, 1t;92 Elsevier Scicncc Publishers t3.V. ,\H n~hts vcsclv~.:d tlnil~;-';~3 u 2 '~l> ii!~
BRES 25474
Numerous SP-positive pyramidal neurons in cat neocortex are glutamate-positive Fiorenzo Conti, Mara Fabri and Andrea Minelli Institute of Human Physiology, University of Ancona, Ancona (Italy) (Accepted 22 September 1992)
Key words': Glutamate; Substance P; Pyramidal neuron; Cerebral cortex
An immunocytochemical technique that allows visualization of two antigens in the same neuron was used to verify the possibility that some neocortical pyramidal neurons contain both glutamate (Glu) and substance P (SP) immunoreactivity. The results show that a large fraction of SP-positive pyramidal neurons are also Glu-positive, and indicate that in a small population of cortical neurons a fast excitatory synaptic transmitter and a slow peptidic modulator coexist.
A large body of anatomical, physiological, biochemical, and pharmacological evidence suggests that the excitatory acidic amino acid glutamate (Glu) is the neurotransmitter of a large fraction of pyramidal neurons, the most distinctive cells of the mammalian cerebral cortex 2. We have recently provided anatomical evidence that few pyramidal cells in the first somatic sensory cortex (SI) of cats are immunoreactive for the undecapeptide substance P (SP) 7. Glu-positive pyramidal neurons are present in all cortical layers, but are more numerous in layers III (mean cross sectional area, m.c.s.a.: 168 + 54/xm 2) and V (m.c.s.a.: 2 2 2 + 8 2 /zm2) 3. SP-positive pyramidal neurons 7 are present in layers III and V and have a m.c.s.a, of 187 + 40 /zm 2. Therefore, based on the laminar distribution and on the soma size of Glu- and SP-positive pyramidal neurons, the possibility exists that some pyramidal neurons contain both Glu and SP. We have addressed this issue by an immunocytochemical technique that allows visualization of different antigens in thin adjacent paraffin sections. Six adult cats were used in this study (Table I). Four of them received colchicine; they were anaesthetized with ketamine (33 m g / k g i.m.) and mounted on a stereotaxic frame. Under aseptic conditions, the skull was opened over the appropriate region and colchicine ( 1 0 0 / z g / t z l in physiological saline) was injected intra-
ventricularly (8/zl) or topically applied. In these cases, a small piece of gelfoam (15 mm × 20 mm wide) soaked with 100 /zl of colchicine (i.e., 0.5 /~l/mm 2) was applied over the pial surface of a cortical area that included: part of the motor cortex, S1, SI1, and part of areas SIV and 5, as judged by the sulcal pattern. Animals receiving colchicine survived 7-48 h. All animals were perfused under Nembutal anaesthesia (30 m g / k g i.p.) through the ascending aorta with physiological saline followed by different fixatives, all made up with 0.1 M phosphate buffer (pH 7.4; see Table 1). All brains were immediately removed from the skull and post-fixed (Table I). Small blocks of SI were obtained from each brain, rinsed in phosphate buffered saline (PBS), dehydrated in ethanol, cleared in toluene, and embedded in paraffin. Several series of two adjacent sections (5/xm thick) were cut from each paraffin block and mounted on subbed slides. Sections were rehydrated in an ethanol series and rinsed in PBS before immunocytochemical processing. Two antisera were used: an anti-Glu serum ~ and an anti-SP serum whose characterization has been reported elsewhere 7. Of each couple of sections, one was reacted for SP (at a dilution of 1:2,000-1:6,000) and one for Glu (at a dilution of 1 : 6,000-1 : 8,000), according to the protocol previously described ~. Paraffin sections alternately reacted with SP and
Correspondence: F. Conti, Istituto di Fisiologia Umana, Universit~ di Ancona, Via Ranieri, Monte d'Ago. 1-60131 Ancona, Italy. Fax: (39) 71-2204652.
141 TABLE I
tions. P h o t o m i c r o g r a p h s
Summary o[' experimental cases
and neurons containing both antigens were identified.
para, paraformaldehyde; gluta, glutaraldehyde; i.v. intraventricular. Animal
Fixation
Post-fixation (h)
Colchicine
c8 Cl0 c12 C13 C14 C15
1% para-4% para 5% acrolein 5% acrolein 4% para-0.5% gluta 4% para-0.5% gluta 4% para
72 (same fixative) 2 (same fixative) 15 (4% para) 39 (same fixative) 42 (same fixative) 96 (same fixative)
topical topical topical i.v.
were taken of these regions,
T h e d i s t r i b u t i o n o f S P - p o s i t i v e n e u r o n s in p a r a f f i n s e c t i o n s as w e l l as t h e e f f e c t s o f c o l c h i c i n e w e r e s i m i l a r to t h o s e o b s e r v e d o n v i b r a t o m e - c u t m a t e r i a l 7, w i t h two n o t a b l e e x c e p t i o n s : (1) l a r g e S P - p o s i t i v e n e u r o n s w e r e m o r e n u m e r o u s in p a r a f f i n s e c t i o n s t h a n in v i b r a t o m e s e c t i o n s a n d w e r e also o b s e r v e d in l a y e r lI; a n d (2) in t h e p r e s e n t m a t e r i a l S P - p o s i t i v e n e u r o n s w e r e easily classifiable
in s m a l l a n d
darkly stained
neurons
vs.
l a r g e a n d lightly s t a i n e d n e u r o n s (Fig. 1). T h e d i s t r i b u t i o n o f G l u - p o s i t i v e n e u r o n s was s i m i l a r to t h a t p r e v i G l u a n t i s e r a w e r e e x a m i n e d u n d e r t h e 40 × o b j e c t i v e
ously r e p o r t e d 3 5. Pairs o f s e c t i o n s f r o m all a n i m a l s
o f a L e i t z m i c r o s c o p e . R e g i o n s o f SI w e r e s e l e c t e d for
w e r e s t u d i e d for c o - l o c a l i z a t i o n , a n d in e a c h c a s e s o m e
d a t a analysis w h e n c o n v e n i e n t l a n d m a r k s , m o s t l y b l o o d
neurons containing both antigens were observed. Nine
vessels, w e r e easily r e c o g n i z a b l e o n t w o a d j a c e n t sec-
p a i r s o f 5 p~m t h i c k a d j a c e n t p a r a f f i n s e c t i o n s f r o m cats
A
"
,h~
~~
m,. - ~
~
Ib
Fig. 1. Two 5 p~m thick adjacent paraffin sections from the first somatic sensory cortex of cat C8 processed for the visualization of Glu- (A) and SP- (B) immunoreactivity. B shows that SP-positive neurons can be either small and darkly stained (cell 1) or large and lightly stained (cell 2). Asterisks indicate the same blood vessels in the two sections, whereas arrow in A and B show two examples of GIu/SP co-localization: one in a pyramidal neuron, and one in an unidentified neuron. Note that both cells are large SP-positive neurons. Bar = 50 ,~m.
142 C8 and C12 were studied in detail, and they represent the basis of the present report. In these two cases, no differences were observed, even though they were perfused with different fixatives. Two hundred eighty two SP-positive neurons were sampled as previously described 3. Layer I was not included in the counts since, although it contained SP-positive neurons, it hardly contained any Glu-positive neurons. The vast majority of SP-immunoreactive neurons were not immunoreactive for Glu, but in all pairs of sections examined, some neurons displayed both SP and Glu immunoreactivity (Fig. 1). Out of 282 SP-positive neurons studied for co-localization, 21 (7.4%: all in layers lI-111 and V) were also Glu-positive. In some cases, it was impossible to define the morphology of immunoreactive neurons; in many cases, however, the presence of a prominent apical dendrite was clear enough to allow identification of these neurons as pyramidal cells (Fig. 1). All double-labelled neurons are amongst the large, lightly stained SP-positive cells (Fig. 1). Controls were performed and included both method and antibody specificity. In the first case, the primary antiserum was omitted and the sections processed as those incubated in the antiSP and antiGlu serum; in the second case, the primary antibody was incubated overnight with an excess of antigen (see Conti et al. 3'7, for details). In all cases there were no immunopositive neurons. The significance of Glu-positive neurons has been discussed in previous papers 3'5"~, and the relationship between cytoplasmatic labelling and the transmitter pool has recently been dealt with 2 and is currently under further investigation ~'. For the purpose of the present paper, we will therefore assume that Glu-immunoreactivity in cortical cell bodies is a marker of Glu-ergic neurons (see also Zhang et al. 2L). In the last ten years many groups have documented the presence of SP-positive neurons in the cerebral cortex, and all reports clearly indicate that the vast majority of SP-positive neurons belong to the nonpyramidal class of cortical neurons (see Conti et al. 7, for data and literature). It has also been shown that many SP-positive neurons are a sub-class of cortical GABAergic neurons 13'~4'~'J. Furthermore, it has been reported that few Glu-positive neurons also contain G A B A 3. Thus, the problem arises whether the neurons containing both Glu and SP immunoreactivity are those cells that stain for G A B A too. This possibility is unlikely since G A B A is contained in the small SP-positive neurons H (which do not co-localize Glu; present observations), whereas Glu is contained in the large SP-positive cells (which do not co-localize G A B N 4 ) . Furthermore, G l u / G A B A neurons are the largest G A B A
cells 3, whereas SP is contained only in small GABApositive neurons ~4. In many cases, neurons immunoreactive for both Glu and SP were easily classifiable as pyramids (scc upper arrow in Fig. 1); in some cases, however, thc poor staining of the dendritic trees of immunoreactivc elements did not allow the precise identification of the morphological type of Glu/SP-positive neurons. Several lines of evidence, however, suggest that these Glu/SP-positive neurons too are pyramidal cells: (1) virtually all Glu-positive cells present in layers II1 and V are pyramidal neurons 2 ~; (2) most Glu-positive non-pyramidal neurons are spiny stellate cells s, which are exceptionally found in layers II1 and V of the somatic sensory cortex 12'~, and do not stain for SpI4; (3) SP-positive pyramidal neurons are present exclusively in layers I I - I I I and V 7 From previous studies ~~'14'~9 and from the present evidence it appears that neocortical SP-positive neurons fall in (at least) two major classes. The first class is composed of non-pyramidal neurons present in all cortical layers; these neurons have a small perikaryal size and most of them contain GABA. This class is by far quantitatively more relevant. The second class of SP-positive neocortical neurons is made up by few pyramidal neurons in layers II-III and V; their cell body is larger than that of neurons belonging to the former class. Many of these neurons contain Glu. Co-localization of Glu and SP has been demonstrated by immunocytochemistry in dorsal root ganglion neurons ~, in primary afferent terminals in the superficial laminae of the spinal cord ~, and in medullo-spinal neurons 2°, suggesting that a fast synaptic transmitter might be co-released with a slow synaptic modulator. The present demonstration of Glu-SP co-localization in few neocortical pyramidal neurons suggests that this p h e n o m e n o n is not restricted to the spinal cord, but is more widespread involving at least the cerebral cortex. The functional significance of G I u / S P co-localization (and, possibly, co-release) is still elusive. However, the recent demonstration that SP modulate the basal and electrically induced release of endogenous Glu in the rat spinal cord-dorsal root ganglion slice preparation 15 suggests that SP might presynaptically modulate excitatory amino acid transmission in the cerebral cortex. Alternative (or complementary) mechanisms can be envisaged on the basis of recent studies of the molecular biology of Glu receptors. It has been shown that ligand-gated ion channels, such as the ionotropic glutamate receptors, are modulated by protein phosphorylation m'~6'w. Since glutamate receptors have several consensus sites for phosphorylation (particularly
143
the intracellular loop of the GIuR 6 kainate receptor'~), it is possible that SP might modulate postsynaptically the functional status of glutamate receptors by acting directly at their t r a n s m e m b r a n e domain. This work was supported by funds from Regione Marche (031118) and C.N.R. (91.00371.CTO4). 1 Battaglia, G. and Rustioni, A., Coexistence of glutamate and substance P in dorsal root ganglion neurons of the rat and monkey., Y. Comp. Neurol.. 277 (1988) 302-312. 2 Conti, F., Toward the anatomical identification of glutamatergic neurons and synapses in the cerebral cortex. In B.S. Meldrum, F. Moroni, R.P. Simon, and J.N. Woods (Eds), Excitatoo, Amino Acid, Raven Press, New York, 1991, pp. 45-53. 3 Conti, F., Rustioni, A.. Petrusz, P. and Towle, A.C., Glutamatepositive neurons in the somatic sensory cortex of rats and monkeys, J. Neurosci., 7 (1987) 485-510. 4 Conti, F., Fabri, M. and Manzoni, T., Glutamate-positive corticocortical neurons in the somatic sensory areas 1 and II of cats, J. Neurosci.. 8 (1988) 2948-2960. 5 Conti, F., DeFelipe, l., Farinas, 1. and Manzoni, T., Glutamatepositive neurons and axon terminals in cat sensory, cortex: a correlative light and electron microscopic study, J. Comp. NeuroL, 290 (1989) 141-153. 6 Conti, F. and Minelli, A., Phosphate-activated glutaminase (PAG) inhibitors abolish glutamate-immunoreactivity in the rat cerebral cortex. I1. The effects of 6-diazo-5-oxo-L-norleucine, Eur. J. Neurosci.. Suppl. 4 (19911 2187. 7 Conti, F., DeBiasi, S., Fabri, M., Abdullah, L., Manzoni, T. and Petrusz, P., Substance P-containing pyramidal neurons in the cat somatic sensory cortex, J. Comp. Neurol., 322 (19921 136 148. 8 DeBiasi. S. and Rustioni, A., Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord, Proc. Natl. Acad. Sci. USA, 85 (t988) 782(/-7824. 9 Egebjerg, J., Bettler B., tlermans-Borgmeyer, 1. and Heinemann, S., Cloning of a e D N A for a glutamate receptor subunit activated by kainate but not A M P A , Nature, 351 (1991) 745-748. 10 Grcengard, P., Jen J.. Nairn, A.C. and Stevens, C.F., Enhancement of the glutamate response by c A M P - d e p e n d e n t protein kinase in hippocampal neurons, Science, 253 ( 1991 ) 1135- 1138.
11 Hepler, J.R., Toomim, C., McCarthy, K.D.. Conti, F.. Banaglia, G., Rustioni, A. and Petrusz, P., Characterization of antisera to glutamate and aspartate, Z Histochem. ~),tochem., 36 (1988) 13 22. 12 Jones, E.G., Varieties and distribution of non-pyramidal ceils in the somatic sensory cortex of the squirrel monkey, J. ('omp. Neurol., 160 (1975) 205-268. 13 Jones, E.G., Hendry, S,H.C. and DeFelipe, J., GABA-peptidc neurons of the primate cerebral cortex. A limited cell class. In E.G. Jones and A. Peters (Eds.), Cerebral Cortex, Vol. 6, Further Aspects of ('ortical Function, including Hippocampus, Plenum, New York, 1987, pp. 237-266. 14 Jones, E.G., DeFelipe, J., Hendry, S.H.C. and Maggio, J.E., A study of tachykinin-immunoreaetive neurons in the monkey cerebral cortex, J. Neurosci., 8 (1988) 1206-1224. 15 Kangrga, 1. and Randic, M. Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice, J. M'urosci., 1{) (199(I) 2026-2038. 16 Knapp, A.G., Schmidt, K.F. and Dowling, J.E., Dopamine modulates the kinetics of ion channels gated by excitatory amino acids in retinal horizontal cells, Proc. Natl. dead. Sci. USA. 87 (1990) 767-771. 17 Liman, E.R., Knapp, A.G. and Dowling, J.E., E n h a n c e m e n t of kainate-gated currents in retinal horizontal cells by cAMP-dependent protein kinase, Brain Res., 487 (19891 399 402. 18 Lund, J.S., Spiny stellate neurons. In A. Peters and E.G. Jones (Eds.) Cerebral Cortex, Vol. l, Celhdar Components of the Cerebral Cortex, Plenum, New York, pp. 255-308. 19 Penny, G.R., Afsharpou, and Kitai. S.T., Substance P-immunoreactive neurons in the neocortex of the rat: A subset of the glutamic acid decarboxylase-immunoreactive neurons, Neurosci. Lett.. 65 (1986) 53 59. 20 Nicholas. A.P., Pieribone, V.A., Arvidsson, U. and Hi-;kfelt, T., Serotonin-, substance P-, and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primatc. Neuroscience, 48 (1992) 545 55~L 21 Zhang, N.F., Walberg, F., Laake, I.N., Meldrum, B.S. and Ottersen, O.P., Aspartateqike and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: a light microscopic and semiquantitative electron microscopic study in rat and baboon (Papio anubis), Neuroscience, 38 (199[)) 6l 80.
BRES 25474
Numerous SP-positive pyramidal neurons in cat neocortex are glutamate-positive Fiorenzo Conti, Mara Fabri and Andrea Minelli Institute of Human Physiology, University of Ancona, Ancona (Italy) (Accepted 22 September 1992)
Key words': Glutamate; Substance P; Pyramidal neuron; Cerebral cortex
An immunocytochemical technique that allows visualization of two antigens in the same neuron was used to verify the possibility that some neocortical pyramidal neurons contain both glutamate (Glu) and substance P (SP) immunoreactivity. The results show that a large fraction of SP-positive pyramidal neurons are also Glu-positive, and indicate that in a small population of cortical neurons a fast excitatory synaptic transmitter and a slow peptidic modulator coexist.
A large body of anatomical, physiological, biochemical, and pharmacological evidence suggests that the excitatory acidic amino acid glutamate (Glu) is the neurotransmitter of a large fraction of pyramidal neurons, the most distinctive cells of the mammalian cerebral cortex 2. We have recently provided anatomical evidence that few pyramidal cells in the first somatic sensory cortex (SI) of cats are immunoreactive for the undecapeptide substance P (SP) 7. Glu-positive pyramidal neurons are present in all cortical layers, but are more numerous in layers III (mean cross sectional area, m.c.s.a.: 168 + 54/xm 2) and V (m.c.s.a.: 2 2 2 + 8 2 /zm2) 3. SP-positive pyramidal neurons 7 are present in layers III and V and have a m.c.s.a, of 187 + 40 /zm 2. Therefore, based on the laminar distribution and on the soma size of Glu- and SP-positive pyramidal neurons, the possibility exists that some pyramidal neurons contain both Glu and SP. We have addressed this issue by an immunocytochemical technique that allows visualization of different antigens in thin adjacent paraffin sections. Six adult cats were used in this study (Table I). Four of them received colchicine; they were anaesthetized with ketamine (33 m g / k g i.m.) and mounted on a stereotaxic frame. Under aseptic conditions, the skull was opened over the appropriate region and colchicine ( 1 0 0 / z g / t z l in physiological saline) was injected intra-
ventricularly (8/zl) or topically applied. In these cases, a small piece of gelfoam (15 mm × 20 mm wide) soaked with 100 /zl of colchicine (i.e., 0.5 /~l/mm 2) was applied over the pial surface of a cortical area that included: part of the motor cortex, S1, SI1, and part of areas SIV and 5, as judged by the sulcal pattern. Animals receiving colchicine survived 7-48 h. All animals were perfused under Nembutal anaesthesia (30 m g / k g i.p.) through the ascending aorta with physiological saline followed by different fixatives, all made up with 0.1 M phosphate buffer (pH 7.4; see Table 1). All brains were immediately removed from the skull and post-fixed (Table I). Small blocks of SI were obtained from each brain, rinsed in phosphate buffered saline (PBS), dehydrated in ethanol, cleared in toluene, and embedded in paraffin. Several series of two adjacent sections (5/xm thick) were cut from each paraffin block and mounted on subbed slides. Sections were rehydrated in an ethanol series and rinsed in PBS before immunocytochemical processing. Two antisera were used: an anti-Glu serum ~ and an anti-SP serum whose characterization has been reported elsewhere 7. Of each couple of sections, one was reacted for SP (at a dilution of 1:2,000-1:6,000) and one for Glu (at a dilution of 1 : 6,000-1 : 8,000), according to the protocol previously described ~. Paraffin sections alternately reacted with SP and
Correspondence: F. Conti, Istituto di Fisiologia Umana, Universit~ di Ancona, Via Ranieri, Monte d'Ago. 1-60131 Ancona, Italy. Fax: (39) 71-2204652.
141 TABLE I
tions. P h o t o m i c r o g r a p h s
Summary o[' experimental cases
and neurons containing both antigens were identified.
para, paraformaldehyde; gluta, glutaraldehyde; i.v. intraventricular. Animal
Fixation
Post-fixation (h)
Colchicine
c8 Cl0 c12 C13 C14 C15
1% para-4% para 5% acrolein 5% acrolein 4% para-0.5% gluta 4% para-0.5% gluta 4% para
72 (same fixative) 2 (same fixative) 15 (4% para) 39 (same fixative) 42 (same fixative) 96 (same fixative)
topical topical topical i.v.
were taken of these regions,
T h e d i s t r i b u t i o n o f S P - p o s i t i v e n e u r o n s in p a r a f f i n s e c t i o n s as w e l l as t h e e f f e c t s o f c o l c h i c i n e w e r e s i m i l a r to t h o s e o b s e r v e d o n v i b r a t o m e - c u t m a t e r i a l 7, w i t h two n o t a b l e e x c e p t i o n s : (1) l a r g e S P - p o s i t i v e n e u r o n s w e r e m o r e n u m e r o u s in p a r a f f i n s e c t i o n s t h a n in v i b r a t o m e s e c t i o n s a n d w e r e also o b s e r v e d in l a y e r lI; a n d (2) in t h e p r e s e n t m a t e r i a l S P - p o s i t i v e n e u r o n s w e r e easily classifiable
in s m a l l a n d
darkly stained
neurons
vs.
l a r g e a n d lightly s t a i n e d n e u r o n s (Fig. 1). T h e d i s t r i b u t i o n o f G l u - p o s i t i v e n e u r o n s was s i m i l a r to t h a t p r e v i G l u a n t i s e r a w e r e e x a m i n e d u n d e r t h e 40 × o b j e c t i v e
ously r e p o r t e d 3 5. Pairs o f s e c t i o n s f r o m all a n i m a l s
o f a L e i t z m i c r o s c o p e . R e g i o n s o f SI w e r e s e l e c t e d for
w e r e s t u d i e d for c o - l o c a l i z a t i o n , a n d in e a c h c a s e s o m e
d a t a analysis w h e n c o n v e n i e n t l a n d m a r k s , m o s t l y b l o o d
neurons containing both antigens were observed. Nine
vessels, w e r e easily r e c o g n i z a b l e o n t w o a d j a c e n t sec-
p a i r s o f 5 p~m t h i c k a d j a c e n t p a r a f f i n s e c t i o n s f r o m cats
A
"
,h~
~~
m,. - ~
~
Ib
Fig. 1. Two 5 p~m thick adjacent paraffin sections from the first somatic sensory cortex of cat C8 processed for the visualization of Glu- (A) and SP- (B) immunoreactivity. B shows that SP-positive neurons can be either small and darkly stained (cell 1) or large and lightly stained (cell 2). Asterisks indicate the same blood vessels in the two sections, whereas arrow in A and B show two examples of GIu/SP co-localization: one in a pyramidal neuron, and one in an unidentified neuron. Note that both cells are large SP-positive neurons. Bar = 50 ,~m.
142 C8 and C12 were studied in detail, and they represent the basis of the present report. In these two cases, no differences were observed, even though they were perfused with different fixatives. Two hundred eighty two SP-positive neurons were sampled as previously described 3. Layer I was not included in the counts since, although it contained SP-positive neurons, it hardly contained any Glu-positive neurons. The vast majority of SP-immunoreactive neurons were not immunoreactive for Glu, but in all pairs of sections examined, some neurons displayed both SP and Glu immunoreactivity (Fig. 1). Out of 282 SP-positive neurons studied for co-localization, 21 (7.4%: all in layers lI-111 and V) were also Glu-positive. In some cases, it was impossible to define the morphology of immunoreactive neurons; in many cases, however, the presence of a prominent apical dendrite was clear enough to allow identification of these neurons as pyramidal cells (Fig. 1). All double-labelled neurons are amongst the large, lightly stained SP-positive cells (Fig. 1). Controls were performed and included both method and antibody specificity. In the first case, the primary antiserum was omitted and the sections processed as those incubated in the antiSP and antiGlu serum; in the second case, the primary antibody was incubated overnight with an excess of antigen (see Conti et al. 3'7, for details). In all cases there were no immunopositive neurons. The significance of Glu-positive neurons has been discussed in previous papers 3'5"~, and the relationship between cytoplasmatic labelling and the transmitter pool has recently been dealt with 2 and is currently under further investigation ~'. For the purpose of the present paper, we will therefore assume that Glu-immunoreactivity in cortical cell bodies is a marker of Glu-ergic neurons (see also Zhang et al. 2L). In the last ten years many groups have documented the presence of SP-positive neurons in the cerebral cortex, and all reports clearly indicate that the vast majority of SP-positive neurons belong to the nonpyramidal class of cortical neurons (see Conti et al. 7, for data and literature). It has also been shown that many SP-positive neurons are a sub-class of cortical GABAergic neurons 13'~4'~'J. Furthermore, it has been reported that few Glu-positive neurons also contain G A B A 3. Thus, the problem arises whether the neurons containing both Glu and SP immunoreactivity are those cells that stain for G A B A too. This possibility is unlikely since G A B A is contained in the small SP-positive neurons H (which do not co-localize Glu; present observations), whereas Glu is contained in the large SP-positive cells (which do not co-localize G A B N 4 ) . Furthermore, G l u / G A B A neurons are the largest G A B A
cells 3, whereas SP is contained only in small GABApositive neurons ~4. In many cases, neurons immunoreactive for both Glu and SP were easily classifiable as pyramids (scc upper arrow in Fig. 1); in some cases, however, thc poor staining of the dendritic trees of immunoreactivc elements did not allow the precise identification of the morphological type of Glu/SP-positive neurons. Several lines of evidence, however, suggest that these Glu/SP-positive neurons too are pyramidal cells: (1) virtually all Glu-positive cells present in layers II1 and V are pyramidal neurons 2 ~; (2) most Glu-positive non-pyramidal neurons are spiny stellate cells s, which are exceptionally found in layers II1 and V of the somatic sensory cortex 12'~, and do not stain for SpI4; (3) SP-positive pyramidal neurons are present exclusively in layers I I - I I I and V 7 From previous studies ~~'14'~9 and from the present evidence it appears that neocortical SP-positive neurons fall in (at least) two major classes. The first class is composed of non-pyramidal neurons present in all cortical layers; these neurons have a small perikaryal size and most of them contain GABA. This class is by far quantitatively more relevant. The second class of SP-positive neocortical neurons is made up by few pyramidal neurons in layers II-III and V; their cell body is larger than that of neurons belonging to the former class. Many of these neurons contain Glu. Co-localization of Glu and SP has been demonstrated by immunocytochemistry in dorsal root ganglion neurons ~, in primary afferent terminals in the superficial laminae of the spinal cord ~, and in medullo-spinal neurons 2°, suggesting that a fast synaptic transmitter might be co-released with a slow synaptic modulator. The present demonstration of Glu-SP co-localization in few neocortical pyramidal neurons suggests that this p h e n o m e n o n is not restricted to the spinal cord, but is more widespread involving at least the cerebral cortex. The functional significance of G I u / S P co-localization (and, possibly, co-release) is still elusive. However, the recent demonstration that SP modulate the basal and electrically induced release of endogenous Glu in the rat spinal cord-dorsal root ganglion slice preparation 15 suggests that SP might presynaptically modulate excitatory amino acid transmission in the cerebral cortex. Alternative (or complementary) mechanisms can be envisaged on the basis of recent studies of the molecular biology of Glu receptors. It has been shown that ligand-gated ion channels, such as the ionotropic glutamate receptors, are modulated by protein phosphorylation m'~6'w. Since glutamate receptors have several consensus sites for phosphorylation (particularly
143
the intracellular loop of the GIuR 6 kainate receptor'~), it is possible that SP might modulate postsynaptically the functional status of glutamate receptors by acting directly at their t r a n s m e m b r a n e domain. This work was supported by funds from Regione Marche (031118) and C.N.R. (91.00371.CTO4). 1 Battaglia, G. and Rustioni, A., Coexistence of glutamate and substance P in dorsal root ganglion neurons of the rat and monkey., Y. Comp. Neurol.. 277 (1988) 302-312. 2 Conti, F., Toward the anatomical identification of glutamatergic neurons and synapses in the cerebral cortex. In B.S. Meldrum, F. Moroni, R.P. Simon, and J.N. Woods (Eds), Excitatoo, Amino Acid, Raven Press, New York, 1991, pp. 45-53. 3 Conti, F., Rustioni, A.. Petrusz, P. and Towle, A.C., Glutamatepositive neurons in the somatic sensory cortex of rats and monkeys, J. Neurosci., 7 (1987) 485-510. 4 Conti, F., Fabri, M. and Manzoni, T., Glutamate-positive corticocortical neurons in the somatic sensory areas 1 and II of cats, J. Neurosci.. 8 (1988) 2948-2960. 5 Conti, F., DeFelipe, l., Farinas, 1. and Manzoni, T., Glutamatepositive neurons and axon terminals in cat sensory, cortex: a correlative light and electron microscopic study, J. Comp. NeuroL, 290 (1989) 141-153. 6 Conti, F. and Minelli, A., Phosphate-activated glutaminase (PAG) inhibitors abolish glutamate-immunoreactivity in the rat cerebral cortex. I1. The effects of 6-diazo-5-oxo-L-norleucine, Eur. J. Neurosci.. Suppl. 4 (19911 2187. 7 Conti, F., DeBiasi, S., Fabri, M., Abdullah, L., Manzoni, T. and Petrusz, P., Substance P-containing pyramidal neurons in the cat somatic sensory cortex, J. Comp. Neurol., 322 (19921 136 148. 8 DeBiasi. S. and Rustioni, A., Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord, Proc. Natl. Acad. Sci. USA, 85 (t988) 782(/-7824. 9 Egebjerg, J., Bettler B., tlermans-Borgmeyer, 1. and Heinemann, S., Cloning of a e D N A for a glutamate receptor subunit activated by kainate but not A M P A , Nature, 351 (1991) 745-748. 10 Grcengard, P., Jen J.. Nairn, A.C. and Stevens, C.F., Enhancement of the glutamate response by c A M P - d e p e n d e n t protein kinase in hippocampal neurons, Science, 253 ( 1991 ) 1135- 1138.
11 Hepler, J.R., Toomim, C., McCarthy, K.D.. Conti, F.. Banaglia, G., Rustioni, A. and Petrusz, P., Characterization of antisera to glutamate and aspartate, Z Histochem. ~),tochem., 36 (1988) 13 22. 12 Jones, E.G., Varieties and distribution of non-pyramidal ceils in the somatic sensory cortex of the squirrel monkey, J. ('omp. Neurol., 160 (1975) 205-268. 13 Jones, E.G., Hendry, S,H.C. and DeFelipe, J., GABA-peptidc neurons of the primate cerebral cortex. A limited cell class. In E.G. Jones and A. Peters (Eds.), Cerebral Cortex, Vol. 6, Further Aspects of ('ortical Function, including Hippocampus, Plenum, New York, 1987, pp. 237-266. 14 Jones, E.G., DeFelipe, J., Hendry, S.H.C. and Maggio, J.E., A study of tachykinin-immunoreaetive neurons in the monkey cerebral cortex, J. Neurosci., 8 (1988) 1206-1224. 15 Kangrga, 1. and Randic, M. Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice, J. M'urosci., 1{) (199(I) 2026-2038. 16 Knapp, A.G., Schmidt, K.F. and Dowling, J.E., Dopamine modulates the kinetics of ion channels gated by excitatory amino acids in retinal horizontal cells, Proc. Natl. dead. Sci. USA. 87 (1990) 767-771. 17 Liman, E.R., Knapp, A.G. and Dowling, J.E., E n h a n c e m e n t of kainate-gated currents in retinal horizontal cells by cAMP-dependent protein kinase, Brain Res., 487 (19891 399 402. 18 Lund, J.S., Spiny stellate neurons. In A. Peters and E.G. Jones (Eds.) Cerebral Cortex, Vol. l, Celhdar Components of the Cerebral Cortex, Plenum, New York, pp. 255-308. 19 Penny, G.R., Afsharpou, and Kitai. S.T., Substance P-immunoreactive neurons in the neocortex of the rat: A subset of the glutamic acid decarboxylase-immunoreactive neurons, Neurosci. Lett.. 65 (1986) 53 59. 20 Nicholas. A.P., Pieribone, V.A., Arvidsson, U. and Hi-;kfelt, T., Serotonin-, substance P-, and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primatc. Neuroscience, 48 (1992) 545 55~L 21 Zhang, N.F., Walberg, F., Laake, I.N., Meldrum, B.S. and Ottersen, O.P., Aspartateqike and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: a light microscopic and semiquantitative electron microscopic study in rat and baboon (Papio anubis), Neuroscience, 38 (199[)) 6l 80.
