Journol of Ncwnrhurnisrrr Vol. 33, pp. 299 to 302 Pergamon PreTs Ltd 1979. Printed in Great Britain 0 International Society Tor Neurochemistry Ltd
LOCALIZATION O F GLUTAMIC ACID DECARBOXYLASE WITHIN LAMINAE OF THE RAT OLFACTORY TUBERCLE NEILR. KRIEGER'and JOHNS. HELLER* 'Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A. and 'Department of Pharmacology, Yale University School of Medicine, New Haven, C T 06510, U.S.A. (Receiued 28 November 1978. Accepted 19 January 1979)
Abstract-There are three histological layers within the rat olfactory tubercle: plexiform, pyramidal and polymorphic. We have assayed glutamic acid decarboxylase (GAD) and GABA in homogenates of frozen sections cut parallel to these layers. Consecutive sections (16 pm) were homogenized in groups and assayed for G A D or GABA. Every seventh section was stained with Toluidine Blue to monitor the depth and orientation of the plane of section. Steep variations in G A D activity (up t o 6-fold) were observed as a function of depth in the tubercle. These were paralleled by corresponding but less marked variations in GABA levels. The lowest values were found in the plexiform layer. This suggests that G A D may play a very limited role there. The highest activities were found in sections from the deepest lamina of the polymorphic layer. This is the only lamina of the tubercle that does not receive a dopaminergic input.
EXPERIMENTAL PROCEDURES THERAT olfactory tubercle is important as a site of Preparation 01tissue. Male Sprague-Dawley rats weighdopaminergic activity and as a possible site of action for antipsychotic drugs (CLEMENT-CORMIER et d., ing approx 300g were killed by decapitation. The hemi1974; HORNet al., 1974). Its richness in the enzymes sected brain was placed on its ventral surface and frozen in powdered dry ice on a Teflon plate (KRIEGERet a/., et al., 1977; TAPPAZ et related to GABA (FONNUM 1977). The tubercle was trimmed t o a pedestal approx al., 1976; KATAOKA et a/., 1975), dopamine (SAAVEDRA 2 mm on each side. Consecutive, tangential sections & ZIVIN,1976; CLEMENT-CORMIER et al., 1974), and (16pm) were cut, homogenized in groups of six and & PALKOVITS. 1974; PAL- assayed for GAD, or in groups of 15 and assa-red for acetylcholine (JACOBOWITZ KOVITZ et a/., 1974) make it particularly suited to GABA. Every seventh section was stained with Toluidine neurochemical study. Furthermore it is arranged in Blue and examined under the microscope to monitor the well defined histological layers that can be analyzed depth and orientation of the plane of section. These et al. methods were pioneered by LINDERSTROM-LANG separately. There is an outer plexiform layer, an intermediate layer of pyramidal cell bodies, and a deep (1935) in their studies of stomach tissue in the 1930's. Two pedestal locations, A and B, were studied (Figs. 1 layer of scattered pyramidal, granule, and polymorphic cell bodies (BECCARI,1910; CAJAL,1955). We and 2). In the caudal tubercle (Pedestal A) the layers are planar and parallel; thus, each tangential section contains have previously suggested that dopamine-sensitive tissue primarily from one layer. In the rostra1 tubercle adenylate cyclase can be localized to the pyramidal (Pedestal B) the layers are corrugated and each section cells of the outer layers of the tubercle (KRIEGERet contains tissue from two or more layers. al., 1977). Assay for yluramir acid decarboxylase. Frozen tissue secTransmitter systems other than dopamine may tions were homogenized in groups of six (40 pg of protein) in 500pl volumes of 2mM-Tris-maleate buffer (pH affect dopaminergic function in the tubercle and may 7.4)-2 mM-ethylene glycol-bis @-amino ethyl e t h e r t N N ' be specifically useful in the study of this function. We have now measured GABA and glutamic acid decar- tetraacetic acid (EGTA). Incubations were carried out without delay. Activity was determined by a microassay based boxylase (GAD, EC 4.1.1.15) in the layers of the o n the amount of l4COz released from the substrate tubercle and report that their distribution is comple[l-t4C]glutamic acid (ALBERS& BRADY,1959) with modifimentary to that of dopamine and dopamine-sensitive cations similar to those described by TAPPAZer ul. (1976). adenylate cyclase: low in the outer layers and highest I4CO, was trapped on a filter paper disc and counted in the deep polymorphic layer. by liquid scintillation spectrometry. The final 55 p1 volume
Abbreuiution used: GAD, glutamic acid decarboxylase.
299
for incubation consisted of 30 pl of incubation mixture, 20p1 of brain homogenate, and 5 p l (1.72pCi) of DL [l-14C]glutamic acid (51.02 mCi/mmol; New England
NEIL R. KRIEGER and JOHNS. HELLER
300
amino acid analyzer. Fifteen consecutive 16 pm sections from approx 250 pm intervals were pooled and homogen. ized in 100 p1 aliquots of 1 m~-CaC1,-50 ~ M - K C ISamples were acidified (pH 2.2) and centrifuged. From the supernatant, 30 or 50pl samples were loaded onto the analyzer column. GABA standards from 0 t o 5 nmol were used. Variations in GABA levels in duplicate samples were less than 10%. Assayfor protein. Protein was determined by the method of LOWRYet al. (1951) with bovine serum albumin as the standard. RESULTS
FIG. 1. Ventral surface of the frozen rat brain. The boundaries of the olfactory tubercles are shown. The location and boundaries of pedestals A and B are indicated on the right half of the diagram (note that the position and the dimensions of the flattened tubercle in the frozen tissue are somewhat different from those in fresh tissue). OB, olfactory bulb; AON, anterior olfactory nucleus; LOT, lateral olfactory tract; OT, olfactory tubercle. Nuclear, Boston, MA). The incubation mixture contained, in mmol/l: potassium phosphate (pH 7.1), 50; EDTA, 0.1; pyridoxal phosphate, 0.05; dithiothreitol, 0.1; L-glutamic acid, 9; with 0.5% (w/v) Triton X-100.Incubations were for 1 h at 37°C and were terminated by the injection of 0.1 ml of 10% (w/v) trichloroacetic acid. An additional hour at 37°C was allowed to ensure complete release of 14C02. Production of I4CO2 was linear with respect to protein and time. Activities were calculated assuming that only the L form of the substrate was decarboxylated and that equal amounts of the D and L forms were present in the ~ ~ - [ t - ' ~ C ] g l u t a macid. ic Assayfor GABA. GABA levels were quantitatively determined with the use of a Durrum high pressure D 500
Steep variations in GAD activity were observed as a function of depth in the tubercle (Fig. 3). For the region in which the layers are parallel (Pedestal A) the figure shows a H o l d variation in GAD activity ranging from 30nmol of CO, formed/h/mg protein in the outermost plexiform layer to 200 nmoles/h/mg protein in the deepest part of the polymorphic cell layer. In the corrugated region (Pedestal B) the pattern is similar, but less steep. GABA levels are shown for the successive layers of pedestal A in Fig. 4. They exhibit the same trend as the values for GAD, lowest in the plexiform layer and highest in the polymorphic layer. In comparison glutamate and glycine levels measured in the same samples were essentially constant across the three layers. DISCUSSION
The highest GAD activities and GABA levels were found in the polymorphic layer of the olfactory tubercle. Marked variations in GAD activity were observed as a function of depth for both pedestal regions of
CP
FIG.2. (a) Coronal section from the rat brain. A 7890 (KONIG & KLIPPEL,1963). (b) Sagittal section from the rat brain. L 950 (KONIG& KLIPPEL,1963). The bars labeled A and B indicate the extent of the intersection of the respective pedestals with these coronal and sagittal sections. Pyr, pyramidal layer of the olfactory tubercle; IsC, Islands of Calleja; MFB, medial forebrain bundle; DB, diagonal band: NA, nucleus accumbens; AC, anterior commissure; CP, caudate putamen.
30 1
Glutamic acid decarboxylase in the olfactory tubercle
r
B
-2 .- 200 m
50
00
50
100 300 500 700 100 300 500 TUBERCLE DEPTH (microns)
700
FIG.3. GAD activity as a function of depth in the olfactory tubercle for the two pedestal locations A and B. The plotted activities are means of specific activities & S.E.M. calculated at each depth from three independent experiments. The depths of the midpoints of the assayed sections are plotted on the abscissa. Plexiform, Pyr. and Poly. indicate the approximate locations of the plexiform, pyramidal,
and polymorphic layers. the tubercle. In the caudal region (Pedestal A) the variation in GAD activity was 6-fold while in the rostral region (Pedestal B) the variation was 2-fold. The shallower gradient seen at B is probably a consequence of the irregular organization of the cell layers in this part of the tubercle and supports the notion that the variations of activity with depth reflect the variations in composition of the layers. Variations in GABA levels were less marked than those for GAD (Pedestal A, Figs. 3 and 4). This may reflect both
the greater interval of tissue used for the assays of GABA and the dependence of GABA levels on degradative as well as synthetic enzymes. In the hippocampus the laminar variation for GABA is also less marked than for GAD (STORM-MATHISEN, 1977). In the plexiform layer the low GAD activity is con(1973) and TAPsistent with the reports of GRAHAM PAZ et u/. (1976) that olfactory tract axom which end in this layer contain little GAD. In the plexiform and pyramidal layers (Fig. 3, the interval from 0 to 400pm) the low GAD activity suggests that the pyramidal cells whose dendrites are distributed over this T interval (PRICE,1975) are not rich in GAD and do not receive dense GABAergic innervation. Since we have previously suggested that these pyramidal cells et ul., 1977), evidence are dopaminoceptive (KRIEGER about their GABAergic innervation is of particular interest. In the deepest part of the polymorphic layer (6W800pm) the G.4D activity is comparable to the highest activities reported for other brain regions. Only the pars reticularis of the substantia nigra (TAPPAZet al., 1976) and the rostra1 medial forebrain bundle (FONNUM et a/., 1977) have been reported to have substantially higher activities. Whether this activity derives from cells intrinsic to the polymorphic 10 * Plexiform Pyr. O Poly. L layer or from entering processes remains to be determined. 100 300 500 700 The distributions of GABA and dopamine in the TU6ERCLE DEQTH laminae of the rat olfactory tubercle appear to be (microns) complementary. Dopamine (FUXE,19650,h), tyrosine FIG. 4. GABA as a function of depth in the olfactory hydroxylase (PICKELet ul., 1977), and doparninetubercle for the A pedestal location. The plotted levels are sensitive adenylate cyclase (KRIEGERet al., 1977) are means & S.E.M. calculated at each depth from four independent experiments. The depths of the midpoints of the more concentrated in the outer two layers and much assayed sections are plotted on the abscissa. Plexiform, less so in the innermost polymorphic layer, while Pyr. and Poly. indicate the approximate locations of the GABA and GAD display the reciprocal pattern. plexiform, pyramidal, and polymorphic layers. Although the actions of neither GABA nor dopamine
R
302
NEILR. KRIEGER and JOHNS. HELLER
have been elucidated in t h e tubercle, this aspect of their distributions is of potential interest and further recommends this region of limbic cortex a s a site for studies concerning t h e actions of these systems.
GRAHAM L. T., JR (1973) Distribution of glutamic acid decarboxylase activity and GABA content in the OlfaCtory bulb. Life Sci. 12, 443-447. HORNA. S., CUELLOA. C. & MILLERR. J. (1974) Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake Acknowledgements-We thank Dr. PAULL. FLETCHER, JR. mechanism and stimulation of adenylate cyclase activity. for assistance with the amino acid and Mr. GARYDAVIS J . Neurochem. 22, 265-270. analyses and Dr. PETERSTERLING for criticizing the JACOBOWITZ D. & PALKOVITS M. (1974) Topographic atlas manuscript. of catecholamine and acetylcholinesterase containing neurons in the rat brain. 1. comp. Neurol. 157, 13 28. KATAOKA K., SORIMACHI M., OKUNOS. & MIZUNON. Note added in proof:. After submitting this paper for pub(1975) Enzymatic evidence for a meso-limbic dopaminerlication the following study came to our attention: OKADA gic innervation in the olfactory tubercle of the rabbit. Y., HOSOYAY. & KUROSAWA F. (1977) High concentration Brain Res. 88, 513-517. of y-aminobutyric acid (GABA) and glutamate decarboxyKONIC J. F. R. & KLIPPELR. A. (1963) The Rut Bruin. lase (GAD) activity in the regions of medial forebrain A Stereotactic Atlas of the Forebrain and Lower Ptirts bundle, diagonal band of Broca and the third layer of the of the Brain Stem. Williams & Wilkins, Baltimore. olfactory tubercle. Proc. Japan Acad. 53B, 236-~240.While G . M. & GREENKRIEGERN. R.,KAUERJ. S., SHEPHERD the approach of these authors differs somewhat from our GARD P. (1977) Dopamine-sensitive adenylate cyclase own, their results and conclusions are similar to ours. within laminae of the olfactory tubercle. Brain Res. 131, 303-312. LINDERSTRBM-LANG K.. HOLTER H. & SOEBORCOHLSEN A. (1935) Studies o n enzymatic histochernistry. XIII. The REFERENCES distribution of enzymes in the stomach of pigs as a funcALBERSR. W. & BRADYR. 0. (1959) The distribution of tion of its histological structure. C. r. Lab Carlsberg 20, glutamate decarboxylase in the nervous system of the 66. N. J., FARR A. L. & RANDALL LOWRY0. H., ROSEBROUGH Rhesus monkey. J . biol. Chem. 234, 926-928. R. J. (1951) Protein measurement with the Folin phenol BECCARIN. (1910) I1 lobo paraolfattorio nei mammiferi. reagent. J . hid. Chem. 193, 265-275. Archo iral. Anat. Embriol. 9, 173-220. M., SAAVEDRA J., KOBAYASHI R. M. & BROWNI. & ZICMONDR. E. (1976) PALKOVITZ BEN-ARI Y., KANAZAWA STEIN M. (1974) Choline acetyltransferase content of limRegional distribution of glutamate decarboxylase and bic nuclei of the rat. Brain Res. 79, 443-450. GABA within the amygdaloid complex and stria terPICKEL V . M., JOH T. H. & REIS D. J. (1977) Regional minalis system of the rat. 1.Neurochem. 26, 1279-1283. and ultrastructural localization of tyrosine hydroxylase CAJALS. R. (1975) Studies on the Cerebrul Cortex (Transby immunochemistry in dopaminergic neurons of the lated by LISBETHM. KRAFT) pp. 70-75. Lloyd-Luke, mesolimbic and nigroneostriatal systems. Adv. Biochem. London. Psychopharmac. 16, 321-329. Y . C., KEBABIAN J. W., PETZOLD G. L. CLEMENT-CORMIER PRICEJ. L. (1975) An autoradiographic study of comple& GREENGARD P. (1974) Dopamine-sensitive adenylate mentary laminar patterns of termination of afferent cyclase in mammalian brain: a possible site of action fibers of the olfactory cortex. J . comp. Neurol. 150, of antipsychotic drugs. Proc. natn. Acad. Sci., U.S.A. 71, 87-108. I 1 13-1 117. SAAVEDRA J. M. & ZIVINJ . (1976) Tyrosine hydroxylase FONNUM F., WALAASI. & IVERSENE. (1977) Localization and dopamine fi hydroxylase: distribution in discrete of GABAergic, cholinergic and aminergic structures in areas of the rat limbic system. Brain Res. 105, 517--524. the mesolimbic system. J . Neurochem. 29, 221-230. J . (1977) Localization of transmitter canSTORM-MATHISEN FUXE K. (19654 The distribution of monoamine terminals didates in the brain: the hippocampal formation as a in the central nervous system. Act0 physiol. scand. 64, Suppl. 247, 37-85. model. Prog. Neurobioi. 8, 119-181. FUXEK. (19656) Evidence for the existence of monoamine TAPPAZM. L., BROWNSTEIN M. J. & PALKOVITS M. (1976) Distribution of glutamate decarboxylase in discrete neurons in the central nervous system, 2. Zellforsch. 65, brain nuclei. Brain Res. 108, 371-379. 573-596.
LOCALIZATION O F GLUTAMIC ACID DECARBOXYLASE WITHIN LAMINAE OF THE RAT OLFACTORY TUBERCLE NEILR. KRIEGER'and JOHNS. HELLER* 'Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A. and 'Department of Pharmacology, Yale University School of Medicine, New Haven, C T 06510, U.S.A. (Receiued 28 November 1978. Accepted 19 January 1979)
Abstract-There are three histological layers within the rat olfactory tubercle: plexiform, pyramidal and polymorphic. We have assayed glutamic acid decarboxylase (GAD) and GABA in homogenates of frozen sections cut parallel to these layers. Consecutive sections (16 pm) were homogenized in groups and assayed for G A D or GABA. Every seventh section was stained with Toluidine Blue to monitor the depth and orientation of the plane of section. Steep variations in G A D activity (up t o 6-fold) were observed as a function of depth in the tubercle. These were paralleled by corresponding but less marked variations in GABA levels. The lowest values were found in the plexiform layer. This suggests that G A D may play a very limited role there. The highest activities were found in sections from the deepest lamina of the polymorphic layer. This is the only lamina of the tubercle that does not receive a dopaminergic input.
EXPERIMENTAL PROCEDURES THERAT olfactory tubercle is important as a site of Preparation 01tissue. Male Sprague-Dawley rats weighdopaminergic activity and as a possible site of action for antipsychotic drugs (CLEMENT-CORMIER et d., ing approx 300g were killed by decapitation. The hemi1974; HORNet al., 1974). Its richness in the enzymes sected brain was placed on its ventral surface and frozen in powdered dry ice on a Teflon plate (KRIEGERet a/., et al., 1977; TAPPAZ et related to GABA (FONNUM 1977). The tubercle was trimmed t o a pedestal approx al., 1976; KATAOKA et a/., 1975), dopamine (SAAVEDRA 2 mm on each side. Consecutive, tangential sections & ZIVIN,1976; CLEMENT-CORMIER et al., 1974), and (16pm) were cut, homogenized in groups of six and & PALKOVITS. 1974; PAL- assayed for GAD, or in groups of 15 and assa-red for acetylcholine (JACOBOWITZ KOVITZ et a/., 1974) make it particularly suited to GABA. Every seventh section was stained with Toluidine neurochemical study. Furthermore it is arranged in Blue and examined under the microscope to monitor the well defined histological layers that can be analyzed depth and orientation of the plane of section. These et al. methods were pioneered by LINDERSTROM-LANG separately. There is an outer plexiform layer, an intermediate layer of pyramidal cell bodies, and a deep (1935) in their studies of stomach tissue in the 1930's. Two pedestal locations, A and B, were studied (Figs. 1 layer of scattered pyramidal, granule, and polymorphic cell bodies (BECCARI,1910; CAJAL,1955). We and 2). In the caudal tubercle (Pedestal A) the layers are planar and parallel; thus, each tangential section contains have previously suggested that dopamine-sensitive tissue primarily from one layer. In the rostra1 tubercle adenylate cyclase can be localized to the pyramidal (Pedestal B) the layers are corrugated and each section cells of the outer layers of the tubercle (KRIEGERet contains tissue from two or more layers. al., 1977). Assay for yluramir acid decarboxylase. Frozen tissue secTransmitter systems other than dopamine may tions were homogenized in groups of six (40 pg of protein) in 500pl volumes of 2mM-Tris-maleate buffer (pH affect dopaminergic function in the tubercle and may 7.4)-2 mM-ethylene glycol-bis @-amino ethyl e t h e r t N N ' be specifically useful in the study of this function. We have now measured GABA and glutamic acid decar- tetraacetic acid (EGTA). Incubations were carried out without delay. Activity was determined by a microassay based boxylase (GAD, EC 4.1.1.15) in the layers of the o n the amount of l4COz released from the substrate tubercle and report that their distribution is comple[l-t4C]glutamic acid (ALBERS& BRADY,1959) with modifimentary to that of dopamine and dopamine-sensitive cations similar to those described by TAPPAZer ul. (1976). adenylate cyclase: low in the outer layers and highest I4CO, was trapped on a filter paper disc and counted in the deep polymorphic layer. by liquid scintillation spectrometry. The final 55 p1 volume
Abbreuiution used: GAD, glutamic acid decarboxylase.
299
for incubation consisted of 30 pl of incubation mixture, 20p1 of brain homogenate, and 5 p l (1.72pCi) of DL [l-14C]glutamic acid (51.02 mCi/mmol; New England
NEIL R. KRIEGER and JOHNS. HELLER
300
amino acid analyzer. Fifteen consecutive 16 pm sections from approx 250 pm intervals were pooled and homogen. ized in 100 p1 aliquots of 1 m~-CaC1,-50 ~ M - K C ISamples were acidified (pH 2.2) and centrifuged. From the supernatant, 30 or 50pl samples were loaded onto the analyzer column. GABA standards from 0 t o 5 nmol were used. Variations in GABA levels in duplicate samples were less than 10%. Assayfor protein. Protein was determined by the method of LOWRYet al. (1951) with bovine serum albumin as the standard. RESULTS
FIG. 1. Ventral surface of the frozen rat brain. The boundaries of the olfactory tubercles are shown. The location and boundaries of pedestals A and B are indicated on the right half of the diagram (note that the position and the dimensions of the flattened tubercle in the frozen tissue are somewhat different from those in fresh tissue). OB, olfactory bulb; AON, anterior olfactory nucleus; LOT, lateral olfactory tract; OT, olfactory tubercle. Nuclear, Boston, MA). The incubation mixture contained, in mmol/l: potassium phosphate (pH 7.1), 50; EDTA, 0.1; pyridoxal phosphate, 0.05; dithiothreitol, 0.1; L-glutamic acid, 9; with 0.5% (w/v) Triton X-100.Incubations were for 1 h at 37°C and were terminated by the injection of 0.1 ml of 10% (w/v) trichloroacetic acid. An additional hour at 37°C was allowed to ensure complete release of 14C02. Production of I4CO2 was linear with respect to protein and time. Activities were calculated assuming that only the L form of the substrate was decarboxylated and that equal amounts of the D and L forms were present in the ~ ~ - [ t - ' ~ C ] g l u t a macid. ic Assayfor GABA. GABA levels were quantitatively determined with the use of a Durrum high pressure D 500
Steep variations in GAD activity were observed as a function of depth in the tubercle (Fig. 3). For the region in which the layers are parallel (Pedestal A) the figure shows a H o l d variation in GAD activity ranging from 30nmol of CO, formed/h/mg protein in the outermost plexiform layer to 200 nmoles/h/mg protein in the deepest part of the polymorphic cell layer. In the corrugated region (Pedestal B) the pattern is similar, but less steep. GABA levels are shown for the successive layers of pedestal A in Fig. 4. They exhibit the same trend as the values for GAD, lowest in the plexiform layer and highest in the polymorphic layer. In comparison glutamate and glycine levels measured in the same samples were essentially constant across the three layers. DISCUSSION
The highest GAD activities and GABA levels were found in the polymorphic layer of the olfactory tubercle. Marked variations in GAD activity were observed as a function of depth for both pedestal regions of
CP
FIG.2. (a) Coronal section from the rat brain. A 7890 (KONIG & KLIPPEL,1963). (b) Sagittal section from the rat brain. L 950 (KONIG& KLIPPEL,1963). The bars labeled A and B indicate the extent of the intersection of the respective pedestals with these coronal and sagittal sections. Pyr, pyramidal layer of the olfactory tubercle; IsC, Islands of Calleja; MFB, medial forebrain bundle; DB, diagonal band: NA, nucleus accumbens; AC, anterior commissure; CP, caudate putamen.
30 1
Glutamic acid decarboxylase in the olfactory tubercle
r
B
-2 .- 200 m
50
00
50
100 300 500 700 100 300 500 TUBERCLE DEPTH (microns)
700
FIG.3. GAD activity as a function of depth in the olfactory tubercle for the two pedestal locations A and B. The plotted activities are means of specific activities & S.E.M. calculated at each depth from three independent experiments. The depths of the midpoints of the assayed sections are plotted on the abscissa. Plexiform, Pyr. and Poly. indicate the approximate locations of the plexiform, pyramidal,
and polymorphic layers. the tubercle. In the caudal region (Pedestal A) the variation in GAD activity was 6-fold while in the rostral region (Pedestal B) the variation was 2-fold. The shallower gradient seen at B is probably a consequence of the irregular organization of the cell layers in this part of the tubercle and supports the notion that the variations of activity with depth reflect the variations in composition of the layers. Variations in GABA levels were less marked than those for GAD (Pedestal A, Figs. 3 and 4). This may reflect both
the greater interval of tissue used for the assays of GABA and the dependence of GABA levels on degradative as well as synthetic enzymes. In the hippocampus the laminar variation for GABA is also less marked than for GAD (STORM-MATHISEN, 1977). In the plexiform layer the low GAD activity is con(1973) and TAPsistent with the reports of GRAHAM PAZ et u/. (1976) that olfactory tract axom which end in this layer contain little GAD. In the plexiform and pyramidal layers (Fig. 3, the interval from 0 to 400pm) the low GAD activity suggests that the pyramidal cells whose dendrites are distributed over this T interval (PRICE,1975) are not rich in GAD and do not receive dense GABAergic innervation. Since we have previously suggested that these pyramidal cells et ul., 1977), evidence are dopaminoceptive (KRIEGER about their GABAergic innervation is of particular interest. In the deepest part of the polymorphic layer (6W800pm) the G.4D activity is comparable to the highest activities reported for other brain regions. Only the pars reticularis of the substantia nigra (TAPPAZet al., 1976) and the rostra1 medial forebrain bundle (FONNUM et a/., 1977) have been reported to have substantially higher activities. Whether this activity derives from cells intrinsic to the polymorphic 10 * Plexiform Pyr. O Poly. L layer or from entering processes remains to be determined. 100 300 500 700 The distributions of GABA and dopamine in the TU6ERCLE DEQTH laminae of the rat olfactory tubercle appear to be (microns) complementary. Dopamine (FUXE,19650,h), tyrosine FIG. 4. GABA as a function of depth in the olfactory hydroxylase (PICKELet ul., 1977), and doparninetubercle for the A pedestal location. The plotted levels are sensitive adenylate cyclase (KRIEGERet al., 1977) are means & S.E.M. calculated at each depth from four independent experiments. The depths of the midpoints of the more concentrated in the outer two layers and much assayed sections are plotted on the abscissa. Plexiform, less so in the innermost polymorphic layer, while Pyr. and Poly. indicate the approximate locations of the GABA and GAD display the reciprocal pattern. plexiform, pyramidal, and polymorphic layers. Although the actions of neither GABA nor dopamine
R
302
NEILR. KRIEGER and JOHNS. HELLER
have been elucidated in t h e tubercle, this aspect of their distributions is of potential interest and further recommends this region of limbic cortex a s a site for studies concerning t h e actions of these systems.
GRAHAM L. T., JR (1973) Distribution of glutamic acid decarboxylase activity and GABA content in the OlfaCtory bulb. Life Sci. 12, 443-447. HORNA. S., CUELLOA. C. & MILLERR. J. (1974) Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake Acknowledgements-We thank Dr. PAULL. FLETCHER, JR. mechanism and stimulation of adenylate cyclase activity. for assistance with the amino acid and Mr. GARYDAVIS J . Neurochem. 22, 265-270. analyses and Dr. PETERSTERLING for criticizing the JACOBOWITZ D. & PALKOVITS M. (1974) Topographic atlas manuscript. of catecholamine and acetylcholinesterase containing neurons in the rat brain. 1. comp. Neurol. 157, 13 28. KATAOKA K., SORIMACHI M., OKUNOS. & MIZUNON. Note added in proof:. After submitting this paper for pub(1975) Enzymatic evidence for a meso-limbic dopaminerlication the following study came to our attention: OKADA gic innervation in the olfactory tubercle of the rabbit. Y., HOSOYAY. & KUROSAWA F. (1977) High concentration Brain Res. 88, 513-517. of y-aminobutyric acid (GABA) and glutamate decarboxyKONIC J. F. R. & KLIPPELR. A. (1963) The Rut Bruin. lase (GAD) activity in the regions of medial forebrain A Stereotactic Atlas of the Forebrain and Lower Ptirts bundle, diagonal band of Broca and the third layer of the of the Brain Stem. Williams & Wilkins, Baltimore. olfactory tubercle. Proc. Japan Acad. 53B, 236-~240.While G . M. & GREENKRIEGERN. R.,KAUERJ. S., SHEPHERD the approach of these authors differs somewhat from our GARD P. (1977) Dopamine-sensitive adenylate cyclase own, their results and conclusions are similar to ours. within laminae of the olfactory tubercle. Brain Res. 131, 303-312. LINDERSTRBM-LANG K.. HOLTER H. & SOEBORCOHLSEN A. (1935) Studies o n enzymatic histochernistry. XIII. The REFERENCES distribution of enzymes in the stomach of pigs as a funcALBERSR. W. & BRADYR. 0. (1959) The distribution of tion of its histological structure. C. r. Lab Carlsberg 20, glutamate decarboxylase in the nervous system of the 66. N. J., FARR A. L. & RANDALL LOWRY0. H., ROSEBROUGH Rhesus monkey. J . biol. Chem. 234, 926-928. R. J. (1951) Protein measurement with the Folin phenol BECCARIN. (1910) I1 lobo paraolfattorio nei mammiferi. reagent. J . hid. Chem. 193, 265-275. Archo iral. Anat. Embriol. 9, 173-220. M., SAAVEDRA J., KOBAYASHI R. M. & BROWNI. & ZICMONDR. E. (1976) PALKOVITZ BEN-ARI Y., KANAZAWA STEIN M. (1974) Choline acetyltransferase content of limRegional distribution of glutamate decarboxylase and bic nuclei of the rat. Brain Res. 79, 443-450. GABA within the amygdaloid complex and stria terPICKEL V . M., JOH T. H. & REIS D. J. (1977) Regional minalis system of the rat. 1.Neurochem. 26, 1279-1283. and ultrastructural localization of tyrosine hydroxylase CAJALS. R. (1975) Studies on the Cerebrul Cortex (Transby immunochemistry in dopaminergic neurons of the lated by LISBETHM. KRAFT) pp. 70-75. Lloyd-Luke, mesolimbic and nigroneostriatal systems. Adv. Biochem. London. Psychopharmac. 16, 321-329. Y . C., KEBABIAN J. W., PETZOLD G. L. CLEMENT-CORMIER PRICEJ. L. (1975) An autoradiographic study of comple& GREENGARD P. (1974) Dopamine-sensitive adenylate mentary laminar patterns of termination of afferent cyclase in mammalian brain: a possible site of action fibers of the olfactory cortex. J . comp. Neurol. 150, of antipsychotic drugs. Proc. natn. Acad. Sci., U.S.A. 71, 87-108. I 1 13-1 117. SAAVEDRA J. M. & ZIVINJ . (1976) Tyrosine hydroxylase FONNUM F., WALAASI. & IVERSENE. (1977) Localization and dopamine fi hydroxylase: distribution in discrete of GABAergic, cholinergic and aminergic structures in areas of the rat limbic system. Brain Res. 105, 517--524. the mesolimbic system. J . Neurochem. 29, 221-230. J . (1977) Localization of transmitter canSTORM-MATHISEN FUXE K. (19654 The distribution of monoamine terminals didates in the brain: the hippocampal formation as a in the central nervous system. Act0 physiol. scand. 64, Suppl. 247, 37-85. model. Prog. Neurobioi. 8, 119-181. FUXEK. (19656) Evidence for the existence of monoamine TAPPAZM. L., BROWNSTEIN M. J. & PALKOVITS M. (1976) Distribution of glutamate decarboxylase in discrete neurons in the central nervous system, 2. Zellforsch. 65, brain nuclei. Brain Res. 108, 371-379. 573-596.
