78
Brain Research, 551 (1991) 78-86 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONIS 0006899391166506
BRES 16650
Galanin and NADPH-diaphorase coexistence in cholinergic neurons of the rat basal forebrain Bryce A. Pasqualotto and Steven R. Vincent Kinsmen Laboratory of Neurological Sciences, Department of Psychiatry, The University of British Columbia, Vancouver, B.C. (Canada) (Accepted 8 January 1991) Key words: Choline acetyltransferase; Galanin; Nicotinamide-adenine dinucleotide phosphate-diaphorase; Co-localization; Basal forebrain; Immunocytochemistry
The distribution of the enzyme NADPH-diaphorase in the rat basal forebrain was examined in relation to the neuropeptide galanin and the neurotransmitter synthetic enzyme choline acetyltransferase. Immunoperoxidase staining permitted camera lucida mapping of galanin and choline acetyltransferase distributions in serial sections through the basal forebrain for comparison with adjacent sections prepared for NADPH-diaphorase histochemistry. Photographs of sections subjected to indirect immunofluorescence for galanin and choline acetyltransferase were compared to photographs of the same sections taken after NADPH-diaphorase histochemistry. This permitted the direct investigation of co-localization within the cholinergic basal forebrain. The distributions of choline acetyltransferase- and galaninimmunoreactive neurons in the basal forebrain agreed with previous descriptions. NADPH-diaphorase histochemistry selectively stained a population of magnocellular basal forebrain neurons with a distribution similar to that observed with galanin immunohistochemistry. Double and triple staining experiments indicated that NADPH-diaphorase labels a majority of the magnocellular cholinergic neurons in the medial septum and diagonal band nuclei. Most of these neurons also contain galanin immunoreactivity. However, small populations of galanin-positive/diaphorase-negative or diaphorase-positive/galanin-negative cholinergic neurons were also observed. In the more caudal portions of the cholinergic basal forebrain, very few galanin or NADPH-diaphorase-positive neurons were observed. Thus, galanin and NADPH-diaphorase coexist in the majority of cholinergic basal forebrain neurons in the regions innervating limbic structures. The neocortically projecting cholinergic cells in the caudal basal forebrain appear to lack these other neurochemical markers. INTRODUCTION
within portions of the cholinergic basal nuclear complex is now w e l l - d o c u m e n t e d in rats ~6'25 and primates a1"15'37.
The m a m m a l i a n basal forebrain contains a large population of cholinergic neurons which in the rat is found in the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the magnocellular preoptic area, the ventral pallidum, the substantia innominata, and the nucleus basalis magnocellularis4"36. This aggregation of cholinergic basal forebrain structures is collectively referred to as the cholinergic basal nuclear complex and has been suggested to be involved in the control of a n u m b e r of higher brain functions 24. The degeneration of these structures has been implicated in a variety of dementias in man. For example, p o s t - m o r t e m studies of A l z h e i m e r ' s patients have d e m o n s t r a t e d degeneration of cholinergic neurons of the medial septum and nucleus basalis 39. Studies of the distribution of the n e u r o p e p t i d e galanin in the rat brain have d e m o n s t r a t e d a high degree of overlap with neurons containing the neurotransmitter acetylcholine in the basal forebrain 14"21'28. The coexistence of galanin and choline acetyltransferase (CHAT)
A l t h o u g h species differences exist regarding the galanin distribution in the basal forebrain, in both m o n k e y and rat the magnocellular neurons of the medial septumnucleus of the diagonal band complex display extensive coexistence of C h A T and galanin 11A5"16'37. Histochemical assays for specific enzymatic activities have p r o v i d e d useful tools for investigating the biochemical and anatomical organization of discrete neuronal populations. N A D P H - d i a p h o r a s e ( N A D P H - d ) is known to be responsible for the enzymatic reduction of tetrazolium salts to insoluble formazans 23. This simple histochemical reaction is especially useful in neuroanatomical studies because N A D P H - d histochemistry can be performed in conjunction with a variety of o t h e r histochemical techniques due to the robust nature of this enzyme. It is also useful in h u m a n neuropathological studies, since Kowall et al. 12 have found the N A D P H - d reaction to be stable for up to 24 h in an animal m o d e l of human autopsy conditions. Vincent et al. 32 have shown that the N A D P H - d reaction provides a selective histochemical
Correspondence: S.R. Vincent, Kinsmen Laboratory of Neurological Sciences, Department of Psychiatry, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, B.C. V6T lW5, Canada.
79 marker for telencephalic neurons which contain both somatostatin and neuropeptide Y. In the midbrain and p o n t i n e t e g m e n t u m N A D P H - d histochemistry has clarified the organization of cholinergic systems which also contain a n u m b e r of neuropeptides 31'33. Thus, this technique has facilitated the histochemical characterization of several unique n e u r o n a l populations and provides a selective marker for specific cell groups which contain various neuroactive compounds. A few reports have indicated that some magnocellular neurons in the basal forebrain were stained with the N A D P H - d histochemical technique in rat 8'35, cat TM, and h u m a n 3'17. This prompted us to examine the coexistence of N A D P H - d activity with galanin in the cholinergic neurons of the rat basal forebrain. MATERIALS AND METHODS
(1) Tissue preparation To permit the accumulation of the neuropeptide galanin to levels detectable by immunofluorescence, 6 young adult male Wistar rats were anesthetized with sodium pentobarbital and administered 80 /~g of colchicine in 20/~1 of saline stereotaxically into the left lateral ventricle. After 48 h the animals were re-anesthetized and perfused through the aorta with 0.02 M phosphate-buffered saline (PBS) pH 6.9 prior to fixation with ice cold 15% saturated picric acid and 4% paraformaldehyde in 0.16 M sodium phosphate buffer (pH 6.9) for 6 min. The brains were than removed and post-fixed for 90 min in the same fixative then stored in 15% sucrose containing 0.01% sodium azide for 48 h at 4 °C. Brains were then blocked, frozen and cut into 15-/~m coronal sections on a cryostat. Free floating serial sections were collected in PBS. Some sections were processed only for galanin immunofluorescence then subjected to NADPH-d histochemistry, whereas other sections were simultaneously processed for galanin and ChAT immunofluorescence then subjected to NADPH-d histochemistry. Another group of serial sections was prepared for camera lucida mapping using the avidin-biotinperoxidase complex (ABC) immunoperoxidase technique7.
(11) Galanin indirect immunofluoresence Sections were incubated in rabbit anti-galanin serum (Peninsula labs) diluted to 1:500 in PBS with 0.3% Triton X-100, 2% normal goat serum (NGS), and 0.01% sodium azide (PBST) for 48 h at 4 °C then washed 3 times for 20 min in fresh PBS. Sections were then incubated in Texas red conjugated goat anti-rabbit IgG (Jackson Labs) diluted to 1:20 in PBST for 1 h at room temperature while shaking. Sections were washed 3 × 20 min in PBS, then mounted on chrom-alum coated slides and dried at room temperature before coverslipping with glycerol-PBS (3:1). Sections were then examined and photographed with a Leitz fluorescence microscope.
(111) ChAT indirect immunofluorescence Free floating sections were incubated in rat anti-ChAT serum (Boehringer Mannheim) diluted to 1:100 in PBST for 48 h at 4 °C then washed 3 times for 20 min in fresh PBS. Sections were then incubated in fluorescein-isothiocyanate (FITC)-conjugated goat anti-rat IgG (Zymed) diluted to 1:20 in PBST for 1 h at room temperature while shaking, then processed as described for galanin immunofluorescence.
(IV) NADPH-d histochemistry After photography the coverslips were removed and sections were rinsed for 10-15 min in 50 mM Tris-Cl buffer pH 8.0 with 0.3% Triton at room temperature. To stain for NADPH-d activity sections were incubated in 10 mi Tris-Cl buffer pH 8.0 containing 0.3%
Triton, 10 mg fl-NADPH, and 1 mg nitro blue tetrazolium at 37 °C for 20 min. Sections were then rinsed in PBS, coverslipped with glycerol:PBS examined and re-photographed under bright field illumination.
(V) lmmunoperoxidase staining procedure The ABC immunoperoxidase technique was employed using commercially available kits (Vector Labs) to facilitate mapping of the distributions of galanin and ChAT-immunoreactive neurons through the basal forebrain of 3 young adult male Wistar rats. Sections were incubated in primary antibody preparations consisting of either rabbit anti-galanin (1:1000) or rat anti-ChAT IgG (1:200) in PBST for 48 h at 4 °C then washed in PBS. These sections were then incubated in secondary antibody preparations of biotinylated goat anti-rabbit or biotinylated rabbit anti-rat IgG (both 1:200 in PBST, Vector Labs) for 60 min at room temperature then washed in PBS and incubated in avidin-biotin-horseradish peroxidase complex preparation (Vector Labs) for 1 h. After washing again in PBS the peroxidase activity was demonstrated in 100 ml of 0.1 M sodium acetate buffer (pH 6.0) containing 2.5 g nickel ammonium sulfate, 50 mg diaminobenzidine tetrahydrochloride, 200 mg rD-glucose, 40 mg ammonium chloride, and 1 mg glucose oxidase27. Sections prepared in this manner were bathed in distilled water for 15 min and allowed to air-dry overnight. Once dry, sections were bathed in xylene for 10 min and coverslipped with permount. These sections were then traced via camera lucida mapping together with adjacent sections subjected to NADPH-d histochemistry which were washed and dehydrated using a similar procedure. RESULTS The distribution of C h A T - i m m u n o r e a c t i v e n e u r o n s as determined with the A B C immunoperoxidase technique (Figs. 1 and 2) concurred with previous reports of cholinergic localization in the rat basal forebrain 4'36. The distribution of galanin-positive n e u r o n s , as determined by the same technique (Figs. 1 and 2) also matches well with previous reports of galanin localization in the rat basal forebrain 14,2x'2s. The distribution of N A D P H - d activity in the rat basal forebrain has not been previously described in detail. A t the rostral end of the basal forebrain the majority of NADPH-d-positive cells are distributed evenly throughout the nucleus accumbens and extend ventrally into the striatal bridges in the olfactory tubercle. These are all medium-sized neurons, easily distinguished from the magnocellular n e u r o n s of the cholinergic basal nuclear complex. Proceeding in a caudal direction, d e a r l y defined NADPH-d-positive magnocellular n e u r o n a l populations were observed in the medial septum and the nuclei of the vertical and horizontal limbs of the diagnonal b a n d (Figs. 1 and 2). Only a few N A D P H d-positive n e u r o n s were found in more caudal regions of the basal forebrain such as the ventral pallidum-substantia innominata/nucleus basalis and the lateral aspect of the lateral preoptic area. A few isolated N A D P H d-positive n e u r o n s were scattered throughout the medial preoptic area, and there were also well defined dense clusters of N A D P H - d - p o s i t i v e cells in the supraoptic and paraventricular nuclei. No N A D P H - d - p o s i t i v e n e u r o n s
80 Cb~.T
GAI_~.NZ N
NADPH-DIAPHO~S
\ S ....!.~.:...:.-I / .. ~,.:i),U it.:: i , ~
.
p
Brain Research, 551 (1991) 78-86 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONIS 0006899391166506
BRES 16650
Galanin and NADPH-diaphorase coexistence in cholinergic neurons of the rat basal forebrain Bryce A. Pasqualotto and Steven R. Vincent Kinsmen Laboratory of Neurological Sciences, Department of Psychiatry, The University of British Columbia, Vancouver, B.C. (Canada) (Accepted 8 January 1991) Key words: Choline acetyltransferase; Galanin; Nicotinamide-adenine dinucleotide phosphate-diaphorase; Co-localization; Basal forebrain; Immunocytochemistry
The distribution of the enzyme NADPH-diaphorase in the rat basal forebrain was examined in relation to the neuropeptide galanin and the neurotransmitter synthetic enzyme choline acetyltransferase. Immunoperoxidase staining permitted camera lucida mapping of galanin and choline acetyltransferase distributions in serial sections through the basal forebrain for comparison with adjacent sections prepared for NADPH-diaphorase histochemistry. Photographs of sections subjected to indirect immunofluorescence for galanin and choline acetyltransferase were compared to photographs of the same sections taken after NADPH-diaphorase histochemistry. This permitted the direct investigation of co-localization within the cholinergic basal forebrain. The distributions of choline acetyltransferase- and galaninimmunoreactive neurons in the basal forebrain agreed with previous descriptions. NADPH-diaphorase histochemistry selectively stained a population of magnocellular basal forebrain neurons with a distribution similar to that observed with galanin immunohistochemistry. Double and triple staining experiments indicated that NADPH-diaphorase labels a majority of the magnocellular cholinergic neurons in the medial septum and diagonal band nuclei. Most of these neurons also contain galanin immunoreactivity. However, small populations of galanin-positive/diaphorase-negative or diaphorase-positive/galanin-negative cholinergic neurons were also observed. In the more caudal portions of the cholinergic basal forebrain, very few galanin or NADPH-diaphorase-positive neurons were observed. Thus, galanin and NADPH-diaphorase coexist in the majority of cholinergic basal forebrain neurons in the regions innervating limbic structures. The neocortically projecting cholinergic cells in the caudal basal forebrain appear to lack these other neurochemical markers. INTRODUCTION
within portions of the cholinergic basal nuclear complex is now w e l l - d o c u m e n t e d in rats ~6'25 and primates a1"15'37.
The m a m m a l i a n basal forebrain contains a large population of cholinergic neurons which in the rat is found in the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the magnocellular preoptic area, the ventral pallidum, the substantia innominata, and the nucleus basalis magnocellularis4"36. This aggregation of cholinergic basal forebrain structures is collectively referred to as the cholinergic basal nuclear complex and has been suggested to be involved in the control of a n u m b e r of higher brain functions 24. The degeneration of these structures has been implicated in a variety of dementias in man. For example, p o s t - m o r t e m studies of A l z h e i m e r ' s patients have d e m o n s t r a t e d degeneration of cholinergic neurons of the medial septum and nucleus basalis 39. Studies of the distribution of the n e u r o p e p t i d e galanin in the rat brain have d e m o n s t r a t e d a high degree of overlap with neurons containing the neurotransmitter acetylcholine in the basal forebrain 14"21'28. The coexistence of galanin and choline acetyltransferase (CHAT)
A l t h o u g h species differences exist regarding the galanin distribution in the basal forebrain, in both m o n k e y and rat the magnocellular neurons of the medial septumnucleus of the diagonal band complex display extensive coexistence of C h A T and galanin 11A5"16'37. Histochemical assays for specific enzymatic activities have p r o v i d e d useful tools for investigating the biochemical and anatomical organization of discrete neuronal populations. N A D P H - d i a p h o r a s e ( N A D P H - d ) is known to be responsible for the enzymatic reduction of tetrazolium salts to insoluble formazans 23. This simple histochemical reaction is especially useful in neuroanatomical studies because N A D P H - d histochemistry can be performed in conjunction with a variety of o t h e r histochemical techniques due to the robust nature of this enzyme. It is also useful in h u m a n neuropathological studies, since Kowall et al. 12 have found the N A D P H - d reaction to be stable for up to 24 h in an animal m o d e l of human autopsy conditions. Vincent et al. 32 have shown that the N A D P H - d reaction provides a selective histochemical
Correspondence: S.R. Vincent, Kinsmen Laboratory of Neurological Sciences, Department of Psychiatry, The University of British Columbia, 2255 Wesbrook Mall, Vancouver, B.C. V6T lW5, Canada.
79 marker for telencephalic neurons which contain both somatostatin and neuropeptide Y. In the midbrain and p o n t i n e t e g m e n t u m N A D P H - d histochemistry has clarified the organization of cholinergic systems which also contain a n u m b e r of neuropeptides 31'33. Thus, this technique has facilitated the histochemical characterization of several unique n e u r o n a l populations and provides a selective marker for specific cell groups which contain various neuroactive compounds. A few reports have indicated that some magnocellular neurons in the basal forebrain were stained with the N A D P H - d histochemical technique in rat 8'35, cat TM, and h u m a n 3'17. This prompted us to examine the coexistence of N A D P H - d activity with galanin in the cholinergic neurons of the rat basal forebrain. MATERIALS AND METHODS
(1) Tissue preparation To permit the accumulation of the neuropeptide galanin to levels detectable by immunofluorescence, 6 young adult male Wistar rats were anesthetized with sodium pentobarbital and administered 80 /~g of colchicine in 20/~1 of saline stereotaxically into the left lateral ventricle. After 48 h the animals were re-anesthetized and perfused through the aorta with 0.02 M phosphate-buffered saline (PBS) pH 6.9 prior to fixation with ice cold 15% saturated picric acid and 4% paraformaldehyde in 0.16 M sodium phosphate buffer (pH 6.9) for 6 min. The brains were than removed and post-fixed for 90 min in the same fixative then stored in 15% sucrose containing 0.01% sodium azide for 48 h at 4 °C. Brains were then blocked, frozen and cut into 15-/~m coronal sections on a cryostat. Free floating serial sections were collected in PBS. Some sections were processed only for galanin immunofluorescence then subjected to NADPH-d histochemistry, whereas other sections were simultaneously processed for galanin and ChAT immunofluorescence then subjected to NADPH-d histochemistry. Another group of serial sections was prepared for camera lucida mapping using the avidin-biotinperoxidase complex (ABC) immunoperoxidase technique7.
(11) Galanin indirect immunofluoresence Sections were incubated in rabbit anti-galanin serum (Peninsula labs) diluted to 1:500 in PBS with 0.3% Triton X-100, 2% normal goat serum (NGS), and 0.01% sodium azide (PBST) for 48 h at 4 °C then washed 3 times for 20 min in fresh PBS. Sections were then incubated in Texas red conjugated goat anti-rabbit IgG (Jackson Labs) diluted to 1:20 in PBST for 1 h at room temperature while shaking. Sections were washed 3 × 20 min in PBS, then mounted on chrom-alum coated slides and dried at room temperature before coverslipping with glycerol-PBS (3:1). Sections were then examined and photographed with a Leitz fluorescence microscope.
(111) ChAT indirect immunofluorescence Free floating sections were incubated in rat anti-ChAT serum (Boehringer Mannheim) diluted to 1:100 in PBST for 48 h at 4 °C then washed 3 times for 20 min in fresh PBS. Sections were then incubated in fluorescein-isothiocyanate (FITC)-conjugated goat anti-rat IgG (Zymed) diluted to 1:20 in PBST for 1 h at room temperature while shaking, then processed as described for galanin immunofluorescence.
(IV) NADPH-d histochemistry After photography the coverslips were removed and sections were rinsed for 10-15 min in 50 mM Tris-Cl buffer pH 8.0 with 0.3% Triton at room temperature. To stain for NADPH-d activity sections were incubated in 10 mi Tris-Cl buffer pH 8.0 containing 0.3%
Triton, 10 mg fl-NADPH, and 1 mg nitro blue tetrazolium at 37 °C for 20 min. Sections were then rinsed in PBS, coverslipped with glycerol:PBS examined and re-photographed under bright field illumination.
(V) lmmunoperoxidase staining procedure The ABC immunoperoxidase technique was employed using commercially available kits (Vector Labs) to facilitate mapping of the distributions of galanin and ChAT-immunoreactive neurons through the basal forebrain of 3 young adult male Wistar rats. Sections were incubated in primary antibody preparations consisting of either rabbit anti-galanin (1:1000) or rat anti-ChAT IgG (1:200) in PBST for 48 h at 4 °C then washed in PBS. These sections were then incubated in secondary antibody preparations of biotinylated goat anti-rabbit or biotinylated rabbit anti-rat IgG (both 1:200 in PBST, Vector Labs) for 60 min at room temperature then washed in PBS and incubated in avidin-biotin-horseradish peroxidase complex preparation (Vector Labs) for 1 h. After washing again in PBS the peroxidase activity was demonstrated in 100 ml of 0.1 M sodium acetate buffer (pH 6.0) containing 2.5 g nickel ammonium sulfate, 50 mg diaminobenzidine tetrahydrochloride, 200 mg rD-glucose, 40 mg ammonium chloride, and 1 mg glucose oxidase27. Sections prepared in this manner were bathed in distilled water for 15 min and allowed to air-dry overnight. Once dry, sections were bathed in xylene for 10 min and coverslipped with permount. These sections were then traced via camera lucida mapping together with adjacent sections subjected to NADPH-d histochemistry which were washed and dehydrated using a similar procedure. RESULTS The distribution of C h A T - i m m u n o r e a c t i v e n e u r o n s as determined with the A B C immunoperoxidase technique (Figs. 1 and 2) concurred with previous reports of cholinergic localization in the rat basal forebrain 4'36. The distribution of galanin-positive n e u r o n s , as determined by the same technique (Figs. 1 and 2) also matches well with previous reports of galanin localization in the rat basal forebrain 14,2x'2s. The distribution of N A D P H - d activity in the rat basal forebrain has not been previously described in detail. A t the rostral end of the basal forebrain the majority of NADPH-d-positive cells are distributed evenly throughout the nucleus accumbens and extend ventrally into the striatal bridges in the olfactory tubercle. These are all medium-sized neurons, easily distinguished from the magnocellular n e u r o n s of the cholinergic basal nuclear complex. Proceeding in a caudal direction, d e a r l y defined NADPH-d-positive magnocellular n e u r o n a l populations were observed in the medial septum and the nuclei of the vertical and horizontal limbs of the diagnonal b a n d (Figs. 1 and 2). Only a few N A D P H d-positive n e u r o n s were found in more caudal regions of the basal forebrain such as the ventral pallidum-substantia innominata/nucleus basalis and the lateral aspect of the lateral preoptic area. A few isolated N A D P H d-positive n e u r o n s were scattered throughout the medial preoptic area, and there were also well defined dense clusters of N A D P H - d - p o s i t i v e cells in the supraoptic and paraventricular nuclei. No N A D P H - d - p o s i t i v e n e u r o n s
80 Cb~.T
GAI_~.NZ N
NADPH-DIAPHO~S
\ S ....!.~.:...:.-I / .. ~,.:i),U it.:: i , ~
.
p
