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Localization of N-acetylaspartylglutamate-like immunoreactivity in selected areas of the rat brain
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Neuroscience Letters, 72 (1986) 14-20 Elsevier Scientific Publishers Ireland Ltd.
NSL 04273
Localization of N-acetylaspartylglutamate-like immunoreactivity in selected areas of the rat brain Kevin J. Anderson l, Daniel T. Monaghan 1, Charles B. Cangro 2, M.A.A. N a m b o o d i r i 2, Joseph H. Neale 2 a n d Carl W. C o t m a n 1 ;Department of Psychobiology, University of California at lrvine, lrvine, CA 92717; and 2Department of Biology, Georgetown University, Washington, DC 20057 (U.S.A.) (Received 9 May 1986; Revised version received 29 July 1986; Accepted 8 August 1986)
Key words': Glutamate; Aspartate; N-Acetylaspartylglutamate (NAAG); Dipeptide; Immunohistochemistry N-acetylaspartylglutamate (NAAG) was detected immunohistochemically in the rat brain using an antiserum which recognizes carbodiimide-fixed NAAG. NAAG-Iike immunoreactivity is described in 5 areas of the brain; olfactory bulb, septal nuclear area, lateral geniculate nucleus, superior colliculus and the entorhinal cortex/hippocampal formation. Mitral cells of the olfactory bulb and neurons concentrated in the medial septum were densely immunostained. A dense population of immunoreactive puncta was found in the superior colliculus and lateral geniculate nucleus (LGN). The LGN also contained immunoreactive neurons. The entorhinal cortex contained numerous immunoreactive cells in layers II-II1 while the hippocampus had few neurons that were NAAG-positive.
N-Acetylaspartylglutamate (NAAG) is a dipeptide that is found in relatively high concentrations in the CNS [4, 8, 14, 15, 20]. N A A G has been reported to depolarize certain populations of neurons when injected into the rat hippocampus in vivo [20] or in the in vitro slice preparation [1]. N A A G also has been shown to have excitatory actions in the primary olfactory cortex [5]. Taken together, these studies suggest that NAAG may be acting as a neurotransmitter at synapses that have been thought to use an excitatory amino acid, such as glutamate or aspartate. Recently, polyclonal antisera directed against N A A G were developed by coupling the hapten to a large protein carrier (thyroglobulin) [2]. We have used one of these antisera to examine the localization of NAAG-Iike immunoreactivity (NAAG-L) in regions of the brain thought to utilize excitatory amino acids as transmitters. Five male Sprague-Dawley rats (21~240 g) were anesthetized with nembutal (50 mg/kg) and perfused transcardially with normal saline followed by 500 ml of a fixative containing 4% paraformaldehyde and 4% l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (carbodiimide, Sigma) in 0.1 M Sorenson's phosphate buffer (pH 7.4). Correspondence: K.J. Anderson, Department of Psychobiology, University of California at lrvine, lrvine, CA 92717, U.S.A. 0304-3940/86/$ 03.50 O 1986 Elsevier Scientific Publishers Ireland Ltd.
15 Additionally, one animal was perfused with buffered 4% paraformaldehyde and another animal was perfused with 4% paraformaldehyde-2% glutaraldehyde. The brains were removed and further fixed for 1 h in fresh buffered 4% paraformaldehyde and cryoprotected overnight in a buffered 30% sucrose solution. The following day the brains were sectioned at 40/tm on a microtome and the sections placed into phosphate-buffered saline (PBS). Sections were rinsed in 3 changes of PBS and incubated for 10 min each in 50% ethanol, 75% ethanol, 100% ethanol, 0.3% H202 in cold 100% methanol, 75% ethanol, 50% ethanol and finally 20 min in PBS containing 1% normal sheep serum (NSS) and 0.1% Triton-X 100. Sections were incubated as free-floating sections on a rotating stage at room temperature. The sections were then incubated in a rabbit antiserum directed against NAAG (R 10, bleeds 9 and 11; diluted 1:500 in PBS containing 1% NSS and 0.1% Triton-X 100) for 24 h. The following day the sections were rinsed 3 x 10 min and then incubated in sheep anti-rabbit serum (Cappel, diluted 1:300 as above) for 1 h, rinsed as above and incubated in rabbit peroxidase-anti-peroxidase serum (Cappel, diluted 1:500 as above) for 1 h. Following an additional rinse, the sections were incubated in 3,3'-diaminobenzidine (DAB, Sigma, 0.5 mg/ml Tris buffer) for 20 min and then reacted with fresh DAB containing 0.01% H202 for 5-10 min. Sections were rinsed thoroughly with PBS, mounted on gelatincoated slides and examined and photographed on an Olympus microscope. For the production of NAAG antisera, NAAG was synthesized and purified via reverse phase and ion exchange HPLC [19]. The dipeptide (24 mg) was coupled to thyroglobulin (10 mg) with carbodiimide (100 mg/ml in H20) and the mixture incubated at 22°C (pH 5-6) for 18 h prior to dialysis against H20 [2]. Two rabbits were immunized initially with multiple intradermal injections each with a total of 2 mg of the conjugate in complete Freund's adjuvant. Animals were subsequently injected every 4--5 weeks with less than 1 mg of the conjugate per boost in incomplete adjuvant. Injections were carried out over 17 months. Specificity controls were as follows. Alternate sections were incubated in 1% preimmune serum in place of the primary antiserum, or antiserum that had been 'absorbed' by pretreatment with 100-600 pm free NAAG (Bachem, Torrance, CA and Peninsula Labs., San Carlos, CA). These sections did not contain immunoreactivity. In contrast, incubation of the diluted antiserum with 5 mM concentrations of the following molecules failed to block the immunoreactivity: glutamate, aspartate, 0c-aspartylglutamate, N-acetylaspartate, 7-glutamylglutamate and N-acetylglutamate. In these absorbtion experiments, NAAG or related molecules were incubated with diluted (1:500) antiserum 24 h prior to use on tissue sections. In addition, conjugated NAAG, glutamate, aspartate, N-acetylaspartate and ~-aspartylglutamate were blotted on millipore filters and the filters processed for NAAG-L. Only those blots of conjugated NAAG stained positively with NAAG antiserum (Fig. 1). Sections from the brains that had been fixed with 4% paraformaldehyde-2% glutaraldehyde without carbodiimide did not immunostain. This is probably due to the fact that NAAG is amino-blocked and is not fixed within cells by glutaraldehyde which cross-links free amine-containing molecules and does not retain N-acetylated molecules.
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@(3 @(3 G Fig. 1. Immunoblots of NAAG and related molecules on a millipore filter disc. Equimolar (50 mM) amounts of these compounds were conjugated to bovine serum albumin (3 mg/ml) with carbodiimide (6 mg/ml) and subsequentlydialyzedagainst distilled H20. Conjugateswere diluted to 2.5 mg/ml (NAA, AG, Asp and Glu) or 0.25 mg/ml (NAAG), blotted on discs, allowedto air-dry and processedfor immunohistochemical localization of NAAG as described in the text. Positive immunoreactivityis seen in the NAAG immunoblot.
Entorhinal cortex/hippocampalformation: the entorhinal cortex had a fairly high density of perikaryal staining (Fig. 2A). These neurons were small (10-20/tm) and concentrated in layers I I - I I I . Staining was most intense within the cell bodies, although a few primary dendrites were immunostained. Immunoreactive neurons seemed to be more dense in the lateral than the medial entorhinal cortex. The hippocampal formation contained few immunoreactive neurons (Fig. 2B). These were concentrated in the hilus fascia dentata and in area CA3. The NAAG-positive neurons were fairly large ( 3 0 - 4 0 / t m ) and appeared to be multipolar. No obvious laminar arrangement of immunoreactive puncta was observed in the hippocampal formation. The dentate granule cells and the pyramidal neurons of the hippocampus did not immunostain. Olfactory bulb: the most striking feature of N A A G - L in this area is the heavy staining of the mitral cells (Fig. 2C). Occasionally, tufted cells within the external plexiform layer were also immunopositive. Olfactory granule cells did not immunostain. Additional dendritic and punctate staining was observed in the glomerular layer, and the internal and external plexiform layers. Septal nuclei: a fairly high density o f perikaryal staining was observed in the septal nuclei (Fig. 2D) with the medial septum containing a higher density of N A A G - L perikaryal staining. Immunoreactive fibers were also seen coursing between the medial and lateral septal nuclei. Lateral geniculate nucleus: a high density of perikaryal and punctate staining was seen in this area outlining the L G N against other adjacent thalamic nuclei (Fig. 3A, B). Immunopositive neurons were small (5-10 pm in diameter) and scattered throughout the substance of the L G N . N A A G - L fiber and punctate staining seemed to outline bundles of fibers coursing through the L G N . Superior colliculus: a dense population of immunoreactive puncta was observed in
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Fig. 2. A: NAAG-L in the medial entorhinal cortex. Fine punctate staining is seen in layer I while stained perikarya are concentrated in layers II-III. B: NAAG-L in neurons of hippocampal area CA3. Note the large immunostained neurons in the stratum oriens (SO). The majority of neurons within the stratum pyramidale (P) are not immunostained. SR, stratum radiatum. C: NAAG-L in the olfactory bulb. The mitral cells (M) are heavily immunostained. Note the fine fibers in the external plexiform (EP) and the glomerular (G1) layers, while the granule cell layer (Gr) is devoid of immunoreactivity. D: NAAG-L in the septal nuclei. Note the stained neurons in the medial (M) septal nucleus relative to the lateral (L) nucleus. Bar = 200 am.
the s u p e r i o r colliculus (Fig. 3C, D). This p u n c t a t e staining a p p e a r e d to be l a m i n a r with the superficial g r a y layer c o n t a i n i n g the highest d e n s i t y o f i m m u n o r e a c t i v e puncta. These results d e m o n s t r a t e t h a t N A A G - L is c o n c e n t r a t e d in a n u m b e r o f n e u r o n a l p o p u l a t i o n s t h r o u g h o u t the r a t brain. These d a t a are c o n s i s t e n t with the findings t h a t N A A G - L can be detected in n e u r o n a l cell b o d i e s o f the d o r s a l r o o t g a n g l i o n [2] a n d t h a t lesions o f afferent p a t h w a y s result in a decrease o f N A A G in specific targets [8]. T h e a n t i s e r u m used in the p r e s e n t e x p e r i m e n t a p p e a r s to be recognizing a N A A G like c o m p o u n d . N A A G a d d e d to the a n t i s e r u m c o m p l e t e l y b l o c k e d i m m u n o s t a i n i n g while tissue fixed with p a r a f o r m a l d e h y d e o r p a r a f o r m a l d e h y d e / g l u t a r a l d e h y d e also d i d n o t i m m u n o s t a i n . This indicates t h a t the a n t i s e r u m is p r o b a b l y n o t c r o s s - r e a c t i n g with free g l u t a m a t e o r a s p a r t a t e which w o u l d be p r e s e n t in tissues fixed in this m a n ner. It is possible t h a t the a n t i s e r u m m a y be recognizing a similar a m i n o - b l o c k e d
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Fig. 3. A: NAAG-L in the thalamus. The lateral geniculate nucleus (LGN) is intensely stained relative to adjacent areas. Rt, Reticular nucleus; Fi, Fimbria. A higher magnification of the LGN is seen in B. Note the small neurons that are immunopositive as well as a fairly dense plexus of fibers and puncta. C: NAAG-L in the superior colliculus. Dense immunoreactive puncta are concentrated in the superficial gray region (Su.G). A higher magnification of these puncta are seen in D. Bars: A and C = 500/~m; B and D = 200 pm. c o m p o u n d ; however, o u r d a t a a n d previous w o r k indicate this a n t i s e r u m has minimal cross-reactivity with N - a c e t y l a s p a r t a t e ( N A A ) , N - a c e t y l g l u t a m a t e , as well as ~a s p a r t y l g l u t a m a t e , 7 - g l u t a m y l g l u t a m a t e , g l u t a m a t e a n d a s p a r t a t e [2]. N A A has been shown to be roughly 10 times higher in c o n c e n t r a t i o n [4, 8, 14] a n d its synthetic enzyme, a s p a r t a t e N - a c e t y l t r a n s f e r a s e , parallels the d i s t r i b u t i o n o f N A A G [18]. W e are currently investigating the d i s t r i b u t i o n o f N A A G a n d N A A in the C N S using i m m u n o h i s t o c h e m i c a l techniques. The p a t t e r n o f N A A G - L in the e n t o r h i n a l c o r t e x / h i p p o c a m p a l f o r m a t i o n is somew h a t surprising. K o l l e r et al. [8] s h o w e d t h a t unilateral kainic acid lesions o f the hipp o c a m p a l f o r m a t i o n resulted in b i l a t e r a l r e d u c t i o n s o f N A A G in the h i p p o c a m p a l f o r m a t i o n a n d septal nucleus. T h e sparse d i s t r i b u t i o n o f N A A G - L in the h i p p o c a m pal f o r m a t i o n , a n d the lack o f staining o f the C A 3 - C A 4 p y r a m i d a l n e u r o n s is not consistent with the idea t h a t N A A G is a m a j o r c o n s t i t u e n t o f c o m m i s s u r a l or h i p p o c a m p a l - s e p t a l p a t h w a y s . T h e intense staining o f layers I I - I I I e n t o r h i n a l cortical neu-
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rons would indicate that NAAG may be contained within neurons giving rise to the perforant and temporoammonic pathways. However, the hippocampal formation was nearly devoid of punctate staining, nor was any lamination of NAAG-L apparent in the terminal zones of these pathways. Moreover, NAAG-L does not parallel acidic amino acid receptor subtypes that have been well characterized in this area [3]. Interestingly, a recent study by Meyerhoff et al. [13] demonstrated that amygdaloid kindled seizures in the rat increased NAAG levels in the entorhinal cortex. The striking NAAG-L in mitral cells of the olfactory bulb is consistent with the idea that NAAG is contained within the olfactory pathway, ffrench-MuUen et al. [5] reported that NAAG is excitatory to pyramidal neurons of the rat piriform cortex, the terminus of the lateral olfactory tract. Furthermore, a reduction of NAAG, aspartate and GABA was observed in this area following unilateral olfactory bulbectomy. The presence of a high concentration of NAAG-L in two visual system-associated nuclei is intriguing. Glutamate and/or aspartate have been suggested as transmitters in the projections from the visual cortex to the lateral geniculate nucleus and to the superficial gray region of the superior colliculus [6, 9, 10, 11] whereas less is known about the transmitter(s) used by the retinotectal and retinogeniculate pathways. There is evidence that transmission at the retinogeniculate synapse is mediated by an excitatory amino acid receptor [7]; however, the retinotectal pathway does not appear to exhibit Ca2+-dependent release of glutamate or aspartate nor retrograde transport of D-[3H]aspartate [12, 17]. In one animal from the present study we sectioned the retina and observed NAAG-L in retinal ganglion cells, suggesting that NAAG may be contained within the primary visual pathway. It is not entirely evident what role molecules such as NAAG and NAA play in the CNS. A body of literature has accumulated implicating NAAG as a possible excitatory neurotransmitter. Other reports have indicated that NAAG is relatively impotent as a physiologically excitatory agent [16, 19]. The data from the present study indicate that NAAG-L is localized to select populations of CNS neurons including two primary sensory systems (olfactory and visual). It is of interest that NAAG-L is associated with two primary sensory pathways; however, further work is needed to define the role of NAAG in CNS function. This work was supported by a program project grant from the NIA (AG-00538 to C.W.C.) and a grant from the NIDA (DA 02297 to J.H.N.) K.J.A. is a recipient of a postdoctoral fellowship from the NINCDS (NS-07627). 1 Bernstein, J., Fisher, R.S., Zaczek, R. and Coyle, J., Dipeptides of glutamate and aspartate may be excitatory neurotransmitters in the rat hippocampal slice, J. Neurosci., 5 (1985) 1429 1433. 2 Cangro, C.B., Garrison, D.E., Luongo, P.A., Truckenmiller, M.E., Namboodiri, M.A.A. and Neale, J.H., First immunohistochemical demonstration of N-acetyl-aspartyl-glutamate in specific neurons, Soc. Neurosci. Abstr., 11 (1985) 108, (Abstract). 3 Cotman, C.W. and Monaghan, D.T., Chemistry and anatomy of excitatory amino acid systems. In H.Y. Meltzer et al. (Eds.), Psychopharmacology: Next Generation of Progress, Raven Press, New York, in press.
20 4 Curatolo, A., d'Archangelo, P. and Lino, A., Distribution of N-acetyl-aspartic and N-acetyl-aspartylglutamic acids in nervous tissue, J. Neurochem., 12 (1965) 339--342. 5 ffrench-Mullen, J.M.H., Koller, K., Zaczek, R., Coyle, J.T., Hori, M. and Carpenter, D.O., N-acetylaspartylglutamate: possible role as the neurotransmitter of the lateral olfactory tract, Proc. Natl. Acad. Sci. USA, 82 (1985) 3897-3900. 6 Fosse, V.M., Heggelund, P., lversen, E. and Fonnum, F., Effects of area 17 ablation on neurotransmitter parameters in efferents to area 18, the lateral geniculate body, pulvinar and superior colliculus in the cat, Neurosci. Lett., 52 (1984) 323-328. 7 Kemp, J.A. and Sillito, A.M., The nature of the excitatory transmitter mediating X and Y cell inputs to the cat dorsal lateral geniculate nucleus, J. Physiol. (London), 323 (1982) 377-391. 8 Koller, K.J., Zaczek, R. and Coyle, J.T., N-acetyl-aspartyl-glutamate: regional levels in rat brain and the effects of brain lesions as determined by a new HPLC method, J. Neurochem., 43 (1984) 1136-1142. 9 Kvale, I. and Fonnum, F., The effects of unilateral removal of visual cortex on transmitter parameters in the adult superior colliculus and lateral geniculate body, Dev. Brain Res., 11 (1983) 261 266. l0 Kvale, I., Fosse, V.M. and Fonnum, F., Postnatal neurochemical development in lateral geniculate body, superior colliculus and visual cortex in the albino rat, Dev. Brain Res., 7 (1983) 137 145. l 1 Lund Karlsen, R. and Fonnum, F., Evidence for glutamate as a neurotransmitter in the corticofugal fibers to the dorsal lateral geniculate body and the superior colliculus in rats, Brain Res., 151 (1978) 457467. 12 Matute, C. and Streit, P., Selective retrograde labeling with D-3H-aspartate in afferents to the mammalian superior colliculus, J Comp. Neurol., 241 (1985) 34-49. 13 Meyerhoff, J.L., Koller, K., Walczak, D.D. and Coyle, J.T., Regional brain levels of N-acetyl-aspartyl glutamate: the effect of kindled seizures, Brain Res., 346 (1985) 392-396. 14 Miyake, M., Kakimoto, Y. and Sorimachi, M., A gas chromatographic method for the determination of N-acetyl-L-aspartic acid, N-acetyl-ct-aspartylglutamic acid and fl-citryl-L-glutamic acid and their distributions in brain and other organs of various species of animals, J. Neurochem., 36 (1981) 804-810. 15 Reichelt, K.L. and Fonnum, F., Subcellular localization of N-acetyl-aspartyl-glutamate, N-acetyl-glutamate, and glutathione in brain, J. Neurochem., 16 (1969) 1409-1416. 16 Riveros, N. and Orrego, F., A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations, Brain Res., 299 (1984) 393-395. 17 Sandberg, M. and Corazzi, L., Release of endogenous amino acids from superior colliculus of the rabbit: in vitro studies after retinal ablation, J. Neurochem., 40 (1983) 917-921. 18 Truckenmiller, M.E., Namboodiri, M.A.A., Brownstein, M.J. and Neale, J.H., N-acetylation of Laspartate in the nervous system: differential distribution of a specific enzyme, J. Neurochem., 45 (1985) 1658 1662. 19 Westbrook, G.L., Mayer, M.L., Namboodiri, M.A.A. and Neale, J.H., High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture, J. Neurosci., in press. 20 Zaczek, R., Koller, K., Cotter, R., Heller, D. and Coyle, J.T., N-acetyl-aspartyl-glutamate: an endogenous peptide with high affinity for a brain 'glutamate' receptor, Proc. Natl. Acad. Sci. USA, 80 (1983) 1116-1119.
Neuroscience Letters, 72 (1986) 14-20 Elsevier Scientific Publishers Ireland Ltd.
NSL 04273
Localization of N-acetylaspartylglutamate-like immunoreactivity in selected areas of the rat brain Kevin J. Anderson l, Daniel T. Monaghan 1, Charles B. Cangro 2, M.A.A. N a m b o o d i r i 2, Joseph H. Neale 2 a n d Carl W. C o t m a n 1 ;Department of Psychobiology, University of California at lrvine, lrvine, CA 92717; and 2Department of Biology, Georgetown University, Washington, DC 20057 (U.S.A.) (Received 9 May 1986; Revised version received 29 July 1986; Accepted 8 August 1986)
Key words': Glutamate; Aspartate; N-Acetylaspartylglutamate (NAAG); Dipeptide; Immunohistochemistry N-acetylaspartylglutamate (NAAG) was detected immunohistochemically in the rat brain using an antiserum which recognizes carbodiimide-fixed NAAG. NAAG-Iike immunoreactivity is described in 5 areas of the brain; olfactory bulb, septal nuclear area, lateral geniculate nucleus, superior colliculus and the entorhinal cortex/hippocampal formation. Mitral cells of the olfactory bulb and neurons concentrated in the medial septum were densely immunostained. A dense population of immunoreactive puncta was found in the superior colliculus and lateral geniculate nucleus (LGN). The LGN also contained immunoreactive neurons. The entorhinal cortex contained numerous immunoreactive cells in layers II-II1 while the hippocampus had few neurons that were NAAG-positive.
N-Acetylaspartylglutamate (NAAG) is a dipeptide that is found in relatively high concentrations in the CNS [4, 8, 14, 15, 20]. N A A G has been reported to depolarize certain populations of neurons when injected into the rat hippocampus in vivo [20] or in the in vitro slice preparation [1]. N A A G also has been shown to have excitatory actions in the primary olfactory cortex [5]. Taken together, these studies suggest that NAAG may be acting as a neurotransmitter at synapses that have been thought to use an excitatory amino acid, such as glutamate or aspartate. Recently, polyclonal antisera directed against N A A G were developed by coupling the hapten to a large protein carrier (thyroglobulin) [2]. We have used one of these antisera to examine the localization of NAAG-Iike immunoreactivity (NAAG-L) in regions of the brain thought to utilize excitatory amino acids as transmitters. Five male Sprague-Dawley rats (21~240 g) were anesthetized with nembutal (50 mg/kg) and perfused transcardially with normal saline followed by 500 ml of a fixative containing 4% paraformaldehyde and 4% l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (carbodiimide, Sigma) in 0.1 M Sorenson's phosphate buffer (pH 7.4). Correspondence: K.J. Anderson, Department of Psychobiology, University of California at lrvine, lrvine, CA 92717, U.S.A. 0304-3940/86/$ 03.50 O 1986 Elsevier Scientific Publishers Ireland Ltd.
15 Additionally, one animal was perfused with buffered 4% paraformaldehyde and another animal was perfused with 4% paraformaldehyde-2% glutaraldehyde. The brains were removed and further fixed for 1 h in fresh buffered 4% paraformaldehyde and cryoprotected overnight in a buffered 30% sucrose solution. The following day the brains were sectioned at 40/tm on a microtome and the sections placed into phosphate-buffered saline (PBS). Sections were rinsed in 3 changes of PBS and incubated for 10 min each in 50% ethanol, 75% ethanol, 100% ethanol, 0.3% H202 in cold 100% methanol, 75% ethanol, 50% ethanol and finally 20 min in PBS containing 1% normal sheep serum (NSS) and 0.1% Triton-X 100. Sections were incubated as free-floating sections on a rotating stage at room temperature. The sections were then incubated in a rabbit antiserum directed against NAAG (R 10, bleeds 9 and 11; diluted 1:500 in PBS containing 1% NSS and 0.1% Triton-X 100) for 24 h. The following day the sections were rinsed 3 x 10 min and then incubated in sheep anti-rabbit serum (Cappel, diluted 1:300 as above) for 1 h, rinsed as above and incubated in rabbit peroxidase-anti-peroxidase serum (Cappel, diluted 1:500 as above) for 1 h. Following an additional rinse, the sections were incubated in 3,3'-diaminobenzidine (DAB, Sigma, 0.5 mg/ml Tris buffer) for 20 min and then reacted with fresh DAB containing 0.01% H202 for 5-10 min. Sections were rinsed thoroughly with PBS, mounted on gelatincoated slides and examined and photographed on an Olympus microscope. For the production of NAAG antisera, NAAG was synthesized and purified via reverse phase and ion exchange HPLC [19]. The dipeptide (24 mg) was coupled to thyroglobulin (10 mg) with carbodiimide (100 mg/ml in H20) and the mixture incubated at 22°C (pH 5-6) for 18 h prior to dialysis against H20 [2]. Two rabbits were immunized initially with multiple intradermal injections each with a total of 2 mg of the conjugate in complete Freund's adjuvant. Animals were subsequently injected every 4--5 weeks with less than 1 mg of the conjugate per boost in incomplete adjuvant. Injections were carried out over 17 months. Specificity controls were as follows. Alternate sections were incubated in 1% preimmune serum in place of the primary antiserum, or antiserum that had been 'absorbed' by pretreatment with 100-600 pm free NAAG (Bachem, Torrance, CA and Peninsula Labs., San Carlos, CA). These sections did not contain immunoreactivity. In contrast, incubation of the diluted antiserum with 5 mM concentrations of the following molecules failed to block the immunoreactivity: glutamate, aspartate, 0c-aspartylglutamate, N-acetylaspartate, 7-glutamylglutamate and N-acetylglutamate. In these absorbtion experiments, NAAG or related molecules were incubated with diluted (1:500) antiserum 24 h prior to use on tissue sections. In addition, conjugated NAAG, glutamate, aspartate, N-acetylaspartate and ~-aspartylglutamate were blotted on millipore filters and the filters processed for NAAG-L. Only those blots of conjugated NAAG stained positively with NAAG antiserum (Fig. 1). Sections from the brains that had been fixed with 4% paraformaldehyde-2% glutaraldehyde without carbodiimide did not immunostain. This is probably due to the fact that NAAG is amino-blocked and is not fixed within cells by glutaraldehyde which cross-links free amine-containing molecules and does not retain N-acetylated molecules.
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@(3 @(3 G Fig. 1. Immunoblots of NAAG and related molecules on a millipore filter disc. Equimolar (50 mM) amounts of these compounds were conjugated to bovine serum albumin (3 mg/ml) with carbodiimide (6 mg/ml) and subsequentlydialyzedagainst distilled H20. Conjugateswere diluted to 2.5 mg/ml (NAA, AG, Asp and Glu) or 0.25 mg/ml (NAAG), blotted on discs, allowedto air-dry and processedfor immunohistochemical localization of NAAG as described in the text. Positive immunoreactivityis seen in the NAAG immunoblot.
Entorhinal cortex/hippocampalformation: the entorhinal cortex had a fairly high density of perikaryal staining (Fig. 2A). These neurons were small (10-20/tm) and concentrated in layers I I - I I I . Staining was most intense within the cell bodies, although a few primary dendrites were immunostained. Immunoreactive neurons seemed to be more dense in the lateral than the medial entorhinal cortex. The hippocampal formation contained few immunoreactive neurons (Fig. 2B). These were concentrated in the hilus fascia dentata and in area CA3. The NAAG-positive neurons were fairly large ( 3 0 - 4 0 / t m ) and appeared to be multipolar. No obvious laminar arrangement of immunoreactive puncta was observed in the hippocampal formation. The dentate granule cells and the pyramidal neurons of the hippocampus did not immunostain. Olfactory bulb: the most striking feature of N A A G - L in this area is the heavy staining of the mitral cells (Fig. 2C). Occasionally, tufted cells within the external plexiform layer were also immunopositive. Olfactory granule cells did not immunostain. Additional dendritic and punctate staining was observed in the glomerular layer, and the internal and external plexiform layers. Septal nuclei: a fairly high density o f perikaryal staining was observed in the septal nuclei (Fig. 2D) with the medial septum containing a higher density of N A A G - L perikaryal staining. Immunoreactive fibers were also seen coursing between the medial and lateral septal nuclei. Lateral geniculate nucleus: a high density of perikaryal and punctate staining was seen in this area outlining the L G N against other adjacent thalamic nuclei (Fig. 3A, B). Immunopositive neurons were small (5-10 pm in diameter) and scattered throughout the substance of the L G N . N A A G - L fiber and punctate staining seemed to outline bundles of fibers coursing through the L G N . Superior colliculus: a dense population of immunoreactive puncta was observed in
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Fig. 2. A: NAAG-L in the medial entorhinal cortex. Fine punctate staining is seen in layer I while stained perikarya are concentrated in layers II-III. B: NAAG-L in neurons of hippocampal area CA3. Note the large immunostained neurons in the stratum oriens (SO). The majority of neurons within the stratum pyramidale (P) are not immunostained. SR, stratum radiatum. C: NAAG-L in the olfactory bulb. The mitral cells (M) are heavily immunostained. Note the fine fibers in the external plexiform (EP) and the glomerular (G1) layers, while the granule cell layer (Gr) is devoid of immunoreactivity. D: NAAG-L in the septal nuclei. Note the stained neurons in the medial (M) septal nucleus relative to the lateral (L) nucleus. Bar = 200 am.
the s u p e r i o r colliculus (Fig. 3C, D). This p u n c t a t e staining a p p e a r e d to be l a m i n a r with the superficial g r a y layer c o n t a i n i n g the highest d e n s i t y o f i m m u n o r e a c t i v e puncta. These results d e m o n s t r a t e t h a t N A A G - L is c o n c e n t r a t e d in a n u m b e r o f n e u r o n a l p o p u l a t i o n s t h r o u g h o u t the r a t brain. These d a t a are c o n s i s t e n t with the findings t h a t N A A G - L can be detected in n e u r o n a l cell b o d i e s o f the d o r s a l r o o t g a n g l i o n [2] a n d t h a t lesions o f afferent p a t h w a y s result in a decrease o f N A A G in specific targets [8]. T h e a n t i s e r u m used in the p r e s e n t e x p e r i m e n t a p p e a r s to be recognizing a N A A G like c o m p o u n d . N A A G a d d e d to the a n t i s e r u m c o m p l e t e l y b l o c k e d i m m u n o s t a i n i n g while tissue fixed with p a r a f o r m a l d e h y d e o r p a r a f o r m a l d e h y d e / g l u t a r a l d e h y d e also d i d n o t i m m u n o s t a i n . This indicates t h a t the a n t i s e r u m is p r o b a b l y n o t c r o s s - r e a c t i n g with free g l u t a m a t e o r a s p a r t a t e which w o u l d be p r e s e n t in tissues fixed in this m a n ner. It is possible t h a t the a n t i s e r u m m a y be recognizing a similar a m i n o - b l o c k e d
18
Fig. 3. A: NAAG-L in the thalamus. The lateral geniculate nucleus (LGN) is intensely stained relative to adjacent areas. Rt, Reticular nucleus; Fi, Fimbria. A higher magnification of the LGN is seen in B. Note the small neurons that are immunopositive as well as a fairly dense plexus of fibers and puncta. C: NAAG-L in the superior colliculus. Dense immunoreactive puncta are concentrated in the superficial gray region (Su.G). A higher magnification of these puncta are seen in D. Bars: A and C = 500/~m; B and D = 200 pm. c o m p o u n d ; however, o u r d a t a a n d previous w o r k indicate this a n t i s e r u m has minimal cross-reactivity with N - a c e t y l a s p a r t a t e ( N A A ) , N - a c e t y l g l u t a m a t e , as well as ~a s p a r t y l g l u t a m a t e , 7 - g l u t a m y l g l u t a m a t e , g l u t a m a t e a n d a s p a r t a t e [2]. N A A has been shown to be roughly 10 times higher in c o n c e n t r a t i o n [4, 8, 14] a n d its synthetic enzyme, a s p a r t a t e N - a c e t y l t r a n s f e r a s e , parallels the d i s t r i b u t i o n o f N A A G [18]. W e are currently investigating the d i s t r i b u t i o n o f N A A G a n d N A A in the C N S using i m m u n o h i s t o c h e m i c a l techniques. The p a t t e r n o f N A A G - L in the e n t o r h i n a l c o r t e x / h i p p o c a m p a l f o r m a t i o n is somew h a t surprising. K o l l e r et al. [8] s h o w e d t h a t unilateral kainic acid lesions o f the hipp o c a m p a l f o r m a t i o n resulted in b i l a t e r a l r e d u c t i o n s o f N A A G in the h i p p o c a m p a l f o r m a t i o n a n d septal nucleus. T h e sparse d i s t r i b u t i o n o f N A A G - L in the h i p p o c a m pal f o r m a t i o n , a n d the lack o f staining o f the C A 3 - C A 4 p y r a m i d a l n e u r o n s is not consistent with the idea t h a t N A A G is a m a j o r c o n s t i t u e n t o f c o m m i s s u r a l or h i p p o c a m p a l - s e p t a l p a t h w a y s . T h e intense staining o f layers I I - I I I e n t o r h i n a l cortical neu-
19
rons would indicate that NAAG may be contained within neurons giving rise to the perforant and temporoammonic pathways. However, the hippocampal formation was nearly devoid of punctate staining, nor was any lamination of NAAG-L apparent in the terminal zones of these pathways. Moreover, NAAG-L does not parallel acidic amino acid receptor subtypes that have been well characterized in this area [3]. Interestingly, a recent study by Meyerhoff et al. [13] demonstrated that amygdaloid kindled seizures in the rat increased NAAG levels in the entorhinal cortex. The striking NAAG-L in mitral cells of the olfactory bulb is consistent with the idea that NAAG is contained within the olfactory pathway, ffrench-MuUen et al. [5] reported that NAAG is excitatory to pyramidal neurons of the rat piriform cortex, the terminus of the lateral olfactory tract. Furthermore, a reduction of NAAG, aspartate and GABA was observed in this area following unilateral olfactory bulbectomy. The presence of a high concentration of NAAG-L in two visual system-associated nuclei is intriguing. Glutamate and/or aspartate have been suggested as transmitters in the projections from the visual cortex to the lateral geniculate nucleus and to the superficial gray region of the superior colliculus [6, 9, 10, 11] whereas less is known about the transmitter(s) used by the retinotectal and retinogeniculate pathways. There is evidence that transmission at the retinogeniculate synapse is mediated by an excitatory amino acid receptor [7]; however, the retinotectal pathway does not appear to exhibit Ca2+-dependent release of glutamate or aspartate nor retrograde transport of D-[3H]aspartate [12, 17]. In one animal from the present study we sectioned the retina and observed NAAG-L in retinal ganglion cells, suggesting that NAAG may be contained within the primary visual pathway. It is not entirely evident what role molecules such as NAAG and NAA play in the CNS. A body of literature has accumulated implicating NAAG as a possible excitatory neurotransmitter. Other reports have indicated that NAAG is relatively impotent as a physiologically excitatory agent [16, 19]. The data from the present study indicate that NAAG-L is localized to select populations of CNS neurons including two primary sensory systems (olfactory and visual). It is of interest that NAAG-L is associated with two primary sensory pathways; however, further work is needed to define the role of NAAG in CNS function. This work was supported by a program project grant from the NIA (AG-00538 to C.W.C.) and a grant from the NIDA (DA 02297 to J.H.N.) K.J.A. is a recipient of a postdoctoral fellowship from the NINCDS (NS-07627). 1 Bernstein, J., Fisher, R.S., Zaczek, R. and Coyle, J., Dipeptides of glutamate and aspartate may be excitatory neurotransmitters in the rat hippocampal slice, J. Neurosci., 5 (1985) 1429 1433. 2 Cangro, C.B., Garrison, D.E., Luongo, P.A., Truckenmiller, M.E., Namboodiri, M.A.A. and Neale, J.H., First immunohistochemical demonstration of N-acetyl-aspartyl-glutamate in specific neurons, Soc. Neurosci. Abstr., 11 (1985) 108, (Abstract). 3 Cotman, C.W. and Monaghan, D.T., Chemistry and anatomy of excitatory amino acid systems. In H.Y. Meltzer et al. (Eds.), Psychopharmacology: Next Generation of Progress, Raven Press, New York, in press.
20 4 Curatolo, A., d'Archangelo, P. and Lino, A., Distribution of N-acetyl-aspartic and N-acetyl-aspartylglutamic acids in nervous tissue, J. Neurochem., 12 (1965) 339--342. 5 ffrench-Mullen, J.M.H., Koller, K., Zaczek, R., Coyle, J.T., Hori, M. and Carpenter, D.O., N-acetylaspartylglutamate: possible role as the neurotransmitter of the lateral olfactory tract, Proc. Natl. Acad. Sci. USA, 82 (1985) 3897-3900. 6 Fosse, V.M., Heggelund, P., lversen, E. and Fonnum, F., Effects of area 17 ablation on neurotransmitter parameters in efferents to area 18, the lateral geniculate body, pulvinar and superior colliculus in the cat, Neurosci. Lett., 52 (1984) 323-328. 7 Kemp, J.A. and Sillito, A.M., The nature of the excitatory transmitter mediating X and Y cell inputs to the cat dorsal lateral geniculate nucleus, J. Physiol. (London), 323 (1982) 377-391. 8 Koller, K.J., Zaczek, R. and Coyle, J.T., N-acetyl-aspartyl-glutamate: regional levels in rat brain and the effects of brain lesions as determined by a new HPLC method, J. Neurochem., 43 (1984) 1136-1142. 9 Kvale, I. and Fonnum, F., The effects of unilateral removal of visual cortex on transmitter parameters in the adult superior colliculus and lateral geniculate body, Dev. Brain Res., 11 (1983) 261 266. l0 Kvale, I., Fosse, V.M. and Fonnum, F., Postnatal neurochemical development in lateral geniculate body, superior colliculus and visual cortex in the albino rat, Dev. Brain Res., 7 (1983) 137 145. l 1 Lund Karlsen, R. and Fonnum, F., Evidence for glutamate as a neurotransmitter in the corticofugal fibers to the dorsal lateral geniculate body and the superior colliculus in rats, Brain Res., 151 (1978) 457467. 12 Matute, C. and Streit, P., Selective retrograde labeling with D-3H-aspartate in afferents to the mammalian superior colliculus, J Comp. Neurol., 241 (1985) 34-49. 13 Meyerhoff, J.L., Koller, K., Walczak, D.D. and Coyle, J.T., Regional brain levels of N-acetyl-aspartyl glutamate: the effect of kindled seizures, Brain Res., 346 (1985) 392-396. 14 Miyake, M., Kakimoto, Y. and Sorimachi, M., A gas chromatographic method for the determination of N-acetyl-L-aspartic acid, N-acetyl-ct-aspartylglutamic acid and fl-citryl-L-glutamic acid and their distributions in brain and other organs of various species of animals, J. Neurochem., 36 (1981) 804-810. 15 Reichelt, K.L. and Fonnum, F., Subcellular localization of N-acetyl-aspartyl-glutamate, N-acetyl-glutamate, and glutathione in brain, J. Neurochem., 16 (1969) 1409-1416. 16 Riveros, N. and Orrego, F., A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations, Brain Res., 299 (1984) 393-395. 17 Sandberg, M. and Corazzi, L., Release of endogenous amino acids from superior colliculus of the rabbit: in vitro studies after retinal ablation, J. Neurochem., 40 (1983) 917-921. 18 Truckenmiller, M.E., Namboodiri, M.A.A., Brownstein, M.J. and Neale, J.H., N-acetylation of Laspartate in the nervous system: differential distribution of a specific enzyme, J. Neurochem., 45 (1985) 1658 1662. 19 Westbrook, G.L., Mayer, M.L., Namboodiri, M.A.A. and Neale, J.H., High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture, J. Neurosci., in press. 20 Zaczek, R., Koller, K., Cotter, R., Heller, D. and Coyle, J.T., N-acetyl-aspartyl-glutamate: an endogenous peptide with high affinity for a brain 'glutamate' receptor, Proc. Natl. Acad. Sci. USA, 80 (1983) 1116-1119.