t h e forebrain. the  The  vast majority  h o r i z o n t a l limb  n e u r o n s were f o u n d diagonal labeled and  band. cells  extended  area.  of  the  of  band.  i n the  laterally  to  levels  of  vertical  the  h o r i z o n t a l limb  basal  of the  limb  of  regions  s u c h as  the  magnocellularis. midline  but  these decreased  substantia  Although the  s t r u c t u r e , the  i n j e c t i o n was  centered  core on  the  side i p s i l a t e r a l  mainly found i p s i l a t e r a l However, a few contralateral  to the  side.  The not  pattern  of  the  f o r e b r a i n n e u r o n s were  injection  site. within  the  limits  of  the  On  the  other  different partially  However, i n c o n t r a s t  sites.  i n the  hand, i n the  in  in  the  sites nucleus,  s e p t a l and two  the  animal  involved  to i n j e c t i o n  lateral  the  labeling in  interpeduncular  somata were o b s e r v e d i n t h e areas.  retrograde  significantly  v e n t r a l t e g m e n t a l a r e a was  preoptic  to  tracer injection  which the  labeled  nucleus  l a b e l e d n e u r o n s were a l s o f o u n d on  f o r e b r a i n was  confined  basal  caudal  nucleus i s a  interpeduncular  hippocampal i n j e c t i o n s , a c c o r d i n g l y  the  nucleus b a s a l i s  interpeduncular  of the  band  preoptic  i n number i n more  i n n o m i n a t a and  the  forebrain, diagonal  i n c l u d e the magnocellular  in  labeled  I n a d d i t i o n , many l a b e l e d somata were o b s e r v e d i n  ventral pallidum,  basal  basal  Occasionally,  i n t h e m e d i a l septum and  found  rostral  t h e s e n e u r o n s were o b s e r v e d  diagonal  A t more c a u d a l  nucleus,  a  few  medial  animals i n which  the i n j e c t i o n s were l i m i t e d to the  interpeduncular  nucleus,  l a b e l e d neurons i n the v e n t r a l tegmental area c o u l d be F i n a l l y , a l l FG  observed.  i n j e c t i o n s r e s u l t e d i n massive r e t r o g r a d e  l a b e l i n g i n the medial habenular n u c l e u s . Neurons p r o j e c t i n g to the hippocampus t h a t were  retrogradely  l a b e l e d were found b i l a t e r a l l y i n the medial septum and 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.  i n the  Very  l a b e l e d c e l l s were observed i n the c a u d a l p o r t i o n of the  few basal  forebrain. D o u b l e - l a b e l e d c e l l s were r a r e l y ( l e s s than 2%) the b a s a l f o r e b r a i n .  A comparison of the d i s t r i b u t i o n of  labeled c e l l s following interpeduncular  i n j e c t i o n s i n the hippocampus or  nucleus r e v e a l e d  of these s t r u c t u r e s  observed i n  generally  t h a t neurons i n n e r v a t i n g  formed separate  populations.  F i r s t , the h o r i z o n t a l limb of the d i a g o n a l band was  the  area t h a t p r o v i d e d s u b s t a n t i a l a f f e r e n t s to both the and  interpeduncular  neurons i n n e r v a t i n g  nucleus. the  Within t h i s region,  interpeduncular  each  only  hippocampus  labeled  nucleus tended to  l o c a t e d d o r s a l t o those l a b e l e d f o l l o w i n g  injections into  be 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 F i g u r e  which shows the  representative  d i s t r i b u t i o n of the  retrogradely  l a b e l e d neurons t h a t were observed through t h r e e c o r o n a l of the b a s a l  forebrain.  17  planes  Figure  17.  The r e p r e s e n t a t i v e  were r e t r o g r a d e l y interpeduncular planes o f the  distribution  labeled following  o f neurons  injections  i n the  n u c l e u s and hippocampus t h r o u g h t h r e e  basal  bregma) c o o r d i n a t e s  forebrain.  The a n t e r o - p o s t e r i o r  f o r each o f the  +0.2mm; -0.3mm a n d -0.8mm.  the interpeduncular  innervating  the  hippocampus.  coronal (from  p l a n e a r e as f o l l o w :  Symbols:. F G - l a b e l e d  innervating  that  nucleus;  neurons  . P i - l a b e l e d neurons  89  DISCUSSION  The major f i n d i n g of the present  experiment was  demonstration t h a t the i n t e r p e d u n c u l a r nucleus hippocampus are independently neurons.  Although negative  the  i n n e r v a t e d by b a s a l f o r e b r a i n  r e s u l t s do not n e c e s s a r i l y mean t h a t  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 , as hypothesized not e x i s t , the p o s s i b i l i t y of undetected unlikely.  and  the  i n t h i s study,  does  c o l l a t e r a l s i s however  F i r s t , 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 appears s u f f i c i e n t l y s e n s i t i v e f o r d e t e c t i n g 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 based on the r e s u l t s of Experiment one.  Secondly, both the i n t e r p e d u n c u l a r nucleus  and  hippocampus  were e x t e n s i v e l y f i l l e d by each of the r e s p e c t i v e t r a c e r s .  In  a d d i t i o n , dye d i f f u s i o n and t r a c e r 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.  I f anything,  these  f a c t o r s should have  enhanced the l i k e l i h o o d of d e t e c t i n g c o l l a t e r a l i z a t i o n . i t would appear reasonable  to conclude t h a t very few  Thus,  basal  f o r e b r a i n neurons have axons t h a t i n n e r v a t e both the hippocampus and  interpeduncular The  nucleus.  p a t t e r n of r e t r o g r a d e  experiment g e n e r a l l y confirmed investigations  l a b e l i n g obtained the r e s u l t s of  (Woolf and Butcher, 1985;  Unreported i n p r e v i o u s  s t u d i e s was  in this  previous  see i n t r o d u c t i o n ) .  the demonstration t h a t  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 b i l a t e r a l l y i n the b a s a l f o r e b r a i n .  are found  A more systematic  i s however r e q u i r e d b e f o r e any f i r m c o n c l u s i o n s can be The  analysis advanced.  r e s u l t s of t h i s study have f u r t h e r e s t a b l i s h e d t h a t b a s a l  f o r e b r a i n neurons i n n e r v a t i n g the hippocampus and interpeduncular  nucleus  are segregated  w i t h i n the r e g i o n s of  o r i g i n s thereby  c o r r o b o r a t i n g the c o n c l u s i o n t h a t these neurons  do not p r o v i d e c o l l a t e r a l s t o the i n t e r p e d u n c u l a r nucleus hippocampus.  and  92  GENERAL DISCUSSION  Anatomical C o n s i d e r a t i o n s  On it  the b a s i s of the r e s u l t s of the present s t u d i e s  i s d i f f i c u l t t o reach an o v e r a l l c o n c l u s i o n  degree t o which b a s a l collateralized.  as to  f o r e b r a i n p r o j e c t i o n systems  Hence, i t was  shown t h a t c h o l i n e r g i c  have axons t h a t branch to i n n e r v a t e  the RTN  not  hippocampus.  interpeduncular  nucleus and  o b t a i n e d i n experiment one existence  of b a s a l  the  are  p o s s i b l y GABAergic neurons o r i g i n a t i n g i n the b a s a l  the  alone,  and  and  forebrain  cortex, The  but  results  p r o v i d e a d d i t i o n a l evidence f o r  the  f o r e b r a i n neurons w i t h c o l l a t e r a l s (see  general i n t r o d u c t i o n ) .  Since both p o s i t i v e and  evidence f o r the e x i s t e n c e as reviewed e a r l i e r , and  negative  of such neurons have been  as o b t a i n e d i n t h i s study,  reported no  g e n e r a l i z a t i o n can be made on the preponderance of c o l l a t e r a l i z e d projections I t can however be  a r i s i n g from the b a s a l  suggested t h a t 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  d i s t i n g u i s h i n g subpopulations of b a s a l contrast,  forebrain.  for  f o r e b r a i n neurons.  By  the demonstration of a c o l l a t e r a l i z e d c h o l i n e r g i c  p r o j e c t i o n a r i s i n g i n the mesopontine tegmentum i n experimemt one  i s i n agreement w i t h the p r o p o s i t i o n  of Woolf and  Butcher  (1985) t h a t these neurons 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 . To t h a t e f f e c t , i t i s i n t e r e s t i n g t o note t h a t , although  not  93  systematically  analyzed, double-labeled  observed i n the had  received  c e l l s were  m e s o p o n t i n e tegmentum o f  injections in anterior  or  control  ventral  also  animals  that  thalamic  nuclei.  Functional  The by  Considerations  demonstration that  the  RTN  and  s i n g l e neurons o r i g i n a t i n g i n the  m e s o p o n t i n e tegmentum may implications.  A  large  basal  have s e v e r a l  body o f  cortex  are  forebrain  interesting  that  and  systems are  mesopontine tegmental c h o l i n e r g i c  s u c h as al.,  f o r e x t r a t h a l a m i c modulation of  arousal  1987;  and  sleep  Stewart et  projections  activity  i t was  n e u r o n s may t h r o u g h an 1987).  a l . , 1986)  and  also  exert  indirect  are  the  activity  (see  Baghdoyan  Ascending  cholinergic  et  known  cortical  indirect basal  RTN  influences Jones,  functions  thereby influence  influence  the  cortical  a l . , 1987;  on  r o u t e i . e v i a the  I t i s thought that  important  transmission  additional  an  forebrain  i n c l u d i n g RTN  suggested that  mediating c h o l i n e r g i c cortical  may  basal  and  m e s o p o n t i n e tegmentum a r e  activities  through t h i s  Recently,  et  a l . , 1984)).  o r i g i n a t i n g i n the  to modulate t h a l a m i c (Kayama e t  (Borst  the  and  functional  electrophysiological  p h a r m a c o l o g i c a l d a t a have e s t a b l i s h e d  substrates  innervated  forebrain cortical RTN  plays  on  1985).  thalamic  route.  cholinergic activity  (Hallanger a critical  et a l . , role  thalamic transmission Of  in and  particular interest  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  pharmacological  94  s t u d i e s w h i c h c o n v e r g e i n e s t a b l i s h i n g a r o l e f o r t h e RTN i n gating  thalamocortical  Jones,  1985).  input  are  to inhibition  o f the spontaneous  a p p l i c a t i o n of acetylcholine  1986).  Since  the GABAergic c e l l s  known t o i n n e r v a t e  inhibitory  (McCormick and o f t h e RTN, w h i c h  the d o r s a l thalamus, a r e b e l i e v e d  v i a t h e RTN l e a d s  r e l a y neurons i n t h e d o r s a l thalamus  transmission  center  (see Jones,  could  Such  of sensory  be f u n c t i o n a l l y r e l a t e d t o s t a t e s o f  Thus, i t would appear t h a t  t h e RTN i s an i m p o r t a n t  f o r i n t e g r a t i n g the information  conveyed by a s c e n d i n g  systems w i t h t h e i n f o r m a t i o n  d o r s a l thalamus. suggests that  The r e s u l t s o b t a i n e d  part  of the information  neocortex from b a s a l cholinergic  1985).  which would then r e s u l t i n m o d u l a t i o n o f c o r t i c a l  that  cholinergic  RTN.  t o be  to d i s i n h i b i t i o n of  a d i s i n h i b i t i o n would r e s u l t i n t h e f a c i l i t a t i o n  arousal.  with  ( s e e Houser e t a l . , 1980), i t has been p r o p o s e d  that c h o l i n e r g i c input  activity  (see  cholinergic  o f t h e GABA n e u r o n s i n t h e RTN, a s r e v e a l e d  iontophoretic Prince,  transmissions  I t has a l s o been e s t a b l i s h e d t h a t  t o t h e RTN l e a d s  activity  and c o r t i c o t h a l a m i c  relayed  i n this  study  transmitted  f o r e b r a i n and mesopontine  neurons i s a l s o simultaneously  through the further  to the  tegmentum  conveyed t o the  T h u s , i t c a n be h y p o t h e s i z e d t h e b a s a l  f o r e b r a i n and  m e s o p o n t i n e tegmentum c h o l i n e r g i c s y s t e m s c a n r e a d i l y influence,  t h r o u g h axon c o l l a t e r a l s ,  thalamocortical RTN.  transmission  Taken a step  collateralized  c o r t i c o t h a l a m i c and  i n t h e d o r s a l thalamus v i a t h e  f u r t h e r , i t c a n be h y p o t h e s i z e d t h a t  p r o j e c t i o n s , by e n a b l i n g  basal  such  f o r e b r a i n and  95  mesopontine tegmental c h o l i n e r g i c systems t o c o n c o m i t a n t l y i n f l u e n c e n e o c o r t i c a l and RTN a c t i v i t i e s , c o n s t i t u t e a powerful anatomical s u b s t r a t e  capable of e x e r t i n g a t i g h t  c o n t r o l over c o r t i c a l t r a n s m i s s i o n  through a s e r i e s of  t h a l a m i c feedback loops. The nature of the f u n c t i o n a l r o l e 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  i d e n t i f i e d remains t o be  established. The  f u n c t i o n a l i m p l i c a t i o n s of a RTN input from PV-  containing  neurons i n the b a s a l f o r e b r a i n remains t o be  established.  Aside from the p o s s i b i l i t y t h a t GABA may be the  neurotransmitter  i n v o l v e d i n these p r o j e c t i o n s and may  c o n s t i t u t e another important neurochemical pathway f o r the i n 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 c l u e s as t o the p h y s i o l o g i c a l p r o p e r t i e s of these b a s a l f o r e b r a i n neurons. Hence, s i n c e parvalbumin i s a c a l c i u m  binding  p r o t e i n , i t has  been suggested t h a t parvalbumin may be a marker of a s e l e c t i v e neuronal p o p u l a t i o n  with calcium  ( C e l i o and Heizmann, 1981).  dependent a c t i o n p o t e n t i a l s  I n t e r e s t i n g l y , i t has been  f u r t h e r suggested t h a t the i n f l u e n c e of PV on i n t r a c e l l u l a r concentrations containing  of c a l c i u m  ions i n neurons such as those  GABA may c o n t r i b u t e  i n enhancing m e t a b o l i c and  e l e c t r i c a l a c t i v i t i e s of these neurons ( C e l i o , 1986). C e r t a i n l y , f u t u r e s t u d i e s on t h i s s u b j e c t rewarding.  should prove  96  REFERENCES  Adams, C.E., C e p e d a , C, B o y l a n , M.K., F i s h e r , R.S., H u l l , C D . , B u c h w a l d , N.A., W a i n e r , B.H. and L e v i n e , M.S. (1986). 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