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Choline acetyltransferase-like immunoreactivity in the forebrain of the red-eared pond turtle (Pseudemys scripta elegans)
103
Brain Re'~eareh, 323 (1984) 1(13-10N Elsevicr BRE ~.04eS ~ ' '
Short Communications
Choline acetyltransferase-like immunoreactivity in the forebrain of the red-eared pond turtle ( Pseudemys scripta elegans) E. J. MUFSON l, P. H. DESAN 2, M. M. MESULAM l, B, H. WAINER3 and A, I. LEVEY ~ 1Bullard and Denny-Brown Laboratories and Behavioral Neurology Section oJthe Harvard Neurology Department and the Charles A. Dana Research Institute of the Beth Israel Hospital, 'The Department of Neurobiology, Harvard Medical SHlool, Boston, MA 02215 and eThe Departments of Pathology and Pediatrics, Joseph P. Kennedy Jr. Mental Retardation Research ( "enter. The University of Chicago, Chicago, IL 60637 (U.S.A.) (Accepted July 5th, 19841 Key words." cholinergic - - choline acetyltransferase - - turtle - - basal forcbrain - - immunohistochemistry
Choline acetyltransferase (CHAT) immunohistochcmistry was used to map the cholinergic neurons in the forebrain of t{seudemys turtles. Cell bodies with ChAT-likc immunoreactivity were seen in the septum, the nucleus of thc diagonal band, und embedded within the medial and lateral forcbruin bundles. The region of the medial and lateral forebrain bundles contained the greatest concentration of ChAT-positive neurons. Virtually no ChAT-like immunoreactivity was seen in the areas composing the reptilian homologue of the mammalian striatum. It is suggested that the turtle basal forebrain cholinergic neurons may represent the evolutionary precursors to the mammalian chotinergic neurons of the basal forebrain and even the striatum. The basal telencephalon of the turtle has been shown histochemically and biochemically to contain acetylcholinesterase (ACHE)
were perfused with physiological saline, then with a
tyltransferase (CHAT)" activity. It has been suggested that neurons of the turtle basal telecephalon
finally with 10% sucrose in 0.1 M phosphate buffer
may provide part of the cholinergic input observed in the turtle cortex '~. C h A T immunohistochemical methods provide the most specific means for determining the location of such neurons. This has not been done in the turtle. In contrast, numerous recent investigations have mapped the distribution of ChAT-positive basal forebrain neurons in mammalian species such as the rat 'j,t2,1
mixture of 4% paraformaldehyde (Fisher) -0.1';:{ glutaraldehyde (TABB, Maidenhead) for 3(1 min and (pH 7.4) according to schedule I1 of Rosene and Mesulam2a. Recently we found that in the rat brain fixation conditions influence the visualization of C h A T immunoreactive stainingI~,,27. Therefore, in a third turtle we changed the fixative to 2(~ paraformaldehyde ( A l d r i c h ) - 0 . t % glutaraldehyde (Kodak) and decreased the fixation time to 20 rain in order to obtain optimal staining:7, All 3 brains were cut at 4()/~m on a fieezing microtome into several adjacent sections. In each brain, one series was processed for C h A T immunohistochemistry using rat monoclonal antibody AB8 I~.te and the peroxidase-antiperoxidase procedure of Sternberger e~'. In the turtle with the 2% paraformald e h y d e - 0 . 1 % glutaraldehyde fixation, the double bridge procedure described by Houser r was used to furthm: increase sensitivity. Monoclonal antibody
Correspondence: E. ,1. Mufson. Neurology Unit, Bcth Israel Hospital. 330 Brookline Avenue, Boston. MA !12215, I J.5,.A. tI00~>8~)93:84$(13.01}@ 1984 Elsevier Science Publishers B.V.
104
A
ndb
Lk f
Fig. 1. Schematic drawings of coronal sections (A-FI illustrating the distribution of ChAT-like neurons ~black circlesl m the basal forebrain of Pseudemys. A is rostral and F is caudal. Abbreviations used in figures arc: ac. anterior commissure: c. area c of Riss ct a1.23; ctx, cortex; d, area d of Riss et al.23; DVR, dorsal ventricular ridge; hy, hypothalamus: OT. optic tract: otb. olfactory tubercle: PA, paleostriatum augmentatum (Powers and Reiner22i: ndb. nucleus of the diagonal band: S. septum: th. thalamus: v. ventricle AB8 binds active to C h A T in a specific double antibody assay, binds specifically to C h A T using the Western blotting system and localized known cholinergic p e r i k a r y a immunohistochemically ti-12,16. In a s e c o n d series, control material was o b t a i n e d by using a non-specific immunoglobulin G instead of AB8. A third series was counterstained with cresyl violet for aid in cytoarchitectonic evaluation. The distribution of C h A T - p o s i t i v e neurons was plotted with the aid of a c a m e r a lucida. A n analysis of the material revealed no essential differences in the distribution of C h A T - l i k e i m m u n o reactive neurons when the two different fixatives and immunohistochemical p r o c e d u r e s were c o m p a r e d
even though the 2% p a r a f o r m a l d e h y d e - 0 . 1 % gtutaraldehyde fixation and the double bridging resulted in a more intense C h A T staining pattern. Neurons containing C h A T - l i k e immunoreactivitv occupied a continuous zone of the basal telencephalon extending from the level of the olfactory tubercule to the level of the amygdala (Fig. 1l. The C h A T positive neuronal staining pattern yielded a brown and diffuse reaction product which filled the cell body. and variable portions of the dendritic ramification (Fig. 2B). We noted a positive immunohistochemical reaction in the presumptive cholinergic neurons m the m o t o r nuclei of the brainstem and spinal cord, although this was noi s t u d i e d in any detail.
105
A
#
L
Fig. 2. A: photomicrograph of cresyl violet stained section of the basal forebrain corresponding to section C of Fig. I. Area outlined with dotted lines indicates the cell sparse region containing the heaviest concentration of ChAT-likc neurons. This arca corresponds to the region of the medial and lateral forebrain bundles of Johnston ~. Bar = 500 t~m. B: photomicrograph of adjacent section to that seen in Fig, 2A which corresponds to Fig. 1C was stained for the immunohistochemical demonstration of choline acetyltransfcrase-like immunoreactivity. Note the large ChAT-positive neurons in the celt sparse zone of the medial and lateral forebrain bundles and in the rcgion of the diagonal band (double black arrows). Bar = 100,urn.
106 Control sections where a non-specific lgG was used instead of the antibody AB8 showed no neuronal staining. Within the basal forebrain, C h A T positive perikarya were found in the septum, the nucleus of diagonal band, and the medial and lateral forebrain bundles. In the septum, a few small oval shaped lightly stained C h A T positive neurons were observed mainly in the more medial sector (Fig. 1 B - C ) . In contrast, numerous ChAT-positive neurons were found in the ventral aspect of the basal telencephalon primarily in the relatively cell sparse region corresponding to the medial and lateral forebrain bundles of Johnston s (Figs. 1 B - F and 2A, B). The relatively large pyramidal and stellate shaped neurons of this region exhibit intense C h A T staining. These cells have few dendrites which travel for long distances in varied directions. Rostrally, a few small oval shaped ChAT-positive neurons were also encroaching upon the border of area c of Riss et al. 23. Another group of ChAT-positive neurons were located in the region termed the nucleus of the diagonal band bv both JohnstonS and Powers and Reiner e2. Yhese perikarya were small and either oval or fusiform in shape, and stained less intensely than cells in the region of the lateral forebrain bundle (Fig. 2A, B). These observations based on monospecific and monoclonal antibody to C h A T showed that cholinergic cell bodies in the basal telencephalon of the redeared pond turtle are confined to the septal region. the medial and lateral forebrain bundles, as well as a few scattered neurons extending to the border of area c of Riss et al.>. The findings of cholinergic neurons in the turtle basal telencephalon supports the recent biochemical data showing high levels of C h A T activity in this region 6. Although negative findings need to be interpreted with extreme caution, our material did not reliably demonstrate C h A T staining in the striatum (caudate or lentiform nuclei of JohnstonS; paleostriatum augmentatum of Powers and Reiner22), the large celled region termed 'globus pallidus' by Powers and Reiner 22, the hypothalamus or in the general cortex. The mammalian telencephalon contains cholinergic cell bodies in the striatum as well as in the basal forebrain m,l~',L~',~7,25. It is interesting that we could not obtain C h A T staining in cell bodies of the striatal homologue even though this area is known to stain heavily for ACHE. Perhaps AChE-rich axons from the basal forebrain choliner-
gic neurons innervate these striatal structures. Pretreatment with the reversible A C h E inhibitor diisopropylphosphorofluoridate (DFP) prior to death may help to establish whether the cell bodies of the turtle striatum exhibit A C h E or il the A C h E is primarily in axons and of extrinsic Ollgm Many of the basal forebrain cholinergic neurons in mammals are m intimate contact with major fiber fascicles le.g. internal capsule, anterior commissure} ~5,~r. Interestingly, the largest concentration of ChAT-positive neurons in the turtle are intermingled within the fiber fascicles of the medial and lateral forebrain bundles. It ts tempting to speculate that the separation of the fiber bundles into more discrete fascicles in the mammal may have influenced the segregation of the eholinergic neurons m higher species into more well delineated subgroup~ The mammalian basal forebram neurons have been subdivided into 4 major groups which we have termed C h l - C h 4 on the basis of their cytochemical and connectivity patterns ~s>. This extensive and continuous system of C h l - C h 4 cell bodies give rise to topographically organized cholinergic projections which innervate the entire mammalian neocortical mantle as well as several limbic and olfactory regions 15. It is conceivable that the ChAT-containing neurons of the turtle basal forebrain also provide a major source of cortical cholinergic innervation. In fact. horseradish peroxidase I H R P t injections into medial and lateral aspects of Pseudemys cortex results m a topographical distribution of labeled neurons within the septum, the nucleus of the diagonal band and the medial and [atera| forebram bundles (Desan. in preparation). Additional studies employing a combined retrograde H R P - C h A T procedure are needed to confirm whether these cholinergm neurons give rise to projections m the cortex and whether these projections are topographically arranged as in mammals. Such information w~mld lend support to the possibility that the cholinergic neurons of the turtle forebrain collectively constitute an undifferentiated analogue for the mammalian C h l - C h 4 neurons. and even perhaps the striatal cholinergic neurons. These neurons may become progressively more differentiated into separate groups in the c~mrse of phylogenetic evolution. Furthermore. the present findings suggest that in addition to receiving noradrenergic input from the locus coeruleus Iv and se-
107 r o t o n i n e r g i c input f r o m the r a p h e nuclei > , the turtle
the turtle basal f o r e b r a i n play a key' role in the ex-
cortex almost certainly r e c e i v e s a cholinergic input
pression of m a n y reptilian b e h a v i o r s . F u r t h e r m o r e ,
from C h A T - p o s i t i v e n e u r o n s in the basal t e l e n c e p h -
because of the c o m p a c t location of the cholinergic
alon.
n e u r o n s in the turtle basal f o r e b r a i n this animal may
R e c e n t investigations have indicated that o t h e r
p r o v i d e a m o d e l investigating the n e u r o b i o l o g i c a l al-
p e p t i d e s are also located in the turtle f o r e b r a i n . F o r
terations that these cholinergic cells u n d e r g o during
e x a m p l e , B e a r and Ebner-' have r e p o r t e d s o m e so-
d e v e l o p m e n t . Such i n f o r m a t i o n may p r o v i d e clues as
matostatin-like
neurons
in the region
designated
to the m e c h a n i s m s underlying s o m e of the changes
globus pallidus by P o w e r s and R e i n e r e-~. W e w e r e un-
seen in cholinergic n e u r o n s of h u m a n patients suffer-
able to identify C h A T - p o s i t i v e n e u r o n s in this re-
ing from A l z h e i m e r ' s Disease.
gion. The axons of the s o m a t o s t a t i n c o n t a i n i n g neurons w e r e o b s e r v e d to p r o j e c t into the region of lateral f o r e b r a i n
b u n d l e 2 suggesting that s o m a t o s t a t i n
We wish to thank Leah Christie, T e r r y Martin, Richard P l o u r d e and M a r c P e l o q u i n for secretarial
n e u r o n s could influence the activity of cholinergic
and technical assistance. This r e s e a r c h was s u p p o r t -
n e u r o n s located in this t e l e n c e p h a l i c region.
ed in part by grants f r o m the Essel F o u n d a t i o n , the
The central cholinergic n e u r o n s of the m a m m a l i a n
M c K n i g h t F o u n d a t i o n , N I H G r a n t s N S - 0 9 2 t l , NS-
brain have b e e n i m p l i c a t e d in a variety of b e h a v -
07011,
iors I,a.> and disease states 5.1s,2s. It is t e m p t i n g to
the Javits N e u r o s c i e n c e I n v e s t i g a t o r A w a r d NS20285 ( M . M . M . ) and the A D R D A Faculty Schohtr
speculate that the cholinergic n e u r o n s o b s e r v e d in
NS-17611,
HD-04583,
T32EY07042-05S1,
Award (E.J.M.). 1 Bartus, R. T., Dean, R. L., III, Beer, B. and Lippa, A. S., The cholinergic hypothesis of geriatric memory dysfunction, £cienee. 217 (1982) 408-417. 2 Bear, M. F. and Ebner, F. F., Somatostatin-like immunoreactivity in the forebrain of Pseudemys turtles, Neuroscie~lee. 9 (19831 297 307. 3 Desan, P. H., Mufson, E. J., Mesulam, M.-M., Wainer, B. H. and Levey, A. 1., Cholinergic neurons in the forebrain of the pond turtle (tTeudernys Scripta elegans) based on monoclonal choline acetyltransferase (CHAT) immunohistochcmistry: cholinergic Pathways III, Soe. Neurosci. Abstr.. 9 (1983) 966. 4 Finger, C.. Dean, D. L. III, Watkins, S. K. and Bartus, R. T., Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to thc cerebral cod rex in thc rat, Pharrnacol. Biochern. BehaP., 18 (1983) 973-981. 5 Hilt, D. C., Uhl. G. R.. Hedreen, J. C., Whitehouse, P. J. and Price, D. L.. Pick's Disease: loss of neurons in the nucleus basalis, Neurology, 32 (19821 A229. 6 Hohmann. C. F., Carroll, P. T. and Ebner, F. F., Acetylcholine levels and choline acetyltransferase activity in turtle cortex, Brain Researeh. 258 (1983) 120-122. 7 Houser, (7. R., Crawford, G. D., Barber, R. P., Salvaterra, P, M. and Vaughn, ,I. E,, Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyhransfcrasc, Brahz Research, 266 (19831 211-215, 8 Johnston, J. B., The cell masses in the forebrain in the turllc {('istudo carolina), ,I. cornp. Neurol., 25 (1915) 393-468. 9 Kimura. H., McGecr, P. L., Peng, J. H. and McGeer, E. G., Choline acetyltransferase-containing neurons in rodent brain demonstrated by immunohistochcmistry, .Science. 2{}8 (1980) 1057-1{)59. 1{} Kimura. H., meGecr, P. L , Peng, J. H. and McGeer, E.
G., The central cholincrgic system studied b\ choline acetyltransferase immunohistochemistrv in the cat, .1. comp. Neurol,. 2{10 {1981 ) 151-2{11. 11 Levey, A. I.. Armstrong, D. M., Atweh, S. F., Tcrry, R. D. and Wainer, B. H.. Monoclonal antibodies to choline acetyltransfcrase: production, specificity and immunohistochemistry, ,/. Ne,rosei.. 3 {19831 1-9. 12 Levey, A. 1., Mufson, E. J., Mesulam, M-M. and Waincr, B. H., Co-localization of acetylcholincstcrasc and choline acetyltransfcrase in thc rat ccrebrum, %'eurovcietlee, {) (1983) 9-22. 13 MacLean, P. D., Why brain rcscarch on lizards? In N. Greenberg and P. D. MacLean (Eds. h YehaPior at~d Ne~rology of Lizards', U.S. Department ot ttcalth and Human Services, Washington, 19811,pp. l - Ill. 14 Meininger, C.. Rye. D. B. and Waincr, B. H., An atlas ol cholinergic structures in the ferret brain demonstrated by immunocytochemical localization of cholinc acetvltransferase: Cholinergic Pathways VII, ,S'oc. :\:earo.r{i. Al~,str., 9 (19831 963. 15 Mesulam, M-M., Mufson, E. J., [,cvc},, A. 1. and Wainer. B. H., Cholincrgic innervation of cortex h_,, the basal forebrain: cytochcmistry and cortical connections of the septal area, diagonal band nuclei, nucleus hasalis (substantia innominata} and hypothalamus in the rhesus monkey, J. comp. Neurol., 214 (1983) 1711-197. 16 Mesulam, M-M,. Mutson, E. J., Waincr, /3. |l. and Levcy, A. 1.. Central cholinergic pathways in the rat: an overview based on an alternative nomenclature {('hl-('h~q, Neuroscie,ee, 10 {1983) 1185- 1201. 17 Mesulam, M-M., Mufson. E. J., Warner, /3. ff. and Lcvey, A. 1., Atlas of cholinergic neurons in the forehram and upper brainstem of the Macaquc based on monochmal cholinc acetyltransferasc immunohistochemistrx and acctylcholinesterase histochemistrv, :\('l~rosci{gl{{,, 12 (1984) 669-686.
108 18 Nakano, I. and Hirano, A., Neuron loss in the nucleus basalis of Meynert in Parkinsonism-Dementia complex of Guam, Ann. Neurol., 13 (1983) 87-91. 19 Ouimet, C. C. and Ebner, F. F., Extrathalamic inputs to the cerebral cortex of the turtle Pseudemys scripta, Anat. Rec., 199 (1981) 189A. 20 Ouimet, C. C., Patrick, R. L. and Ebner, F. F., An ultrastructural and biochemical analysis of norepinephrine-containing varicosities in the cerebral cortex of the turtle Pseudemys, J. comp. Neurol., 195 (1981) 289-304. 21 Parent, A. and Oliver, A., Comparative histochemical study of the corpus striatum, J. Hirnforsch., 12 (1972) 73-81. 22 Powers, A. S. and Reiner, A., A stereotaxic atlas of the forebrain and midbrain of the eastern painted turtle (Chrysemys picta picta), J. Hirnforsch., 12 (1980) 125-159. 23 Riss, W., Halpern, M. and Scalia, F., The quest for clues to forebrain evolution - - the study of reptiles, Brain Behav. Evol., 2 (1969) 1-50, 24 Rosene, D. L. and Mesulam, M-M., Fixation variables in horesradish peroxidase neurohistochemistry. I. The effects
25
26 27
28
29
of fixation time and perfusion procedures upon enzyme activity. J. Histochem. Cytochem.. 26 (1978] 28-39. Sofroniew. M. V.. Eckenstein F.. Thoenen. H. and Cuello. A. C.. Topography of choline acetyltransferase-containing neurons m the forebrain of the r~t ~/eurosci. I.ett. 33 f 19821 7-12 Sternberger, L. A.. lmmunocytochemtso T, John Witev and Sons. New York. 1979, pp. 1(14-169 Wainer. B. El.. Levey, A. 1.. Mufson. E J. and Mesulam. M-M.. Cholinerglc systems m mammalian brain identified with antibodies against choline acet~ltransferase. Neurochem. Int.. fi 11984] 297-307 Whitehouse. P. J.. Price. D. L.. Strublc, R G., Clark. A W . Coylc, J. ['. and DeLong, M. R.. Alzheimer's Disease and senile dementia: toss of neurons in the basal forebrain Science. 215 f 1982) 1237-1239 Yoshimura. H. and Ucki, S.. Biochemical correlates tn mouse-killing behavior of the rat: protonges isolation and brain cholinerglc function. Pharmacoi Biochem. Behav.. ~ ~lt)771 193-106
Brain Re'~eareh, 323 (1984) 1(13-10N Elsevicr BRE ~.04eS ~ ' '
Short Communications
Choline acetyltransferase-like immunoreactivity in the forebrain of the red-eared pond turtle ( Pseudemys scripta elegans) E. J. MUFSON l, P. H. DESAN 2, M. M. MESULAM l, B, H. WAINER3 and A, I. LEVEY ~ 1Bullard and Denny-Brown Laboratories and Behavioral Neurology Section oJthe Harvard Neurology Department and the Charles A. Dana Research Institute of the Beth Israel Hospital, 'The Department of Neurobiology, Harvard Medical SHlool, Boston, MA 02215 and eThe Departments of Pathology and Pediatrics, Joseph P. Kennedy Jr. Mental Retardation Research ( "enter. The University of Chicago, Chicago, IL 60637 (U.S.A.) (Accepted July 5th, 19841 Key words." cholinergic - - choline acetyltransferase - - turtle - - basal forcbrain - - immunohistochemistry
Choline acetyltransferase (CHAT) immunohistochcmistry was used to map the cholinergic neurons in the forebrain of t{seudemys turtles. Cell bodies with ChAT-likc immunoreactivity were seen in the septum, the nucleus of thc diagonal band, und embedded within the medial and lateral forcbruin bundles. The region of the medial and lateral forebrain bundles contained the greatest concentration of ChAT-positive neurons. Virtually no ChAT-like immunoreactivity was seen in the areas composing the reptilian homologue of the mammalian striatum. It is suggested that the turtle basal forebrain cholinergic neurons may represent the evolutionary precursors to the mammalian chotinergic neurons of the basal forebrain and even the striatum. The basal telencephalon of the turtle has been shown histochemically and biochemically to contain acetylcholinesterase (ACHE)
were perfused with physiological saline, then with a
tyltransferase (CHAT)" activity. It has been suggested that neurons of the turtle basal telecephalon
finally with 10% sucrose in 0.1 M phosphate buffer
may provide part of the cholinergic input observed in the turtle cortex '~. C h A T immunohistochemical methods provide the most specific means for determining the location of such neurons. This has not been done in the turtle. In contrast, numerous recent investigations have mapped the distribution of ChAT-positive basal forebrain neurons in mammalian species such as the rat 'j,t2,1
mixture of 4% paraformaldehyde (Fisher) -0.1';:{ glutaraldehyde (TABB, Maidenhead) for 3(1 min and (pH 7.4) according to schedule I1 of Rosene and Mesulam2a. Recently we found that in the rat brain fixation conditions influence the visualization of C h A T immunoreactive stainingI~,,27. Therefore, in a third turtle we changed the fixative to 2(~ paraformaldehyde ( A l d r i c h ) - 0 . t % glutaraldehyde (Kodak) and decreased the fixation time to 20 rain in order to obtain optimal staining:7, All 3 brains were cut at 4()/~m on a fieezing microtome into several adjacent sections. In each brain, one series was processed for C h A T immunohistochemistry using rat monoclonal antibody AB8 I~.te and the peroxidase-antiperoxidase procedure of Sternberger e~'. In the turtle with the 2% paraformald e h y d e - 0 . 1 % glutaraldehyde fixation, the double bridge procedure described by Houser r was used to furthm: increase sensitivity. Monoclonal antibody
Correspondence: E. ,1. Mufson. Neurology Unit, Bcth Israel Hospital. 330 Brookline Avenue, Boston. MA !12215, I J.5,.A. tI00~>8~)93:84$(13.01}@ 1984 Elsevier Science Publishers B.V.
104
A
ndb
Lk f
Fig. 1. Schematic drawings of coronal sections (A-FI illustrating the distribution of ChAT-like neurons ~black circlesl m the basal forebrain of Pseudemys. A is rostral and F is caudal. Abbreviations used in figures arc: ac. anterior commissure: c. area c of Riss ct a1.23; ctx, cortex; d, area d of Riss et al.23; DVR, dorsal ventricular ridge; hy, hypothalamus: OT. optic tract: otb. olfactory tubercle: PA, paleostriatum augmentatum (Powers and Reiner22i: ndb. nucleus of the diagonal band: S. septum: th. thalamus: v. ventricle AB8 binds active to C h A T in a specific double antibody assay, binds specifically to C h A T using the Western blotting system and localized known cholinergic p e r i k a r y a immunohistochemically ti-12,16. In a s e c o n d series, control material was o b t a i n e d by using a non-specific immunoglobulin G instead of AB8. A third series was counterstained with cresyl violet for aid in cytoarchitectonic evaluation. The distribution of C h A T - p o s i t i v e neurons was plotted with the aid of a c a m e r a lucida. A n analysis of the material revealed no essential differences in the distribution of C h A T - l i k e i m m u n o reactive neurons when the two different fixatives and immunohistochemical p r o c e d u r e s were c o m p a r e d
even though the 2% p a r a f o r m a l d e h y d e - 0 . 1 % gtutaraldehyde fixation and the double bridging resulted in a more intense C h A T staining pattern. Neurons containing C h A T - l i k e immunoreactivitv occupied a continuous zone of the basal telencephalon extending from the level of the olfactory tubercule to the level of the amygdala (Fig. 1l. The C h A T positive neuronal staining pattern yielded a brown and diffuse reaction product which filled the cell body. and variable portions of the dendritic ramification (Fig. 2B). We noted a positive immunohistochemical reaction in the presumptive cholinergic neurons m the m o t o r nuclei of the brainstem and spinal cord, although this was noi s t u d i e d in any detail.
105
A
#
L
Fig. 2. A: photomicrograph of cresyl violet stained section of the basal forebrain corresponding to section C of Fig. I. Area outlined with dotted lines indicates the cell sparse region containing the heaviest concentration of ChAT-likc neurons. This arca corresponds to the region of the medial and lateral forebrain bundles of Johnston ~. Bar = 500 t~m. B: photomicrograph of adjacent section to that seen in Fig, 2A which corresponds to Fig. 1C was stained for the immunohistochemical demonstration of choline acetyltransfcrase-like immunoreactivity. Note the large ChAT-positive neurons in the celt sparse zone of the medial and lateral forebrain bundles and in the rcgion of the diagonal band (double black arrows). Bar = 100,urn.
106 Control sections where a non-specific lgG was used instead of the antibody AB8 showed no neuronal staining. Within the basal forebrain, C h A T positive perikarya were found in the septum, the nucleus of diagonal band, and the medial and lateral forebrain bundles. In the septum, a few small oval shaped lightly stained C h A T positive neurons were observed mainly in the more medial sector (Fig. 1 B - C ) . In contrast, numerous ChAT-positive neurons were found in the ventral aspect of the basal telencephalon primarily in the relatively cell sparse region corresponding to the medial and lateral forebrain bundles of Johnston s (Figs. 1 B - F and 2A, B). The relatively large pyramidal and stellate shaped neurons of this region exhibit intense C h A T staining. These cells have few dendrites which travel for long distances in varied directions. Rostrally, a few small oval shaped ChAT-positive neurons were also encroaching upon the border of area c of Riss et al. 23. Another group of ChAT-positive neurons were located in the region termed the nucleus of the diagonal band bv both JohnstonS and Powers and Reiner e2. Yhese perikarya were small and either oval or fusiform in shape, and stained less intensely than cells in the region of the lateral forebrain bundle (Fig. 2A, B). These observations based on monospecific and monoclonal antibody to C h A T showed that cholinergic cell bodies in the basal telencephalon of the redeared pond turtle are confined to the septal region. the medial and lateral forebrain bundles, as well as a few scattered neurons extending to the border of area c of Riss et al.>. The findings of cholinergic neurons in the turtle basal telencephalon supports the recent biochemical data showing high levels of C h A T activity in this region 6. Although negative findings need to be interpreted with extreme caution, our material did not reliably demonstrate C h A T staining in the striatum (caudate or lentiform nuclei of JohnstonS; paleostriatum augmentatum of Powers and Reiner22), the large celled region termed 'globus pallidus' by Powers and Reiner 22, the hypothalamus or in the general cortex. The mammalian telencephalon contains cholinergic cell bodies in the striatum as well as in the basal forebrain m,l~',L~',~7,25. It is interesting that we could not obtain C h A T staining in cell bodies of the striatal homologue even though this area is known to stain heavily for ACHE. Perhaps AChE-rich axons from the basal forebrain choliner-
gic neurons innervate these striatal structures. Pretreatment with the reversible A C h E inhibitor diisopropylphosphorofluoridate (DFP) prior to death may help to establish whether the cell bodies of the turtle striatum exhibit A C h E or il the A C h E is primarily in axons and of extrinsic Ollgm Many of the basal forebrain cholinergic neurons in mammals are m intimate contact with major fiber fascicles le.g. internal capsule, anterior commissure} ~5,~r. Interestingly, the largest concentration of ChAT-positive neurons in the turtle are intermingled within the fiber fascicles of the medial and lateral forebrain bundles. It ts tempting to speculate that the separation of the fiber bundles into more discrete fascicles in the mammal may have influenced the segregation of the eholinergic neurons m higher species into more well delineated subgroup~ The mammalian basal forebram neurons have been subdivided into 4 major groups which we have termed C h l - C h 4 on the basis of their cytochemical and connectivity patterns ~s>. This extensive and continuous system of C h l - C h 4 cell bodies give rise to topographically organized cholinergic projections which innervate the entire mammalian neocortical mantle as well as several limbic and olfactory regions 15. It is conceivable that the ChAT-containing neurons of the turtle basal forebrain also provide a major source of cortical cholinergic innervation. In fact. horseradish peroxidase I H R P t injections into medial and lateral aspects of Pseudemys cortex results m a topographical distribution of labeled neurons within the septum, the nucleus of the diagonal band and the medial and [atera| forebram bundles (Desan. in preparation). Additional studies employing a combined retrograde H R P - C h A T procedure are needed to confirm whether these cholinergm neurons give rise to projections m the cortex and whether these projections are topographically arranged as in mammals. Such information w~mld lend support to the possibility that the cholinergic neurons of the turtle forebrain collectively constitute an undifferentiated analogue for the mammalian C h l - C h 4 neurons. and even perhaps the striatal cholinergic neurons. These neurons may become progressively more differentiated into separate groups in the c~mrse of phylogenetic evolution. Furthermore. the present findings suggest that in addition to receiving noradrenergic input from the locus coeruleus Iv and se-
107 r o t o n i n e r g i c input f r o m the r a p h e nuclei > , the turtle
the turtle basal f o r e b r a i n play a key' role in the ex-
cortex almost certainly r e c e i v e s a cholinergic input
pression of m a n y reptilian b e h a v i o r s . F u r t h e r m o r e ,
from C h A T - p o s i t i v e n e u r o n s in the basal t e l e n c e p h -
because of the c o m p a c t location of the cholinergic
alon.
n e u r o n s in the turtle basal f o r e b r a i n this animal may
R e c e n t investigations have indicated that o t h e r
p r o v i d e a m o d e l investigating the n e u r o b i o l o g i c a l al-
p e p t i d e s are also located in the turtle f o r e b r a i n . F o r
terations that these cholinergic cells u n d e r g o during
e x a m p l e , B e a r and Ebner-' have r e p o r t e d s o m e so-
d e v e l o p m e n t . Such i n f o r m a t i o n may p r o v i d e clues as
matostatin-like
neurons
in the region
designated
to the m e c h a n i s m s underlying s o m e of the changes
globus pallidus by P o w e r s and R e i n e r e-~. W e w e r e un-
seen in cholinergic n e u r o n s of h u m a n patients suffer-
able to identify C h A T - p o s i t i v e n e u r o n s in this re-
ing from A l z h e i m e r ' s Disease.
gion. The axons of the s o m a t o s t a t i n c o n t a i n i n g neurons w e r e o b s e r v e d to p r o j e c t into the region of lateral f o r e b r a i n
b u n d l e 2 suggesting that s o m a t o s t a t i n
We wish to thank Leah Christie, T e r r y Martin, Richard P l o u r d e and M a r c P e l o q u i n for secretarial
n e u r o n s could influence the activity of cholinergic
and technical assistance. This r e s e a r c h was s u p p o r t -
n e u r o n s located in this t e l e n c e p h a l i c region.
ed in part by grants f r o m the Essel F o u n d a t i o n , the
The central cholinergic n e u r o n s of the m a m m a l i a n
M c K n i g h t F o u n d a t i o n , N I H G r a n t s N S - 0 9 2 t l , NS-
brain have b e e n i m p l i c a t e d in a variety of b e h a v -
07011,
iors I,a.> and disease states 5.1s,2s. It is t e m p t i n g to
the Javits N e u r o s c i e n c e I n v e s t i g a t o r A w a r d NS20285 ( M . M . M . ) and the A D R D A Faculty Schohtr
speculate that the cholinergic n e u r o n s o b s e r v e d in
NS-17611,
HD-04583,
T32EY07042-05S1,
Award (E.J.M.). 1 Bartus, R. T., Dean, R. L., III, Beer, B. and Lippa, A. S., The cholinergic hypothesis of geriatric memory dysfunction, £cienee. 217 (1982) 408-417. 2 Bear, M. F. and Ebner, F. F., Somatostatin-like immunoreactivity in the forebrain of Pseudemys turtles, Neuroscie~lee. 9 (19831 297 307. 3 Desan, P. H., Mufson, E. J., Mesulam, M.-M., Wainer, B. H. and Levey, A. 1., Cholinergic neurons in the forebrain of the pond turtle (tTeudernys Scripta elegans) based on monoclonal choline acetyltransferase (CHAT) immunohistochcmistry: cholinergic Pathways III, Soe. Neurosci. Abstr.. 9 (1983) 966. 4 Finger, C.. Dean, D. L. III, Watkins, S. K. and Bartus, R. T., Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to thc cerebral cod rex in thc rat, Pharrnacol. Biochern. BehaP., 18 (1983) 973-981. 5 Hilt, D. C., Uhl. G. R.. Hedreen, J. C., Whitehouse, P. J. and Price, D. L.. Pick's Disease: loss of neurons in the nucleus basalis, Neurology, 32 (19821 A229. 6 Hohmann. C. F., Carroll, P. T. and Ebner, F. F., Acetylcholine levels and choline acetyltransferase activity in turtle cortex, Brain Researeh. 258 (1983) 120-122. 7 Houser, (7. R., Crawford, G. D., Barber, R. P., Salvaterra, P, M. and Vaughn, ,I. E,, Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyhransfcrasc, Brahz Research, 266 (19831 211-215, 8 Johnston, J. B., The cell masses in the forebrain in the turllc {('istudo carolina), ,I. cornp. Neurol., 25 (1915) 393-468. 9 Kimura. H., McGecr, P. L., Peng, J. H. and McGeer, E. G., Choline acetyltransferase-containing neurons in rodent brain demonstrated by immunohistochcmistry, .Science. 2{}8 (1980) 1057-1{)59. 1{} Kimura. H., meGecr, P. L , Peng, J. H. and McGeer, E.
G., The central cholincrgic system studied b\ choline acetyltransferase immunohistochemistrv in the cat, .1. comp. Neurol,. 2{10 {1981 ) 151-2{11. 11 Levey, A. I.. Armstrong, D. M., Atweh, S. F., Tcrry, R. D. and Wainer, B. H.. Monoclonal antibodies to choline acetyltransfcrase: production, specificity and immunohistochemistry, ,/. Ne,rosei.. 3 {19831 1-9. 12 Levey, A. 1., Mufson, E. J., Mesulam, M-M. and Waincr, B. H., Co-localization of acetylcholincstcrasc and choline acetyltransfcrase in thc rat ccrebrum, %'eurovcietlee, {) (1983) 9-22. 13 MacLean, P. D., Why brain rcscarch on lizards? In N. Greenberg and P. D. MacLean (Eds. h YehaPior at~d Ne~rology of Lizards', U.S. Department ot ttcalth and Human Services, Washington, 19811,pp. l - Ill. 14 Meininger, C.. Rye. D. B. and Waincr, B. H., An atlas ol cholinergic structures in the ferret brain demonstrated by immunocytochemical localization of cholinc acetvltransferase: Cholinergic Pathways VII, ,S'oc. :\:earo.r{i. Al~,str., 9 (19831 963. 15 Mesulam, M-M., Mufson, E. J., [,cvc},, A. 1. and Wainer. B. H., Cholincrgic innervation of cortex h_,, the basal forebrain: cytochcmistry and cortical connections of the septal area, diagonal band nuclei, nucleus hasalis (substantia innominata} and hypothalamus in the rhesus monkey, J. comp. Neurol., 214 (1983) 1711-197. 16 Mesulam, M-M,. Mutson, E. J., Waincr, /3. |l. and Levcy, A. 1.. Central cholinergic pathways in the rat: an overview based on an alternative nomenclature {('hl-('h~q, Neuroscie,ee, 10 {1983) 1185- 1201. 17 Mesulam, M-M., Mufson. E. J., Warner, /3. ff. and Lcvey, A. 1., Atlas of cholinergic neurons in the forehram and upper brainstem of the Macaquc based on monochmal cholinc acetyltransferasc immunohistochemistrx and acctylcholinesterase histochemistrv, :\('l~rosci{gl{{,, 12 (1984) 669-686.
108 18 Nakano, I. and Hirano, A., Neuron loss in the nucleus basalis of Meynert in Parkinsonism-Dementia complex of Guam, Ann. Neurol., 13 (1983) 87-91. 19 Ouimet, C. C. and Ebner, F. F., Extrathalamic inputs to the cerebral cortex of the turtle Pseudemys scripta, Anat. Rec., 199 (1981) 189A. 20 Ouimet, C. C., Patrick, R. L. and Ebner, F. F., An ultrastructural and biochemical analysis of norepinephrine-containing varicosities in the cerebral cortex of the turtle Pseudemys, J. comp. Neurol., 195 (1981) 289-304. 21 Parent, A. and Oliver, A., Comparative histochemical study of the corpus striatum, J. Hirnforsch., 12 (1972) 73-81. 22 Powers, A. S. and Reiner, A., A stereotaxic atlas of the forebrain and midbrain of the eastern painted turtle (Chrysemys picta picta), J. Hirnforsch., 12 (1980) 125-159. 23 Riss, W., Halpern, M. and Scalia, F., The quest for clues to forebrain evolution - - the study of reptiles, Brain Behav. Evol., 2 (1969) 1-50, 24 Rosene, D. L. and Mesulam, M-M., Fixation variables in horesradish peroxidase neurohistochemistry. I. The effects
25
26 27
28
29
of fixation time and perfusion procedures upon enzyme activity. J. Histochem. Cytochem.. 26 (1978] 28-39. Sofroniew. M. V.. Eckenstein F.. Thoenen. H. and Cuello. A. C.. Topography of choline acetyltransferase-containing neurons m the forebrain of the r~t ~/eurosci. I.ett. 33 f 19821 7-12 Sternberger, L. A.. lmmunocytochemtso T, John Witev and Sons. New York. 1979, pp. 1(14-169 Wainer. B. El.. Levey, A. 1.. Mufson. E J. and Mesulam. M-M.. Cholinerglc systems m mammalian brain identified with antibodies against choline acet~ltransferase. Neurochem. Int.. fi 11984] 297-307 Whitehouse. P. J.. Price. D. L.. Strublc, R G., Clark. A W . Coylc, J. ['. and DeLong, M. R.. Alzheimer's Disease and senile dementia: toss of neurons in the basal forebrain Science. 215 f 1982) 1237-1239 Yoshimura. H. and Ucki, S.. Biochemical correlates tn mouse-killing behavior of the rat: protonges isolation and brain cholinerglc function. Pharmacoi Biochem. Behav.. ~ ~lt)771 193-106