nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin,...

8
Brain Research 863 (2000) 205–212 www.elsevier.com / locate / bres Research report Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells * John F. Smiley , John P. McGinnis, Daniel C. Javitt The Program in Cognitive Neurology and Schizophrenia, Nathan Kline Institute, Bldng. 39, 140 Old Orangeburg Rd., Orangeburg, NY 10962, USA Accepted 7 February 2000 Abstract Neuronal nitric oxide synthase (nNOS) is expressed most densely in two types of cortical interneurons. Large nitric oxide (LNOS) interneurons express dense somatostatin- and neuropeptide Y-immunoreactivity, whereas small (SNOS) interneurons are calbindin- immunoreactive. We used double labeling methods in several areas of the monkey cerebral cortex to determine how precisely these neurochemical labels define the two types of nNOS interneurons. The LNOS cells were essentially always (.99%) immunoreactive for somatostatin and neuropeptide Y, but did not express calbindin. The LNOS cells comprised about 30% of the somatostatin cells and about 60% of the neuropeptide Y cells. The SNOS cells were nearly always (87–98%) calbindin-immunoreactive, and were rarely or never labeled with antibodies to somatostatin or neuropeptide Y. The SNOS cells accounted for about 20% of all of the calbindin cells. The findings demonstrate that the two types of nNOS cells can be distinguished by antibodies to calbindin, somatostatin and neuropeptide Y, but none of these markers is found exclusively in nNOS cells. Nevertheless, neuropeptide Y-immunoreactivity provides a useful marker for LNOS cells, because it is very dense in these cells and only light in the interneurons that lack nNOS. 2000 Elsevier Science B.V. All rights reserved. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Other neurotransmitters Keywords: Nitric oxide; Calbindin; Somatostatin; Neuropeptide Y; GABA; Primate 1. Introduction precisely these features describe the nNOS-containing cells, because there is evidence that both somatostatin and The neurons with the most obvious nNOS-immuno- NPY are present in other neurons besides nNOS cells reactivity in the cerebral cortex are those which also [3,6,18–21]. contain intense immunoreactivity for the neuropeptides More recently it was shown that nNOS is also present in somatostatin and neuropeptide Y (NPY) [25,26]. In the rat a smaller and morphologically distinct type of interneuron cerebral cortex many somatostatin-immunoreactive inter- in the cortex of monkeys, but apparently not in that of rats neurons are Martinotti and wide arbor cells which have [1,12,27,28]. In contrast to the large nNOS cells, these characteristic physiological responses [15,16]. Axons im- small nNOS interneurons express the calcium binding munoreactive for somatostatin or NPY synapse mainly on protein calbindin [27,28]. In the monkey cortex it is the distal dendrites of pyramidal cells in both the rat and unclear whether the small nNOS cells express somatostatin monkey cortex, suggesting that this may be a preferred and NPY, as do the large nNOS cells. The purpose of this target of nNOS axons [8,13]. However, it is not clear how study in the monkey cortex was to determine how reliably immunoreactivity for somatostatin, neuropeptide Y, and calbindin can be used to differentiate the large and small *Corresponding author. Tel.: 11-914-398-6601; fax: 11-914-398- nNOS cells, and to determine to what extent these markers 5531. E-mail address: [email protected] (J.F. Smiley) are found in other neurons besides the nNOS interneurons. 0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993(00)02136-3

Upload: john-f-smiley

Post on 14-Sep-2016

214 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

Brain Research 863 (2000) 205–212www.elsevier.com/ locate /bres

Research report

Nitric oxide synthase interneurons in the monkey cerebral cortex aresubsets of the somatostatin, neuropeptide Y, and calbindin cells

*John F. Smiley , John P. McGinnis, Daniel C. JavittThe Program in Cognitive Neurology and Schizophrenia, Nathan Kline Institute, Bldng. 39, 140 Old Orangeburg Rd., Orangeburg, NY 10962, USA

Accepted 7 February 2000

Abstract

Neuronal nitric oxide synthase (nNOS) is expressed most densely in two types of cortical interneurons. Large nitric oxide (LNOS)interneurons express dense somatostatin- and neuropeptide Y-immunoreactivity, whereas small (SNOS) interneurons are calbindin-immunoreactive. We used double labeling methods in several areas of the monkey cerebral cortex to determine how precisely theseneurochemical labels define the two types of nNOS interneurons. The LNOS cells were essentially always (.99%) immunoreactive forsomatostatin and neuropeptide Y, but did not express calbindin. The LNOS cells comprised about 30% of the somatostatin cells and about60% of the neuropeptide Y cells. The SNOS cells were nearly always (87–98%) calbindin-immunoreactive, and were rarely or neverlabeled with antibodies to somatostatin or neuropeptide Y. The SNOS cells accounted for about 20% of all of the calbindin cells. Thefindings demonstrate that the two types of nNOS cells can be distinguished by antibodies to calbindin, somatostatin and neuropeptide Y,but none of these markers is found exclusively in nNOS cells. Nevertheless, neuropeptide Y-immunoreactivity provides a useful markerfor LNOS cells, because it is very dense in these cells and only light in the interneurons that lack nNOS. 2000 Elsevier Science B.V.All rights reserved.

Themes: Neurotransmitters, modulators, transporters, and receptors

Topics: Other neurotransmitters

Keywords: Nitric oxide; Calbindin; Somatostatin; Neuropeptide Y; GABA; Primate

1. Introduction precisely these features describe the nNOS-containingcells, because there is evidence that both somatostatin and

The neurons with the most obvious nNOS-immuno- NPY are present in other neurons besides nNOS cellsreactivity in the cerebral cortex are those which also [3,6,18–21].contain intense immunoreactivity for the neuropeptides More recently it was shown that nNOS is also present insomatostatin and neuropeptide Y (NPY) [25,26]. In the rat a smaller and morphologically distinct type of interneuroncerebral cortex many somatostatin-immunoreactive inter- in the cortex of monkeys, but apparently not in that of ratsneurons are Martinotti and wide arbor cells which have [1,12,27,28]. In contrast to the large nNOS cells, thesecharacteristic physiological responses [15,16]. Axons im- small nNOS interneurons express the calcium bindingmunoreactive for somatostatin or NPY synapse mainly on protein calbindin [27,28]. In the monkey cortex it isthe distal dendrites of pyramidal cells in both the rat and unclear whether the small nNOS cells express somatostatinmonkey cortex, suggesting that this may be a preferred and NPY, as do the large nNOS cells. The purpose of thistarget of nNOS axons [8,13]. However, it is not clear how study in the monkey cortex was to determine how reliably

immunoreactivity for somatostatin, neuropeptide Y, andcalbindin can be used to differentiate the large and small*Corresponding author. Tel.: 11-914-398-6601; fax: 11-914-398-nNOS cells, and to determine to what extent these markers5531.

E-mail address: [email protected] (J.F. Smiley) are found in other neurons besides the nNOS interneurons.

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02136-3

Page 2: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

206 J.F. Smiley et al. / Brain Research 863 (2000) 205 –212

2. Materials and methods the other with a biotinylated secondary antibody linked tostreptavidin–Texas Red. Control experiments, which omit-

Two Macaca nemestrina and one Macaca fascicularis ted the primary antibodies, were routinely processed withmonkeys were used. The nemestrina monkeys were re- all single and double labeling procedures.ceived from the Regional Primate Research Center at the Analysis of the cortical distribution of single and doubleUniversity of Washington, and the fascicularis monkey labeled cells was done with an X–Y plotter electronicallywas housed at the Nathan Kline Institute in accordance coupled to a microscope stage. Quantifications of thewith NIH guidelines. All monkeys were transcardially extent of double labeling were done by systematicallyperfused using 4% paraformaldehyde as a fixative as sampling labeled cells across the depth of the cortical graypreviously described [24]. The right cerebral hemispheres matter and the infracortical white matter. All doublewere cryoprotected and sectioned at 40 or 80 mm thickness labeling experiments were done in at least two monkeys,with a sliding microtome, or alternatively blocks of tissue and comparisons between animals showed nearly identicalfrom the left hemispheres were sectioned on a vibratome at results. Measurements of the sizes of labeled cells were50 mm thickness and stored frozen in buffered sucrose. taken from microscopic video images using the NIH ImageTissue processed in either way gave comparable results in software (written by Wayne Rasband). Measurements areimmunohistochemical and histochemical procedures. expressed as mean6standard deviation, with n 5 50 to 60

Every 10th or 20th section through the right hemisphere for each cell type.was processed for NADPHd histochemistry and Nisslstaining. In one monkey, an additional series of sectionswas processed for nNOS-immunocytochemistry. All dou- 3. Resultsble labeling fluorescence experiments were processed infive areas of cerebral cortex, which included two areas of Two distinct types of interneurons were nNOS-immuno-association cortex (the principle sulcus of the prefrontal reactive, and these are referred to as LNOS and SNOScortex and the superior temporal sulcus), two areas of neurons (Fig. 1A and B). The LNOS cells were largesensory or sensory /motor cortex (the primary visual cortex (196666 m2) densely labeled multipolar or bipolar cellsfrom the calcarine sulcus, and the sensory and motor which typically had two to six thick dendrites projectingcortex from the central sulcus from the dorso-lateral brain from their cell body. They were especially abundant in theconvexity) and one area of paralimbic cortex (the para- infracortical white matter, and they were also presenthippocampal gyrus). throughout the cortical gray matter (Fig. 2B). The SNOS

Immunolabeling of nNOS was visualized either with a cells were consistently smaller (65616 m2) and usuallymouse monoclonal antibody or with a rabbit polyclonal more lightly labeled than the LNOS cells. They had fineantibody, both from RBI (Natick, MA, USA) used at diameter dendrites which emerged from multiple sites on1:2000 and 1:10,000, respectively. Somatostatin-14-im- their approximately round cell bodies (Fig. 1A and B). Themunoreactivity was visualized with a rabbit polyclonal SNOS cells were numerous in layers II–V, with a slightlyantibody from DiaSorin (Clearwater, MN, USA) used at greater density in layer II. They were less numerous in1:1500. Neuropeptide Y was visualized with a rabbit layer VI, sparsely distributed in the infracortical whitepolyclonal antibody from RBI used at a final concentration matter and only occasionally found in layer I (Fig. 2A).of 1:10,000. The monoclonal anti-calbindin antibody was The density of SNOS cells was seven to nine times that ofpurchased from RBI and used at 1:2000, and was char- LNOS cells, in single sections through the gray and whiteacterized by Celio et al. [5]. The monoclonal anti-parval- matter of the lateral temporal lobe (n 5 3 sections). Inspec-bumin antibody was purchased from RBI, used at 1:5000, tion of coronal sections throughout the cerebral hemisphereand was characterized by Celio et al. [4]. Calretinin was did not reveal obvious variations in the distribution ofvisualized with a mouse monoclonal antibody from SNOS and LNOS cells across cytoarchitectonic areas (e.g.,Chemicon (Temucula, CA, USA) used at 1:2000. Fig. 2).

Immunolabeling was done as previously described [24].For bright field microscopy, tissue was processed using the 3.1. Double labeling of nNOS and neuropeptidesavidin–biotin–peroxidase complex (ABC) method, usingdiaminobenzidine as a chromogen. As previously de- Double labeling results were equivalent in the fivescribed, some immunolabeled sections were silver inten- cortical areas sampled in each of two monkeys. Immuno-sified to increase the contrast, and some were subsequently fluorescence experiments combined antibodies to nNOSlabeled for NADPHd histochemistry [24]. Double labeling with antibodies to NPY or somatostatin. These experimentsimmunofluorescence experiments always combined pri- demonstrated that essentially all (.99%) of the LNOSmary antibodies from different species, and these were cells contained dense immunoreactivity for both of theseconcurrently applied to the tissue for 2 days. One of the neuropeptides (Fig. 3A–D). In contrast, less than 2% ofprimary antibodies was visualized with a secondary anti- the SNOS cells contained NPY, and less than 5% containedbody conjugated to fluorescein isothiocyanate (FITC), and somatostatin. It is possible that all of the SNOS cells lack

Page 3: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

J.F. Smiley et al. / Brain Research 863 (2000) 205 –212 207

Fig. 1. Distinct types of interneurons express nNOS, somatostatin, and NPY in the monkey neocortex. (A) Immunoreactivity for nNOS is seen in two typesof interneurons. The LNOS cells (arrow) were the most densely labeled. The SNOS cells (arrowhead) were more lightly labeled and more numerous. (B)The appearance of cells labeled with NADPHd histochemistry was similar to nNOS-immunoreactivity. The labeling was more intense in LNOS cells(straight arrows) compared to the more numerous SNOS cells (arrowhead). (C) NPY-immunoreactivity was found in two distinct cell types. The large cells(straight arrow) were identical to the LNOS cells. Smaller and more lightly labeled cells were also present (curved arrow) and these did not express nNOS(see text). (D) Somatostatin-immunoreactivity was also densely expressed in LNOS cells (straight arrow). Numerous smaller somatostatin cells were alsopresent (curved arrow). Scale bar 20 mm ((A)–(D)).

Page 4: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

208 J.F. Smiley et al. / Brain Research 863 (2000) 205 –212

Fig. 2. The SNOS and LNOS cells were evenly distributed across cytoarchitectonic areas. An example of their distribution is shown in a coronal sectionthrough the temporal lobe. The inset shows the level of section of the temporal lobe. Each dot indicates a single cell. (A) The SNOS cells are especiallyabundant in layers II–V, and less numerous in layer VI. They were occasionally found in layer I and in the white matter. The borders of the primaryauditory cortex (arrows) were identified with parvalbumin-immunoreactivity in an adjacent section. (B) The LNOS cells were especially concentrated in theinfracortical white matter. Cells were labeled with NADPHd histochemistry in 80 mm thick frozen sections. Abbreviations: sts, superior temporal sulcus;phg, parahippocampal gyrus.

Page 5: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

J.F. Smiley et al. / Brain Research 863 (2000) 205 –212 209

Fig. 3. Double labeling immunofluorescence demonstrated that the LNOS and SNOS cells could be differentiated by their expression of neuropeptides andcalbindin. (A,B) Double labeling demonstrated that the LNOS cells (arrow) were densely immunoreactive for NPY. In contrast, the SNOS cells(arrowheads) did not express NPY. (C,D) The LNOS cells (arrow) expressed somatostatin (SOM), whereas the SNOS cells (arrowhead) did not. Additionalsomatostatin cells which did not express nNOS were also present (double arrowhead). (E,F) An LNOS cell (arrow) is seen which does not expresscalbindin (Calb)-immunoreactivity. A nearby SNOS cell (arrowhead) is calbindin-immunoreactive. (G,H) Three SNOS cells (arrowheads) are also labeledwith calbindin-immunoreactivity. Additional calbindin cells (e.g., double arrowhead) which do not express nNOS are also present. (I,J) Somatostatin-immunoreactive cells (arrowhead) were often colocalized with calbindin-immunoreactivity. Additional calbindin cells which lack somatostatin-immuno-reactivity (double arrowhead) are also present. All micrographs were taken with 35 mm color slide film, and were subsequently scanned into a computerand converted to gray scale images. Scale bar 20 mm and applies to all micrographs.

these neuropeptides, but that an occasional LNOS cell was peroxidase labeling and NADPHd histochemistry (i.e., amistakenly identified as an SNOS cell. marker for nNOS) allowed the concurrent visualization of

Double labeling immunofluorescence also demonstrated the lightly immunoreactive NPY cells and the denselythe presence of additional NPY and somatostatin cells immunoreactive NPY cells that also contained thewhich lacked nNOS. The NPY cells which lacked nNOS NADPHd reaction product. The NPY cells which lackedwere lightly NPY-immunoreactive. They were better vis- nNOS were found mainly in layer VI, but were occasion-ualized using the more sensitive immunoperoxidase meth- ally encountered in other layers (Fig. 4C). Their soma sizeod (Fig. 1C). Experiments which combined NPY-immuno- (120629 m2) was smaller than the LNOS cells.

Page 6: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

210 J.F. Smiley et al. / Brain Research 863 (2000) 205 –212

Immunofluorescence experiments also showed thatmany of the somatostatin cells lacked nNOS-immuno-reactivity (Fig. 3C and D). Immunoperoxidase labelingcombined with NADPHd histochemistry showed that 65–74% of the somatostatin cells lacked nNOS. The compara-tively small (83622 m2) somatostatin cells were found inall layers of the cortex, and occasionally in the infracorticalwhite matter (Fig. 4B). Double labeling showed that,unlike the LNOS cells, the additional somatostatin cellswere often calbindin-immunoreactive (Fig. 3I and J).

3.2. Double labeling of nNOS and calcium bindingproteins

Double immunofluorescence of nNOS and calciumbinding proteins demonstrated that 87–98% of the SNOScells were calbindin-immunoreactive, whereas the LNOScells lacked calbindin (Fig. 3E–H). Comparison with thetotal population of calbindin cells indicated that 18–22%of the calbindin cells were nNOS-immunoreactive.

Double labeling of nNOS with calretinin or with parval-bumin did not demonstrate any double labeled cells (datanot shown).

4. Discussion

The findings demonstrate that the two types of nNOSinterneurons can be distinguished by the differential ex-pression of calbindin, somatostatin and NPY, but that thesemarkers are not found exclusively in nNOS cells. Theneurochemical features and distribution of nNOS cells didnot vary across cortical areas, suggesting that these cellsare a constant component of the cortical circuitry.

Some NPY cells did not contain nNOS-immunoreactivi-ty. These cells had light NPY-immunoreactivity comparedto the LNOS cells. In our best preparations, the NPY-positive /nNOS-negative cells were nearly as numerous asthe NPY-positive /nNOS-positive cells. The presence ofmultiple morphological types of NPY cells was previouslynoted in the monkey and human cerebral cortex [6,20].Studies of the rat neocortex also showed that only about

Fig. 4. The distribution of LNOS cells (A), which contained somatostatin half of the NPY cells express nNOS [19,26]. Althoughand NPY, is compared to the distribution of somatostatin (B) and NPY

NPY is not found exclusively in nNOS cells, it is(C) cells that lacked nNOS. In this example, the distribution of theseespecially intense in these cells, and our preliminarydifferent cell types is shown in plots of 50 mm thick vibratome sections

through the superior temporal sulcus. Each dot represents one cell. Tissue experiments with confocal microscopy suggested that mostsections were first labeled for either somatostatin or NPY, using a brown of the nNOS axons in the cortex also contain intenseimmunoperoxidase label, and then processed for NADPHd histochemis- NPY-immunoreactivity. It is therefore possible that NPYtry, which gave a blue reaction product. (A) The LNOS cells were

will provide a useful marker for LNOS axons in futureidentified by their dense blue NADPHd label, which was easily dis-studies.tinguished even in the presence of brown peroxidase labeling for NPY

and somatostatin which was also present. (B) The somatostatin cells that Approximately 70% of the somatostatin cells did notlacked NADPHd labeling were distributed throughout the gray matter and contain nNOS. In contrast to the LNOS/somatostatin cells,occasionally in the infracortical white matter. (C) The NPY cells which many of the remaining somatostatin cells also expressedlacked NADPHd labeling were less abundant than the LNOS cells, and

calbindin. This situation in the monkey is similar to that ofwere usually encountered in layer VI near the border of the white matter.the rat where 10–20% of the somatostatin cells colocalizedThey were occasionally found in other layers of the gray matter and in the

infracortical white matter. with nNOS, and 85% of the somatostatin cells contained

Page 7: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

J.F. Smiley et al. / Brain Research 863 (2000) 205 –212 211

[2] G. Bertini, Z.-C. Peng, M. Bentivoglio, The chemical heterogeneitycalbindin [19,23]. Somatostatin cells in the monkey cortexof cortical interneurons: nitric oxide synthase vs. calbindin andwere previously shown to have heterogeneous mor-parvalbumin immunoreactivity in the rat, Brain Res. Bull. 39 (1996)

phologies [3,21]. 261–266.We visualized calbindin in 87–98% of the SNOS cells. [3] M.J. Campbell, D.A. Lewis, R. Benoit, J.H. Morrison, Regional

It is possible that all SNOS cells contain calbindin, but we heterogeneity in the distribution of somatostatin-28- andsomatostatin-28(1–12)-immunoreactive profiles in monkey neocor-cannot exclude the presence of a small subpopulationtex, J. Neurosci. 7 (1987) 1133–1144.which lacks calbindin. We estimated that the SNOS cells

[4] M.R. Celio, W. Baier, L. Scharer, P.A. de Viragh, C. Gerday,comprised approximately 20% of all of the calbindin cells. Monoclonal antibodies directed against the calcium binding proteinThe finding in the monkey that the SNOS cells contain parvalbumin, Cell Calcium 9 (1988) 81–86.calbindin, and that they had non-pyramidal morphologies, [5] M.R. Celio, W. Baier, L. Scharer, H.J. Gregersen, P.A. de Viragh,

A.W. Norman, Monoclonal antibodies directed against the calciumsuggests that they are GABAergic interneurons. Previousbinding protein calbindin D-28k, Cell Calcium 11 (1990) 599–602.colocalization studies also concluded that nearly all of the

[6] V. Chan-Palay, C. Kohler, U. Haesler, W. Lang, G. Yasargh,SNOS cells express calbindin and GABA [27,28].Distribution of neurons and axons immunoreactive with antisera

In the rat cortex, a cell type corresponding to the SNOS against neuropeptide Y in the normal human hippocampus, J. Comp.cells has not been demonstrated [26,27]. Double labeling Neurol. 248 (1986) 360–375.

[7] F. Conde, J.S. Lund, D.M. Jacobowitz, K.G. Baimbridge, D.A.studies in the rat cortex also demonstrated additionalLewis, Local circuit neurons immunoreactive for calretinin, calbind-differences. Whereas all of the nNOS cells containing D-28k or parvalbumin in monkey prefrontal cortex: distributionsomatostatin and NPY, some of them also contain calbin-and morphology, J. Comp. Neurol. 341 (1994) 95–116.

din [2,9,11,19,23,26]. [8] A.D. De Lima, J.H. Morrison, Ultrastructural analysis of somatos-At present the synaptic connections of these nNOS cells tatin-immunoreactive neurons and synapses in the temporal and

occipital cortex of the macaque monkey, J. Comp. Neurol. 283are only partially understood. In the rat cortex some(1989) 212–227.somatostatin cells of the deep cortical layers are Martinotti

[9] N.J. Dun, R. Huang, S.L. Dun, U. Forstermann, Infrequent co-cells whose axons ascend to the cortical surface. Theselocalization of nitric oxide synthase and calcium binding proteins

cells have a characteristic spiking pattern [15,16] and are immunoreactivity in rat neocortical neurons, Brain Res. 666 (1994)especially excited by local pyramidal cells, to which they 289–294.

[10] J.R. Gibson, M. Beierlein, B.W. Connors, Two networks of electri-provide inhibitory feedback [10]. In the monkey, at leastcally coupled inhibitory neurons in neocortex, Nature 402 (1999)some of the LNOS cells cortex are Martinotti cells [22]75–79.and electron microscopic studies suggest that nNOS axons

[11] Y. Gonchar, A. Burkhalter, Three distinct families of GABAergicsynapse mainly onto pyramidal cell dendrites [1,8,13]. neurons in rat visual cortex, Cereb. Cortex 7 (1997) 347–358.Therefore, it is possible that the LNOS cells in the monkey [12] T. Hashikawa, M.G. Leggio, R. Hattori, Y. Yui, Nitric oxide synthase

immunoreactivity colocalized with NADPH-diaphorase histochemis-are local feedback neurons like the Martinotti cells in thetry in monkey cerebral cortex, Brain Res. 641 (1994) 341–349.rat. An additional source of input to LNOS cells, at least in

[13] H.C. Hendry, E.G. Jones, P.C. Emson, Morphology, distribution,primates, is from the cholinergic basal forebrain [24]. Aand synaptic relations of somatostatin- and neuropeptide Y-immuno-

similar input may exist in the rat, where cholinergic reactive neurons in rat and monkey cortex, J. Neurosci. 4 (1984)agonists reliably excite somatostatinergic Martinotti cells 2497–2517.

[14] Y. Kawaguchi, Selective cholinergic modulation of cortical[14].GABAergic cell subtypes, J. Neurophys. 78 (1997) 1743–1747.The morphology of SNOS cells is similar to that of

[15] Y. Kawaguchi, Y. Kubota, Physiological and morphological identifi-neuroglioform cells previously described with Golgication of somatostatin- or vasoactive intestinal polypeptide-con-

studies and calbindin-immunoreactivity (for discussion, see taining cells among GABAergic cell subtypes in rat frontal cortex, J.Ref. [7]). Golgi preparations showed that these cells have Neurosci. 16 (1996) 2701–2715.

[16] Y. Kawaguchi, T. Shindou, Noradrenergic excitation and inhibitionlocally restricted axonal and dendritic arbors, and that theyof GABAergic cell types in rat frontal cortex, J. Neurosci. 18 (1998)synapse on pyramidal cell dendrites [7,17]. The different6963–6976.morphologies of the SNOS and LNOS cells predict they

[17] Z.F. Kisvarday, A. Gulyas, D. Beroukas, J.B. North, I.W. Chubb, P.have different functions. The fact that the two populations Somogyis, Synapses, axonal and dendritic patterns of GABA-im-can be discriminated based upon calcium binding protein munoreactive neurons in human cerebral cortex, Brain 113 (1990)

793–812.and neuropeptide immunoreactivity provides a potential[18] N.W. Kowall, M.F. Beal, Cortical somatostatin, neuropeptide Y, andmethod for identifying the source of the nNOS-positive

NADPH diaphorase neurons: normal anatomy and alterations inaxons and synaptic terminals within cortex.Alzheimer’s disease, Ann. Neurol. 23 (1988) 105–114.

[19] Y. Kubota, R. Hattori, Y. Yui, Three distinct subpopulations ofGABAergic neurons in rat frontal agranular cortex, Brain Res. 649(1994) 159–173.

References [20] R.O. Kuljis, P. Rakic, Multiple types of neuropeptide Y-containingneurons in primate neocortex, J. Comp. Neurol. 280 (1989) 393–

[1] C. Aoki, S. Fenstemaker, M. Lubin, C.-G. Go, Nitric oxide synthase 409.in the visual cortex of monocular monkeys as revealed by light and [21] D.A. Lewis, M.J. Campbell, J.H. Morrison, An immunohistochemi-electron microscopic immunocytochemistry, Brain Res. 620 (1993) cal characterization of somatostatin-28 and somatostatin-28(1–12)97–113. in monkey prefrontal cortex, J. Comp. Neurol. 248 (1986) 1–18.

Page 8: Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells

212 J.F. Smiley et al. / Brain Research 863 (2000) 205 –212

[22] H.-J. Luth, A. Hedlich, H. Hilbig, E. Winkelmann, B. Mayer, [26] S.R. Vincent, O. Johansson, T. Hokfelt, L. Skirboll, R.P. Elde, L.Morphological analyses of NADPH-diaphorase /nitric oxide synth- Terenius, J. Kimmel, M. Goldstein, NADPH-diaphorase: a selectivease positive structures in human visual cortex, J. Neurocytol. 23 histochemical marker for striatal neurons containing both somatos-(1994) 770–782. tatin- and avian pancreatic polypeptide (APP)-like immuno-

[23] J.H. Rogers, Immunohistochemical markers in rat cortex: co-locali- reactivities, J. Comp. Neurol. 217 (1983) 252–263.zation of calretinin and calbindin-D28k with neuropeptides and [27] X.X. Yan, L.J. Garey, Morphological diversity of nitric oxideGABA, Brain Res. 587 (1992) 147–157. synthesising neurons in mammalian cerebral cortex, J. Hirnforsch.

[24] J.F. Smiley, A.I. Levey, M.-M. Mesulam, Infracortical interstitial 38 (1997) 165–172.cells concurrently expressing m2-muscarinic receptors, AChE, and [28] X.X. Yan, L.S. Jen, L.J. Garey, NADPH-diaphorase-positive neu-NADPH-d in the human and monkey cerebral cortex, Neuroscience rons in primate cerebral cortex colocalize with GABA and calcium84 (1998) 755–769. binding proteins, Cereb. Cortex 6 (1996) 524–529.

[25] S.R. Vincent, Nitric oxide: a radical neurotransmitter in the centralnervous system, Prog. Neurobiol. 42 (1994) 129–160.