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Loss of GABAergic neurons in medial septum after fimbria-fornix transection
Neuroscience Leller,~, 76 !1987) 140 144 Elsevier Scientific Publishers Ireland kid
I40
NSL 04548
Loss of GABAergic neurons in medial septum after fimbria-fornix transection G a r y M. Peterson 3, Lawrence R. Williams 1, Silvio V a r o n 2 and Fred H. G a g e 1 Departments of JNeurosciences and 2Biology, University of California, San Diego, School of Medicine, La Jolla. CA 92093 (U.S.A.) and ~Department o[ Anatomy, East Carolina University, School Of Medicine. Greenville, NC 27834 (U.S.A.) (Received 1 October 1986; Revised version received 22 December 1986: Accepted 23 December 1986)
Key words: Cell death; 7-Aminobutyric acid (GABA); Medial septum; Fimbria-fornix; Hippocampus: Substance P: Rat Neurons in the medial septum of the rat brain undergo retrograde degeneration after transection of their projection to the hippocampal formation, the fimbria-fornix. This cell death has been characterized for both Nissl-stained neurons and acetylcholinesterase-stained neurons. The major cell type in the medial septum is GABAergic, and many of these GABAergic neurons project to the hippocampal formation. Because the fimbria-fornix transection causes more neuronal death than can be accounted for by the loss of cholinergic neurons, we have sought to determine if" the GABAergic neurons undergo a cell death similar to thai reported for the cholinergic neurons. We report here that GABAergic neurons are indeed losl after the transection but the time course is considerably slower than that for the cholinergic neurons.
Neuronal death following axotomy is a common phenomenon in both the peripheral and central nervous system. At present it is not known why some neurons die and are digested following damage, while others survive. One possibility is that death or survival is dependent on the distance of the axotomy from the cell body and the remaining collateral branches, since the closer the damage to the cell body and the fewer sustained collaterals, the greater the probability of cell death. A related possibility is that some neurons are dependent for survival on the retrograde transport of neuronotrophic factors from target areas to the cell bodies [5, 15]. Blockade or damage to this transport system by axotomy could result in cell death. Damage to part of the transport system would be proportionally less deleterious. Within the CNS the septo-hippocampal system has been studied as a model system of retrograde degeneration after axotomy. In this system neurons within the medial septum (MS) and vertical limb of the diagonal band of Broca (VDB) die following transection of the fimbria-fornix (FF) and the supracallosal stria. About 45% of the Correspondence: F.H, Gage, Department of Neurosciences (M-024), U C S D School of Medicine, La Jolla, CA 92093, U.S,A.
141
Nissl-stained neurons die within the MS and VDB by 2 weeks following the transection [3, 4, 9]. To date the cholinergic neurons are the only chemically identified neuronal population that undergoes degeneration following FF transection [4]. However, the cholinergic neurons account for less than 50% of the neurons that project through the FF to the hippocampal formation (HF) [1, 14]. Cholinergic neurons also account for less than 50% of the cells that die following the FF transection [4]. GABAergic neurons tire the only other identified neurons that have been shown to project to the HF [6], although other neurons in the MS-VDB could also be potential contributors. Neurons containing substance P (SP) [9], galanin [10], and somatostatin and neurotensin [7] till exist in the MS-VDB region, but only galanin has been shown to be co-localized to any extent with a hippocampal-projecting neuronal system which is cholinergic. A septo-hippocampal projection by SP neurons has been suggested based on decreased levels of SP in hippocampus after lesions of the MS [13]. The objectives of this study were to determine (1) if the GABAergic system m the MS-VDB region also undergoes retrograde degeneration following FF transection, and (2) what similarities and differences may occur in response to injury by GABAergic and cholinergic systems following axotomy. Seventeen female Sprague Dawley rats weighing 225 250 g were used in the study. Eleven animals received a unilateral aspirative transection of the supracallosal stria, the FF, and the overlying cortex at a point near the rostral pole of the HF on the right side [4]. Animals where sacrificed 1 week ( n = 5 ) or 6 weeks ( n = 6 ) after the lesion. To enhance the localization of glutamic acid decarboxylase (GAD) all animals received bilateral stercotaxic injections of colchicine (10 #g//tl in 10 Ill saline) into the lateral ventricles. Six age-matched animals received only colchicine injections and served as unlesioned controls. One day after the colchicine the animals were killed by transcardiac perfusion with 50 ml of physiologic saline tk~llowed by 500 ml of icecold 4% paratk~rmaldehyde in sodium phosphate buffer (0.1 M, pH 7.4). Brains were postfixed overnight at 4'C in the same fixative with 25% sucrose added as a cryoprotectant. The following day the brains were transferred to 25% sucrose in phosphate buffer and stored at 4 C until they sank. Frozen sections were cut at 30 ira1 in the coronal plane through the rostrocaudal extent of the septum. A one-in-three series was collected for Nissl staining (Cresyl violet), a second set stained tk~r SP, and the third set of adjacent sections was prepared for G A D immunocytochemistry. The irnmunocytochemical processing basically followed the method described by Oertel et al. [1 I]. Briefly, free-floating sections were incubated in a solution of Tris-buffcred saline (TBS, 0.05 M, pH 7.6) with 10% normal rabbit serum and 0.1 M D,i.-lysine for 30 min and then transferred to the G A D antiserum (kindly provided b\. D.E. Schmechel) diluted 1:2000 in TBS plus 1% normal rabbit serum, or to the SP antiserum. Sections were incubated in the primary antiserum for 3 days at 4 ('. With several rinses in TBS separating each subsequent step, the sections were incubated in biotinylated rabbit anti-sheep serum (1:200) and avidin-biotin peroxidase complex (ABC, Vector Labs., Burlingame, CA) at a dilution of I:100. Sections were stained by incubation in a solution of diaminobenzidine tetrahydrochloride (0.05%), nickel chloride (0.04%) and hydrogen peroxide (0.02%). Sections were then rinsed in TBS,
142
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Fig. 1. Photomicrogruph of GAD-immunoreactivc cells in the MS. A: intact non-lesioned animal. B: I week following a unil;.ttcra] 1717transecticm. (': 6 weeks following a unilateral FF transection. Bar - I00 /ma. Arrows indicate midline. m o u n t e d o n t o g e l a t i n - s u b b e d slides, dried, c l e a r e d , a n d c o v e r e d . All G A D - i m m u n o r e a c t i v e
n e u r o n s w e r e c o u n t e d in the M S at o n e level ipsilateral
and c o n t r a l a t e r a l to the t r a n s e c t i o n using an e y e p i e c e reticule at x 200 m a g n i f i c a t i o n . T h e selected s e c t i o n was identified as the m o s t r o s t r a l s e c t i o n in w h i c h the M S was clearly s e p a r a t e f r o m the V D B . T h i s was a p p r o x i m a t e l y 1 8 0 / , m rostral to the crossing o f the a n t e r i o r c o m m i s s u r e . A t this level, the M S has a r a t h e r o v o i d s h a p e a n d contains
many
GAD-immunoreactive
neurons
as well as c h o l i n e r g i c
neurons.
C o u n t e d cells w e r e d e f i n e d as b e i n g g r e a t e r t h a n 12/2m in d i a m e t e r w i t h a c l e a r nucleus. D u e to v e r y l o w b a c k g r o u n d
s t a i n i n g in the i m m u n o c y t o c h e m i c a l
sections,
b o t h d a r k l y a n d lightly s t a i n e d n e u r o n s w e r e c o u n t e d . T w o a n i m a l s in the o n e w e e k g r o u p h a d o v e r a l l s u b o p t i m a l s t a i n i n g for G A D , w h i c h was a t t r i b u t e d to m i s p l a c e d
300
1' ""
~E
Z Z
j
1so
I ,oo
t
O~
CONTROL
TIME
1 WEEK
6 WEEK
POST-LESION
Fig. 2. Histogram of the total number of GAD-immunoreactive cells at the same level (as shown in Fig. 1) of the left and right MS. The 3 groups of animals are (I) normal controls, (2) I week following FF transection, (3) 6 weeks following FF transection. White bars correspond to the left septum, which was on the side contralateral to the lesion, and solid black bars correspond to the right septum, which was ipsilateral to the lesion,
143
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MS
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Fig. 3. Photomicrograph of SP-immunoreaclive cells in the MS 6 weeks following an FF transection. This section was adjacent to the section pholographed for GAD-immunoreactivity in Fig. 1. A: inlacl. B: F|: lesion. Bar 200/tm. Arrows show midline. colchicine injection, and thus these two animals were not used for the quantitative G A D antllysis. At the level o f the MS examined in this study (see Fig. 1A), there were on the average 410 Nissl-stained cells, 54 A C h E positively stained cells, 238 G A D - i m n l u n o r e a c rive cells, and 13 SP-immunoreactive cells. Thus at this level the G A D neurons accounted t'or 58% o f the neurons, and the cholinergic neurons for only' 137, o f the total neurons, whereas the SP cells only accounted for 3% o f the total neural population. The remaining 26% of the cells in the MS was not accounted for in the present study. N o difference in G A D cells was found between the left and right side of the septum in the unoperated animals. In addition, the n u m b e r o f cells in the septum contralateral to a lesion (left side) did not significantly differ from control septum tit 1 or 6 weeks following FF lesion. However, tt clear statistical difl'crence was observed between contralateral septum and ipsilateral septum of FF-lesioned animals tit either 1 week ( P < 0 . 0 2 5 ) or 6 weeks ( P < 0 . 0 0 5 ) (see Figs. I and 2). At I week the G A D cell n u m b e r was reduced by 19% on the lesion side, whereas tit (~ weeks the G A D cell loss a m o u n t e d to 58%, o f the contralateral population. Because o f the small n u m b e r of SP cells counted and the variability of staining. the changes in SP cells were only qualitatively evaluated. Fig. 3B shows the effect of an F F lesion on SP cells in the septum 6 weeks following the lesion. In all 5 animals examined there appeared to be a near complete loss o f SP cells on the side ipsilateral to the lesion relative to the contralateral septum. G A D - i m m u n o r e a c t i v e cells constitute one of the principal cell types in the medial septal area and diagonal band [2, 12]. Some, yet unknown, p r o p o r t i o n of these cells projects to the HF. However, it has been estimated that about 30% o f all projection neurons from the M S - V D B are G A D - p o s i t i v e [6]. FF transection restllts in a loss in n u m b e r of G A D neurons c o m p a r a b l e in proportion to the loss of cholinergic neurons 6 weeks post-transection (60%) [4]. However, the loss o f G A D cells is considerably lower tit 1 week post-transection than previously observed for cholinergic neurons, i.e. 19 and 60%, respectively. This difference in time course suggests a potentially different loss mechanism or reactivity to transection for the G A D cells than for the cholinergic cells•
144
The loss of GAD-positive neurons following FF lesions could be a result of (1~ traumatic cell death due to the elimination of all collaterals at a location too close to the soma, (2) loss of retrograde transport of some neuronotrophic factor, a n d o r (3) transneuronal death as a function of the dependence of G A D cells on the integrity of other septal neurons. At present we do not know which is accurate. We conclude that FF transection causes death of GABAergic neurons in a similar proportion to that of cholinergic neurons in the MS. However, the time of maximal cell loss is different for each of the two populations, with the GABAergic neurons taking longer to disappear than the cholinergic neurons. Future studies will determine similarities and differences between the response to axotomy of these two specified neuron types in the MS. We thank Jan Berglund and Lee Vahlsing for excellent technical assistance, and Sheryl Christenson lbr typing the manuscript, and drawing the figures. This research was supported by the Office of Naval Research, and the Margaret and Herbert Hoover Jr. Foundation (to F.H.G,) and NSF BNS-85-01677 (to S.V.). I Amaral, D.C;. and Kurz, J., An analysis of the origins of the cholinergic and noncholinergic scptal projections to the hippocampal formation of the rat, J. Comp. Neurol., 240 (1985) 37 59. 2 Brashear, HR., Zaborszky, L. and l-:leimer, L., Distribution of GABAergic and cholinergic neurons in the rat diagonal band, Neuroscicnce, 17 (1986) 439 451. 3 Daitz, H.M. and Powell, T.P.S., Studies of the connexions of the fornix system, J. Neurol. Neurosurg. Psychiatr.. 17 (1954) 75 82. 4 (}age, F.H., Wictorm, K., Fisher, W,, Williams, L.R., Varon, S. and Bjorklund, A., Retrograde cell changes in medial septum and diagonal band following timbria-fornix transection: quantitative temporal analysis, Neurosciencc, I (1986) 241 255. 5 Hefti, F., Nerve growth factor (NGF) promotes survival of septal cholinergic neurons after injury, J. Neurosci., 6 (1986) 2155 2162. 6 Kohler, C., Chan-Palay, V. and Wu, J.W., Seplal neurons containing glutamic acid decarboxylase immunoreactivity project to the hippocampal region in the rat brain, Anat. Embryol., 169 (1984) 41 44. 7 Kohler. C. and Eriksson, L.G., An immunohistochemical study ofsomatostatin and neurotensin posilive neurons in the septal nuclei of the rat brain, Anat. Embryol., 170 (1984) 1 10. 8 Ljungdahl, A., H6kfelt. T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861 943. 9 McLardy, T., Observations of the fornix of the monkey, J. Comp. Neurol., 103 (1955) 305. 10 Mclander, T., Staines, W.A,. H6kfelt, T., Rokaeus, A., Eckenstein, F., Salvaterra, P.M. and Wainer, B.H., Galanin-like immunoreactivity m cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat. Brain Res.. 360 (1985) 130 138. 11 Oertel, W.H.. Mugnaini, E., Schmechel, D.E., Tappaz, M.L. and Kopin, l.J., The immunocytochemical demonstration ofgamma-aminobutyric acidergic neurons methods and application. In V. ChanPalay and S.L. Paley (Eds.), Cytochemical Methods in Neuroanatomy, Alan R. Liss, New York, 1982, pp. 297 329. 12 Panula, P., Revuelta, A.V., Cheney, D.L., Wu, J.Y. and Costa, E., An immunohistochemical study on the location of GABAergic neurons in rat septum. J. Comp. Neurol., 222 (1984) 69 80. 13 Vincent, S.R. and McGeer, E.G., A substance P projection to the hippocampus, Brain Res.. 215 ( 1981 ) 349 351. 14 Wainer, B.H., Levey, A.I., Rye, D.B., Mesulam, M.M. and Mufsom E.J., Cholinergic and non-cholinergic septohippocampal pathways, Neurosci. Lett.. 54 (1985) 45 52. 15 Williams, LR., Varon. S.. Pelcrson, G.M., Wictorin, K., Fischer, W., Bjorklund, A. and Gage, F.H., Continuous infusion of nerve growth litctor prevents basal forebrain neuronal death after fimbriafornixtransection, Proc. Natl. Acad. Sci. llSA, 83(1986) 9231 9235.
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NSL 04548
Loss of GABAergic neurons in medial septum after fimbria-fornix transection G a r y M. Peterson 3, Lawrence R. Williams 1, Silvio V a r o n 2 and Fred H. G a g e 1 Departments of JNeurosciences and 2Biology, University of California, San Diego, School of Medicine, La Jolla. CA 92093 (U.S.A.) and ~Department o[ Anatomy, East Carolina University, School Of Medicine. Greenville, NC 27834 (U.S.A.) (Received 1 October 1986; Revised version received 22 December 1986: Accepted 23 December 1986)
Key words: Cell death; 7-Aminobutyric acid (GABA); Medial septum; Fimbria-fornix; Hippocampus: Substance P: Rat Neurons in the medial septum of the rat brain undergo retrograde degeneration after transection of their projection to the hippocampal formation, the fimbria-fornix. This cell death has been characterized for both Nissl-stained neurons and acetylcholinesterase-stained neurons. The major cell type in the medial septum is GABAergic, and many of these GABAergic neurons project to the hippocampal formation. Because the fimbria-fornix transection causes more neuronal death than can be accounted for by the loss of cholinergic neurons, we have sought to determine if" the GABAergic neurons undergo a cell death similar to thai reported for the cholinergic neurons. We report here that GABAergic neurons are indeed losl after the transection but the time course is considerably slower than that for the cholinergic neurons.
Neuronal death following axotomy is a common phenomenon in both the peripheral and central nervous system. At present it is not known why some neurons die and are digested following damage, while others survive. One possibility is that death or survival is dependent on the distance of the axotomy from the cell body and the remaining collateral branches, since the closer the damage to the cell body and the fewer sustained collaterals, the greater the probability of cell death. A related possibility is that some neurons are dependent for survival on the retrograde transport of neuronotrophic factors from target areas to the cell bodies [5, 15]. Blockade or damage to this transport system by axotomy could result in cell death. Damage to part of the transport system would be proportionally less deleterious. Within the CNS the septo-hippocampal system has been studied as a model system of retrograde degeneration after axotomy. In this system neurons within the medial septum (MS) and vertical limb of the diagonal band of Broca (VDB) die following transection of the fimbria-fornix (FF) and the supracallosal stria. About 45% of the Correspondence: F.H, Gage, Department of Neurosciences (M-024), U C S D School of Medicine, La Jolla, CA 92093, U.S,A.
141
Nissl-stained neurons die within the MS and VDB by 2 weeks following the transection [3, 4, 9]. To date the cholinergic neurons are the only chemically identified neuronal population that undergoes degeneration following FF transection [4]. However, the cholinergic neurons account for less than 50% of the neurons that project through the FF to the hippocampal formation (HF) [1, 14]. Cholinergic neurons also account for less than 50% of the cells that die following the FF transection [4]. GABAergic neurons tire the only other identified neurons that have been shown to project to the HF [6], although other neurons in the MS-VDB could also be potential contributors. Neurons containing substance P (SP) [9], galanin [10], and somatostatin and neurotensin [7] till exist in the MS-VDB region, but only galanin has been shown to be co-localized to any extent with a hippocampal-projecting neuronal system which is cholinergic. A septo-hippocampal projection by SP neurons has been suggested based on decreased levels of SP in hippocampus after lesions of the MS [13]. The objectives of this study were to determine (1) if the GABAergic system m the MS-VDB region also undergoes retrograde degeneration following FF transection, and (2) what similarities and differences may occur in response to injury by GABAergic and cholinergic systems following axotomy. Seventeen female Sprague Dawley rats weighing 225 250 g were used in the study. Eleven animals received a unilateral aspirative transection of the supracallosal stria, the FF, and the overlying cortex at a point near the rostral pole of the HF on the right side [4]. Animals where sacrificed 1 week ( n = 5 ) or 6 weeks ( n = 6 ) after the lesion. To enhance the localization of glutamic acid decarboxylase (GAD) all animals received bilateral stercotaxic injections of colchicine (10 #g//tl in 10 Ill saline) into the lateral ventricles. Six age-matched animals received only colchicine injections and served as unlesioned controls. One day after the colchicine the animals were killed by transcardiac perfusion with 50 ml of physiologic saline tk~llowed by 500 ml of icecold 4% paratk~rmaldehyde in sodium phosphate buffer (0.1 M, pH 7.4). Brains were postfixed overnight at 4'C in the same fixative with 25% sucrose added as a cryoprotectant. The following day the brains were transferred to 25% sucrose in phosphate buffer and stored at 4 C until they sank. Frozen sections were cut at 30 ira1 in the coronal plane through the rostrocaudal extent of the septum. A one-in-three series was collected for Nissl staining (Cresyl violet), a second set stained tk~r SP, and the third set of adjacent sections was prepared for G A D immunocytochemistry. The irnmunocytochemical processing basically followed the method described by Oertel et al. [1 I]. Briefly, free-floating sections were incubated in a solution of Tris-buffcred saline (TBS, 0.05 M, pH 7.6) with 10% normal rabbit serum and 0.1 M D,i.-lysine for 30 min and then transferred to the G A D antiserum (kindly provided b\. D.E. Schmechel) diluted 1:2000 in TBS plus 1% normal rabbit serum, or to the SP antiserum. Sections were incubated in the primary antiserum for 3 days at 4 ('. With several rinses in TBS separating each subsequent step, the sections were incubated in biotinylated rabbit anti-sheep serum (1:200) and avidin-biotin peroxidase complex (ABC, Vector Labs., Burlingame, CA) at a dilution of I:100. Sections were stained by incubation in a solution of diaminobenzidine tetrahydrochloride (0.05%), nickel chloride (0.04%) and hydrogen peroxide (0.02%). Sections were then rinsed in TBS,
142
e
D
!,
.k,
~c r !
Fig. 1. Photomicrogruph of GAD-immunoreactivc cells in the MS. A: intact non-lesioned animal. B: I week following a unil;.ttcra] 1717transecticm. (': 6 weeks following a unilateral FF transection. Bar - I00 /ma. Arrows indicate midline. m o u n t e d o n t o g e l a t i n - s u b b e d slides, dried, c l e a r e d , a n d c o v e r e d . All G A D - i m m u n o r e a c t i v e
n e u r o n s w e r e c o u n t e d in the M S at o n e level ipsilateral
and c o n t r a l a t e r a l to the t r a n s e c t i o n using an e y e p i e c e reticule at x 200 m a g n i f i c a t i o n . T h e selected s e c t i o n was identified as the m o s t r o s t r a l s e c t i o n in w h i c h the M S was clearly s e p a r a t e f r o m the V D B . T h i s was a p p r o x i m a t e l y 1 8 0 / , m rostral to the crossing o f the a n t e r i o r c o m m i s s u r e . A t this level, the M S has a r a t h e r o v o i d s h a p e a n d contains
many
GAD-immunoreactive
neurons
as well as c h o l i n e r g i c
neurons.
C o u n t e d cells w e r e d e f i n e d as b e i n g g r e a t e r t h a n 12/2m in d i a m e t e r w i t h a c l e a r nucleus. D u e to v e r y l o w b a c k g r o u n d
s t a i n i n g in the i m m u n o c y t o c h e m i c a l
sections,
b o t h d a r k l y a n d lightly s t a i n e d n e u r o n s w e r e c o u n t e d . T w o a n i m a l s in the o n e w e e k g r o u p h a d o v e r a l l s u b o p t i m a l s t a i n i n g for G A D , w h i c h was a t t r i b u t e d to m i s p l a c e d
300
1' ""
~E
Z Z
j
1so
I ,oo
t
O~
CONTROL
TIME
1 WEEK
6 WEEK
POST-LESION
Fig. 2. Histogram of the total number of GAD-immunoreactive cells at the same level (as shown in Fig. 1) of the left and right MS. The 3 groups of animals are (I) normal controls, (2) I week following FF transection, (3) 6 weeks following FF transection. White bars correspond to the left septum, which was on the side contralateral to the lesion, and solid black bars correspond to the right septum, which was ipsilateral to the lesion,
143
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MS
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Fig. 3. Photomicrograph of SP-immunoreaclive cells in the MS 6 weeks following an FF transection. This section was adjacent to the section pholographed for GAD-immunoreactivity in Fig. 1. A: inlacl. B: F|: lesion. Bar 200/tm. Arrows show midline. colchicine injection, and thus these two animals were not used for the quantitative G A D antllysis. At the level o f the MS examined in this study (see Fig. 1A), there were on the average 410 Nissl-stained cells, 54 A C h E positively stained cells, 238 G A D - i m n l u n o r e a c rive cells, and 13 SP-immunoreactive cells. Thus at this level the G A D neurons accounted t'or 58% o f the neurons, and the cholinergic neurons for only' 137, o f the total neurons, whereas the SP cells only accounted for 3% o f the total neural population. The remaining 26% of the cells in the MS was not accounted for in the present study. N o difference in G A D cells was found between the left and right side of the septum in the unoperated animals. In addition, the n u m b e r o f cells in the septum contralateral to a lesion (left side) did not significantly differ from control septum tit 1 or 6 weeks following FF lesion. However, tt clear statistical difl'crence was observed between contralateral septum and ipsilateral septum of FF-lesioned animals tit either 1 week ( P < 0 . 0 2 5 ) or 6 weeks ( P < 0 . 0 0 5 ) (see Figs. I and 2). At I week the G A D cell n u m b e r was reduced by 19% on the lesion side, whereas tit (~ weeks the G A D cell loss a m o u n t e d to 58%, o f the contralateral population. Because o f the small n u m b e r of SP cells counted and the variability of staining. the changes in SP cells were only qualitatively evaluated. Fig. 3B shows the effect of an F F lesion on SP cells in the septum 6 weeks following the lesion. In all 5 animals examined there appeared to be a near complete loss o f SP cells on the side ipsilateral to the lesion relative to the contralateral septum. G A D - i m m u n o r e a c t i v e cells constitute one of the principal cell types in the medial septal area and diagonal band [2, 12]. Some, yet unknown, p r o p o r t i o n of these cells projects to the HF. However, it has been estimated that about 30% o f all projection neurons from the M S - V D B are G A D - p o s i t i v e [6]. FF transection restllts in a loss in n u m b e r of G A D neurons c o m p a r a b l e in proportion to the loss of cholinergic neurons 6 weeks post-transection (60%) [4]. However, the loss o f G A D cells is considerably lower tit 1 week post-transection than previously observed for cholinergic neurons, i.e. 19 and 60%, respectively. This difference in time course suggests a potentially different loss mechanism or reactivity to transection for the G A D cells than for the cholinergic cells•
144
The loss of GAD-positive neurons following FF lesions could be a result of (1~ traumatic cell death due to the elimination of all collaterals at a location too close to the soma, (2) loss of retrograde transport of some neuronotrophic factor, a n d o r (3) transneuronal death as a function of the dependence of G A D cells on the integrity of other septal neurons. At present we do not know which is accurate. We conclude that FF transection causes death of GABAergic neurons in a similar proportion to that of cholinergic neurons in the MS. However, the time of maximal cell loss is different for each of the two populations, with the GABAergic neurons taking longer to disappear than the cholinergic neurons. Future studies will determine similarities and differences between the response to axotomy of these two specified neuron types in the MS. We thank Jan Berglund and Lee Vahlsing for excellent technical assistance, and Sheryl Christenson lbr typing the manuscript, and drawing the figures. This research was supported by the Office of Naval Research, and the Margaret and Herbert Hoover Jr. Foundation (to F.H.G,) and NSF BNS-85-01677 (to S.V.). I Amaral, D.C;. and Kurz, J., An analysis of the origins of the cholinergic and noncholinergic scptal projections to the hippocampal formation of the rat, J. Comp. Neurol., 240 (1985) 37 59. 2 Brashear, HR., Zaborszky, L. and l-:leimer, L., Distribution of GABAergic and cholinergic neurons in the rat diagonal band, Neuroscicnce, 17 (1986) 439 451. 3 Daitz, H.M. and Powell, T.P.S., Studies of the connexions of the fornix system, J. Neurol. Neurosurg. Psychiatr.. 17 (1954) 75 82. 4 (}age, F.H., Wictorm, K., Fisher, W,, Williams, L.R., Varon, S. and Bjorklund, A., Retrograde cell changes in medial septum and diagonal band following timbria-fornix transection: quantitative temporal analysis, Neurosciencc, I (1986) 241 255. 5 Hefti, F., Nerve growth factor (NGF) promotes survival of septal cholinergic neurons after injury, J. Neurosci., 6 (1986) 2155 2162. 6 Kohler, C., Chan-Palay, V. and Wu, J.W., Seplal neurons containing glutamic acid decarboxylase immunoreactivity project to the hippocampal region in the rat brain, Anat. Embryol., 169 (1984) 41 44. 7 Kohler. C. and Eriksson, L.G., An immunohistochemical study ofsomatostatin and neurotensin posilive neurons in the septal nuclei of the rat brain, Anat. Embryol., 170 (1984) 1 10. 8 Ljungdahl, A., H6kfelt. T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861 943. 9 McLardy, T., Observations of the fornix of the monkey, J. Comp. Neurol., 103 (1955) 305. 10 Mclander, T., Staines, W.A,. H6kfelt, T., Rokaeus, A., Eckenstein, F., Salvaterra, P.M. and Wainer, B.H., Galanin-like immunoreactivity m cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat. Brain Res.. 360 (1985) 130 138. 11 Oertel, W.H.. Mugnaini, E., Schmechel, D.E., Tappaz, M.L. and Kopin, l.J., The immunocytochemical demonstration ofgamma-aminobutyric acidergic neurons methods and application. In V. ChanPalay and S.L. Paley (Eds.), Cytochemical Methods in Neuroanatomy, Alan R. Liss, New York, 1982, pp. 297 329. 12 Panula, P., Revuelta, A.V., Cheney, D.L., Wu, J.Y. and Costa, E., An immunohistochemical study on the location of GABAergic neurons in rat septum. J. Comp. Neurol., 222 (1984) 69 80. 13 Vincent, S.R. and McGeer, E.G., A substance P projection to the hippocampus, Brain Res.. 215 ( 1981 ) 349 351. 14 Wainer, B.H., Levey, A.I., Rye, D.B., Mesulam, M.M. and Mufsom E.J., Cholinergic and non-cholinergic septohippocampal pathways, Neurosci. Lett.. 54 (1985) 45 52. 15 Williams, LR., Varon. S.. Pelcrson, G.M., Wictorin, K., Fischer, W., Bjorklund, A. and Gage, F.H., Continuous infusion of nerve growth litctor prevents basal forebrain neuronal death after fimbriafornixtransection, Proc. Natl. Acad. Sci. llSA, 83(1986) 9231 9235.