The Distribution of NADPH Diaphorase and Nitric Oxide Synthetase (NOS) in Relation to the Functional Compartments of Areas V1 and V2 of Primate Visual Cortex

A.E. Wiencken1 and V.A. Casagrande1,2,3

1 Departments of Cell Biology, , 2 Psychology and , 3 Ophthalmology and Visual Sciences, Vanderbilt University, Nashville, TN 37232-2175, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The primary visual cortex (V1) of primates receives visual signals from cells in the koniocellular (K), magnocellular (M) and parvocellular (P) layers of the lateral geniculate nucleus (LGN). The functional role of the K pathway is unknown, but one proposal is that it modulates visual activity locally via release of nitric oxide (NO). One goal of this study was to examine the distribution of nitric oxide synthetase (NOS), the enzyme that produces NO, using immunocytochemistry for brain NOS (bNOS) or histochemistry for nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity in the V1 target cells of the K pathway and within the LGN itself. A second goal was to examine bNOS and NADPH diaphorase activity within proposed functional compartments in the second visual area (V2). We examined the LGN, V1 and V2 in squirrel monkeys, owl monkeys and bushbabies. In V1 and V2, we found that dense neuropil staining for NADPH diaphorase mirrored the pattern of high metabolic activity shown with cytochrome oxidase (CO) staining but did not necessarily mirror the pattern of immunolabeling seen with antibodies against NOS. The smooth stellate cells stained for NADPH diaphorase or bNOS were sparse and did not colocalize with LGN recipient zones in V1 or with the CO compartments in V2. LGN cells projecting to V1, including K, M and P cells, were negative for bNOS and NADPH diaphorase. Therefore, high levels of NOS are not limited to the K pathway. Instead, dense NOS activity is present in interneurons and within the neuropil of V1 and V2 that exhibit high metabolic demand.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Because of their utility in the identification of specific classes of cells or types of functional modules, neurochemical markers have contributed to our understanding of the functional organization of the visual system. The discovery that a regular array of patches (the ‘blobs’) in primary visual cortex (V1) and stripes in the second visual area (V2) of primates stain darkly for the mitochondrial enzyme cytochrome oxidase (CO) (Hendrickson et al., 1981Go; Horton and Hubel, 1981Go; Carroll and Wong-Riley, 1984Go) led to a revolution in how we view the functional architecture of V1 and V2. We now know that CO blobs and interblobs in V1 connect to different CO stripe compartments in V2. In all primates examined, these compartments in V1 and V2 send signals into a hierarchy of other areas concerned with either form or motion perception (Casagrande and Kaas, 1994Go). Several issues, however, remain unresolved concerning the role of the CO-defined modules in V1 and V2. In diurnal primates, some investigators have argued that CO blobs in V1 and their anatomical targets in V2, the CO thin stripes, carry signals relevant to color vision (Hubel and Livingstone, 1987Go; Zeki, 1993Go). However, some nocturnal primates (owl monkeys and bushbabies) have only one cone type, likely lack color vision, and yet have well-developed CO blobs and stripes (Horton, 1984Go; Wikler and Rakic, 1990Go; Kemp and Jacobson, 1991Go; Jacobs et al., 1993Go; Jacobs, 1996Go). The CO blobs in V1 and thin stripes in V2 must, therefore, play either a more general role in vision than segregation of color signals or additional unidentified roles.

The CO blobs in primate V1 receive direct input from a unique class of lateral geniculate nucleus (LGN) cells, the koniocellular (K) cells, as well as indirect input from the magnocellular (M) and parvocellular (P) pathways of the LGN (Casagrande and DeBruyn, 1982Go; Livingstone and Hubel, 1982Go; Fitzpatrick et al., 1983Go; Weber et al., 1983Go; Diamond et al., 1985Go; Casagrande et al., 1992Go; Lachica and Casagrande, 1992Go; Lachica et al., 1992Go, 1993Go; Hendry and Yoshioka, 1994Go; Ding and Casagrande, 1997Go). A variety of other enzymes and transmitter-related molecules are differentially localized to the CO blobs and interblobs in V1 and stripes within V2 (Hockfield et al., 1983Go; Hendry et al., 1984Go; Deyoe et al., 1990Go; Johnson and Casagrande, 1995Go; Kaminska et al., 1997Go). For example, nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase colocalizes with the CO blobs in macaque monkeys (Sandell, 1986Go). NADPH diaphorase in fixed tissue subsequently was identified as nitric oxide synthetase (NOS) (Hope et al., 1991Go; Matsumoto et al., 1993Go). Nitric oxide synthetase is the enzyme that produces the proposed neuromodulator, nitric oxide (NO). Nitric oxide is of particular interest because it is the first proposed gaseous neurotransmitter (Vincent and Hope, 1992Go). It does not have a receptor in the postsynaptic membrane, but diffuses across membranes freely to activate guanylate cyclase (Arnold et al., 1977Go; Miki et al., 1977Go).

The colocalization of NADPH diaphorase and CO in V1 led to the proposal that the K pathway might locally regulate neural activity in V1 by releasing the transmitter NO (Casagrande, 1994Go). One objective of the current study was to address this hypothesis at the histochemical and immunocytochemical levels by examining the distribution of NOS in the K LGN cells and within the CO blob target cells of this pathway in V1. A related objective was to determine if the thick stripe, thin stripe and interstripe compartments of area V2 contained different levels of NADPH diaphorase activity. Second, we wanted to determine whether NOS immunostaining colocalizes specifically with CO dense compartments in V1 and V2, as would be expected if NO plays some special role in these visual modules. Finally, we wished to determine if the distribution of NADPH diaphorase activity in V1 and V2 exhibits a common pattern across primates.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Subjects and Tissue Preparation

We examined tissue from three squirrel monkeys, Saimiri sciureus, eight owl monkeys, Aotus trivirgatus, and ten bushbabies, Galago crassicaudatus garnetti (reclassified as Otolemur garnetti). All animals used for the study were adults. In all cases, we obtained one hemisphere from an animal used in another compatible experiment. All animals were handled according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the Vanderbilt University Animal Care Committee.

For perfusion, all animals were given 6.7 units of heparin, sacrificed with a lethal dose of sodium pentobarbital and then perfused trans-cardially with a saline rinse, followed by an aldehyde fixative in 0.1 M phosphate buffer (pH 7.4), and finally, the same fixative containing 10% sucrose. The fixative solution contained 2–4% paraformaldehyde, 0–0.2% glutaraldehyde and 0 or 0.2% saturated picric acid. In nine hemispheres (two bushbabies, four owl monkeys and three squirrel monkeys) the occipital lobe was dissected from the rest of the brain, the cortex unfolded and flattened between glass slides, and the tissue post-fixed for 4–6 h at 25°C in 0.1 M phosphate buffer containing 4% paraformaldehyde. All of the brains were cryoprotected and then frozen quickly on dry ice.

Histochemistry and Immunocytochemistry

Brain tissue was sectioned on a freezing microtome in either the coronal or the parasaggital plane except in the flattened cases, which were cut tangentially. Sections destined for CO or NADPH diaphorase histochemistry were cut at 40 µm, while those for immunocytochemistry were cut at 15 or 20 µm.

We used two methods to stain for NADPH diaphorase (both slight modifications of the methods used by Sandell, 1986). Incubating tissue slices in a solution containing 1 mg/ml ßNADPH (Sigma, N-1630), 0.25 mg/ml nitro blue tetrazolium (NBT, Sigma, N-6876 ) and 0.5% Triton X-100 in 0.1 M Tris buffer, pH 8.0, for 1 to 4 h at 37°C optimized cell and blood vessel staining. Incubating sections in 0.1 M Tris buffer (pH 8.0) containing 1 mM ßNADP (Sigma, N-3886), 0.2 mM NBT, 30 mM L-malic acid (Sigma, M-1125) and 0.2% TweeN-80 for 2–6 h at 37°C, sometimes followed by overnight incubation at 4°C, optimized neuropil staining. In control sections, either ßNADPH or ßNADP was left out of the incubation solution, and no staining was observed.

For immunocytochemistry, tissue sections were incubated in 60% methanol in Tris-buffered saline (TBS) for 30 min at 25°C on a rocker table, washed, then incubated in 2% H2O2 in TBS for 30 min. At this point, some sections were microwaved for 3 min (800 W) in 10 mM sodium citrate buffer, pH 6.0, to enhance antigen retrieval (Shi et al., 1993Go). Tissue was then washed and blocked with 10% normal serum (usually donkey or horse), 1% bovine serum albumin (BSA) and 0.2% Triton X-100 in TBS for at least 2 h at 25°C. The primary antibody was applied in the blocking buffer overnight at 4°C. Polyclonal bNOS antibody (Transduction Labs) was diluted to 1:500, although a second lot of the bNOS antibody was inferior and could only be used with microwaving at a dilution of 1:100. Sections were incubated in the secondary antibody in blocking buffer for 2 h at 25°C. When DAB was used as a chromogen, a donkey {alpha}-rabbit biotinylated antibody (Chemicon) was used at a dilution of 1:200. Washes and incubation in avidin–biotin complex (Vector Elite) solution followed this step, and then visualization with DAB.

Tissue sections were reacted for CO activity as previously reported (Boyd and Matsubara, 1996Go). All sections (excluding those used for fluorescence immunocytochemistry) were mounted on glass slides, dehydrated and cleared in xylene. The primary antibody was left out of the incubation solution for control sections. No labeling was found in any control sections where antibodies were recognized with DAB. The contrast level of images of blobs in V1 and stripes in V2 was enhanced using Adobe Photoshop.

Western Blots

To evaluate the specificity of the bNOS antibody, several Western blots were prepared as follows. An owl monkey was given 0.06 mg/kg atropine and anesthetized with 20 mg/kg ketamine and 5 mg/kg xylazine. After the initial dose of anaesthesia, the animal was maintained at half the original dose as needed. The skull was opened, the meninges were reflected and small slices of brain tissue were removed with a scalpel, being careful to avoid major blood vessels. The tissue was frozen immediately by wrapping it in parafilm and lowering it into a slurry of dry ice and ethanol.

Frozen tissue was placed into harvest buffer consisting of 50 mM Tris, 2 mM EDTA, 1 mM EGTA, 1 mM DTT, 2 µg/ml aprotinin, 1 mM AESBF and 1 µM pepstatin A. In this buffer, the tissue was homogenized in a glass sleeve with 50 strokes. The homogenate was centrifuged for 30 min at 16 000 g at 4°C. The supernatant was removed and the pellet was resuspended in harvest buffer plus 0.1% Triton-X 100. The protein concentration of the samples was determined using a Bradford Assay (Biorad). The positive control was a sample of rat pituitary tissue. SDS sample buffer was added to the samples and they were stored at –20°C until the gel was run.

Western blots were performed using standard methods. Either 8.0 or 7.5% acrylamide gels were run overnight at 80 V. Proteins were transferred onto a nitrocellulose membrane overnight with 250 mA constant current at 4°C. Tris–glycine transfer buffer without methanol facilitated the transfer of high molecular weight proteins. The membrane was then blocked using 5% nonfat milk, 3% BSA and 0.2% TweeN-20 in TBS for 6 h at 25°C. The bNOS (Transduction Labs) antibody was diluted at 1:1000 in blocking buffer and incubated on the membrane overnight at 4°C. The membrane was rinsed then placed into donkey {alpha}-rabbit biotinylated antibody at 1:200 for 2 h at 25°C, followed by incubation in avidin–biotin complex solution for 2 h. After washing, the signal was visualized with DAB and H2O2. The membrane was washed in water, air dried and photographed.

The Transduction Labs bNOS antibody was made against a synthetic peptide located in the carboxyl terminal region of the protein. Several splice variants of bNOS have been described (Ogura et al., 1993Go; Hall et al., 1994Go). One of these variants, bNOSß, is present and functional in vivo in the mouse (Eliasson et al., 1997Go). We see two major bands at ~160 and 150 kDa, corresponding to the expected molecular weights in humans of the traditional bNOS variants, bNOS{alpha} and bNOSß, respectively (Hall et al., 1994Go). Brain NOSß is thought to be cytoplasmic, while bNOS{alpha} is associated with membranes, and this is mirrored in the fact that we see bNOS{alpha} mainly in the pellet fraction, while bNOSß is in the supernatant fraction as well (see Fig. 1Go). These data support our assumption that the polyclonal bNOS antibody is specific for bNOS in the owl monkey and that it detects both splice variants. It was surprising to see that bNOSß is the form of bNOS predominantly expressed in our owl monkey tissue samples. Because of our tissue collection protocol, our samples probably contained mainly the upper layers of cortex. Further studies will be required to determine whether bNOSß is the predominant form of bNOS expressed in owl monkey brain generally, or whether this is only the case in the upper layers of owl monkey cortex.



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Figure 1.  A representative Western blot depicting bNOS (detected with a polyclonal antibody from Transduction Labs) in owl monkey cortex. Numbers to the left indicate the position of molecular weight standards in kilodaltons. The positive control sample (C) consisted of rat pituitary tissue. The owl monkey tissue sample was crudely separated into membrane (P) and soluble (S) fractions. Although this was a crude particulate preparation, most of the two splice variants were found in the membrane fraction. In contrast to rat pituitary, in owl monkey cortex the major form of bNOS expressed is the lower molecular weight form (bNOSß, 150 kDa), whereas bNOS{alpha} (160 kDa) appears to be sparse.

 
Analysis

To determine the relationship between CO staining and the position of NADPH diaphorase positive cells, darkly labeled cells were plotted by hand using a camera lucida drawing tube. The adjacent CO section was then aligned with the cell survey of NADPH diaphorase positive cells using the blood vessel pattern. We counted and plotted both darkly and lightly labeled cells with the aid of a computer-controlled microscope with stage encoders (Bioquant, R & M Biometrics). This system was used to arrange the serial CO and NADPH diaphorase sections, and to adjust for tissue shrinkage in the comparisons between sections. The CO blobs were drawn by hand on the computer. However, comparisons were made with the computer's thresholding in areas of the sections where the blobs were well differentiated. There was good agreement between the detection of CO blobs by eye and by thresholding.

The finest and most consistent cellular and neuropil labeling with NADPH diaphorase was observed in the squirrel monkey tissue, so this tissue was used for further computer analysis of darkly and lightly labeled cells. We used a chi-square analysis to decide whether the darkly labeled cells were preferentially located inside or outside blob compartments. In this analysis, cells were considered inside the blob compartments if the point representing the cell was inside a circle representing the blob. We counted any cell touching the edge of the circle that defined a blob as a separate population divided in half, half assigned to blob and half to interblob compartments.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The results are presented in three parts. First we describe the laminar distribution of NADPH diaphorase positive neuropil in V1 and V2. We compare NADPH diaphorase positive neuropil in the compartments of V1 and V2 with the CO pattern in these areas. We next compare the NADPH diaphorase positive neuropil distribution with the distribution and morphology of neurons positive for both NADPH diaphorase and bNOS. Finally, we examine the NADPH diaphorase and bNOS labeling in LGN cells which project to V1.

NADPH Diaphorase Stained Neuropil in the Cortex

Neuropil staining with CO and NADPH diaphorase colocalized in V1 and V2 of all three primate species examined; NADPH diaphorase activity mirrored both laminar and compartmental patterns of CO activity in V1 and V2. To compare the same layers without confusion across species, we used a modification of Hässler's (Hässler, 1967Go) terminology to refer to the cortical layers. The key differences from the more commonly used terminology of Brodmann (Brodmann, 1909Go) are that all layers above Brodmann's layer IVC are considered in this paper to be subdivisions of layer III and Brodmann's layer IVC is referred to as layer IV. See Lachica et al. (Lachica et al., 1993Go) and Casagrande and Kaas (Casagrande and Kaas, 1994Go) for a complete explanation of the usefulness of this nomenclature.

Two types of staining were observed in the cortical neuropil after NADPH diaphorase histochemistry. Fibers with boutons en passant could be seen coursing throughout the cortex and in the white matter below it. Additionally, a more amorphous pattern of staining, which was impossible to assign to any particular cellular structure at the light microscopic level, was observed. These two forms of neuropil staining have also been observed in human postmortem tissue (Luth et al., 1994Go). One possible explanation for the more amorphous staining is that it reflects the location of mitochondria in cells that are negative for cytoplasmic staining with NADPH diaphorase. Such mitochondrial labeling with NADPH diaphorase has been observed at the electron microscopic level (Wolf et al., 1992Go). Qualitatively, the two types of neuropil staining were observed in the same layers with the exception that only NADPH diaphorase positive fibers were evident in the white matter. Also, cortical layers that contained high amorphous NADPH diaphorase activity also contained a denser NADPH diaphorase positive fiber plexus than neighboring regions.

The laminar pattern of NADPH diaphorase staining was very similar to the laminar pattern of CO staining in the neuropil of visual cortex. In V1 of all three primate species, layer IV was stained most darkly. In the squirrel monkey, layer IVß was stained more darkly than IV{alpha} and layer IIIBß also was densely stained (see Fig. 2Go). The neuropil of layer VI had the next highest level of NADPH diaphorase activity. Layers V and II/III had the least NADPH diaphorase staining, but greater levels of activity were detected in patches in layer III. Except for the NADPH diaphorase positive fibers, the white matter was negative for NADPH diaphorase.



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Figure 2.  In all species examined, CO and NADPH diaphorase showed similar staining patterns. Adjacent coronal sections through V1 in the squirrel monkey were stained with CO (A) and NADPH diaphorase (B). Roman numerals on the left side of the CO section indicate cortical layers [a modification of Hässler's terminology (Hässler, 1967Go)]. The patterns of staining using both CO and NADPH diaphorase are identical. Layer IIIBß and IV contain the most densely stained neuropil. The neuropil in layer IVß appears more darkly stained than that in IV{alpha}. Notice the patchy staining with both histochemical reactions in layer IIIB{alpha} representing the blobs (this is more apparent in tangential sections). An arrowhead marks the same blood vessel in both sections. The scale bar is 200 µm.

 
Examination of tangential sections of flattened cortex revealed that NADPH diaphorase activity was coincident with patches of dense CO activity that extended through the cortical layers and was most obvious in the blobs in the supragranular layers of V1 in all three primate species (see Fig. 3Go). The higher NADPH diaphorase activity in the blobs was not accompanied by a greater number of stained fibers, but instead there appeared to be a higher level of the amorphous staining in the blobs. The NADPH diaphorase positive blobs were the same size as the CO blobs in each species.



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Figure 3.  NADPH diaphorase activity (B, D, F) is coincident with dense CO activity (A, C, E) in the blobs in tangential sections through area V1 in three species of primates. Black arrows indicate representative blobs stained darkly with both histochemical reactions in the bushbaby (A, B). The scale bar is 5 mm. White arrows indicate representative blobs densely stained for both NADPH diaphorase and CO in the owl monkey (C, D) and the squirrel monkey (E, F). Note the dense endothelial cell staining in NADPH diaphorase stained tissue representing activity from the endothelial form of NOS. The scale bar is 500 µm.

 
In area V2 of all three species, the staining of layers IV and VI was lighter than that seen in the same layers in areas V1, highlighting the border between these two cortical areas. The neuropil of the supragranular layers was most densely stained for NADPH diaphorase activity in V2. As in V1, the laminar pattern of NADPH diaphorase positive neuropil in V2 paralleled the CO laminar pattern (see Fig. 4Go).



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Figure 4.  Adjacent coronal sections in squirrel monkey tissue through the V1/V2 border (indicated by an arrowhead) stained with CO (A) and NADPH diaphorase (B). The patterns of staining at the V1/V2 border with both CO and NADPH diaphorase histochemistry are identical. The more densely stained layers IV and VI in area V1 disappear in area V2. The scale bar is 300 µm.

 
In the owl monkey and the squirrel monkey, the highest NADPH diaphorase activity was coincident with CO activity in the stripes of area V2 (see Fig. 5Go). In agreement with previous studies (Franca et al., 1997Go), dense labeling by both CO and NADPH diaphorase was found in both the thick and thin stripe compartments in squirrel monkey V2. In the owl monkey, each CO dense stripe also was darkly stained for NADPH diaphorase. Although differences between thick and thin stripes were not always evident in owl monkeys, all stripes were labeled equally densely for both CO and NADPH diaphorase. In the bushbaby, stripes in V2 were not apparent using either CO or NADPH diaphorase histochemistry. In general, high NADPH diaphorase activity is not associated with a specific visual processing stream. Rather, high NADPH diaphorase activity exists in compartments and layers in both V1 and V2 that have high overall activity and glucose metabolism.



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Figure 5.  Adjacent tangential sections through area V2 of the owl monkey (A, B) and the squirrel monkey (C, D) reveal colocalization of NADPH diaphorase activity (B, D) with CO activity (A, C) in the stripes (indicated by arrows). In the squirrel monkey, histochemical staining with both enzymes was found in both thick (indicated with large arrows) and thin (indicated with small arrows) stripes. Note the dense endothelial cell staining in NADPH diaphorase stained tissue representing activity from the endothelial form of NOS. The scale bar is 1 mm.

 
Immunocytochemistry with a polyclonal bNOS antibody did not reveal any neuropil staining in layers of or in the CO blob compartments of V1 or the stripe compartments of V2 (data not shown). However, immunocytochemistry also did not reveal complete cell morphology comparable to that seen with histochemistry (see Fig. 6Go and compare with Fig. 8Go). Additionally, the immunocytochemistry did not label thin processes, even in the most darkly labeled cells. Therefore, the NOS positive neuropil may not be effectively labeled with immunocytochemistry due to the small size of fibers or possibly incomplete penetration of the bNOS antibodies.



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Figure 6.  NADPH diaphorase positive type 1 cell between layers V and VI in area V1 of the squirrel monkey. This cell was partially reconstructed using a camera lucida drawing tube. The axon (thinnest line) emerges from a primary dendrite below the left side of the soma and courses mainly in lower layer V. A dashed line indicates the layer V/VI border. The dendrites of type 1 cells are sparsely spiny (arrowhead indicates a spine in the drawing and the inset photomicrograph) and are often beaded. The scale bar is 20 µm; the inset scale bar is 10 µm.

 


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Figure 8.  Two types of cells are also seen when cortical tissue is stained with a bNOS antibody. Panel A shows a coronal section of layers I-III in area V1. Brain NOS positive type 1 and type 2 cells are present. Panel B shows a tangential section through the subadjacent white matter of area V1, where type 1 cells are more numerous. The scale bar is 50 µm.

 
Cellular Staining with bNOS and NADPH Diaphorase in the Cortex

We next examined the morphology of cells in areas V1 and V2 stained with either NADPH diaphorase histochemistry or bNOS immunocytochemistry. We found two populations of cells, one lightly stained and the other darkly stained, neither of which were strong candidates for the source of the dense neuropil staining in the CO compartments of V1. First we describe the characteristics of the NOS positive cells and then the laminar locations and compartmental distributions of those cells.

Both bNOS immunocytochemistry and NADPH diaphorase histochemistry revealed two populations of cells. One set was more sparsely distributed and very darkly stained, while the second was less completely labeled and more numerous. In many darkly stained cells, the complete dendritic tree and sometimes the axon was evident. Axons were most often labeled in the squirrel monkey tissue stained for NADPH diaphorase (see Fig. 6Go). The darkly labeled cells were smooth or sparsely spiny and some had dendritic swellings. These cells have been called type 1 cells while the lightly labeled cells are called type 2 (Sandell, 1986Go; Aoki et al., 1993Go; Luth et al., 1994Go; Tomimoto et al., 1994Go; Yan et al., 1996Go; Franca et al., 1997Go; Wong-Riley et al., 1998Go). In the squirrel monkey, most of the type 1 cells were sparsely spiny, but this was not so in the bushbaby, where most type 1 cells appeared smooth. Based upon morphology and the colocalization of GABA and bNOS or NADPH diaphorase, these cells are likely to be interneurons (Spike et al., 1993Go; Valtschanoff et al., 1993Go; Gabbott and Bacon, 1996Go; Yan et al., 1996Go; Wong-Riley et al., 1998Go). Some type 1 cell processes were intimately entwined with blood vessels in the cortex (see Fig. 7CGo). This relationship might have implications for the function of NO in the cortex, given that NO is a potent vasodilator and that the endothelial form of NOS is abundant in astrocytes (Wiencken and Casagrande, 1999Go). The association between NOS containing cells and the vasculature suggests that NOS positive cells control local blood flow in the cortex. This issue is considered in more detail in the discussion.



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Figure 7.  Two types of cells are found in the NADPH diaphorase stained cortical tissue of the primate. Type 1 cells are darkly stained, are more numerous in the white matter (tangential section in B) than in the gray matter (tangential section of layer III shown in A), and are sometimes found in close apposition to blood vessels (C). Type 2 cells are never found in the white matter, but are spread throughout the gray matter (compare A and B), and are most numerous in the supragranular layers. The scale bar is 50 µm in (A, B), 25 µm in (C).

 
In both V1 and V2, most of the darkly labeled type 1 cells were found in the white matter below layer VI, while fewer were located in layers II/III, V and VI (see Figs 7, 8GoGo). The fact that type 1 cells were most numerous in the white matter below layer VI suggests that these cells are some of the oldest cells in the cortex, since the first cells to become postmitotic and migrate in development form the topmost and bottommost layers of developing cortex (Rakic, 1974Go). Very few type 1 cells were found in layers I or IV of V1 [also reported in (Aoki et al., 1993Go)]. In V2, however, qualitative examination revealed that type 1 cells were more likely to be found in layer IV in all three primate species examined. This suggests that layer IV of V1 is unique in lacking bNOS positive cells as compared with layer IV in V2. Lightly labeled type 2 cells were found in all layers of the cortex in V1 and V2, but more were seen supragranularly. These lightly labeled cells were not present in the white matter below layer VI in any of the primates studied.

To decide whether cortical NADPH diaphorase containing cells are responsible for the dark neuropil staining in the CO blobs, we did surveys of type 1 cells in coronal sections. The blob compartments were identified using adjacent tissue sections stained for CO. As shown in Figure 9AGo, we found that the type 1 cells were not more numerous within the blob compartments; instead, they appeared to lie mainly outside these compartments with their dendrites sometimes reaching into the blobs.



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Figure 9.  Type 1 cells are not more numerous in the blob compartments. Panel A shows a survey of type 1 cells in a section through area V1. Although type 1 cells are not concentrated in the CO blob compartments, some of their dendrites reach into the blobs. The scale bar is 200 µm. In panel B, the results of a chi-square analysis of 191 type 1 cells in relation to blob compartments are shown (P = 0.20).

 
To more thoroughly examine the type 1 NADPH diaphorase cells in relationship to the blob and interblob compartments of area V1, these cells were plotted in flattened sections of squirrel monkey cortex and their positions compared with a plot of CO blobs on an adjacent CO stained tissue section (see Fig. 9BGo). Our prediction was that the distribution of type 1 cells would be uniform across CO blob and interblob compartments. Chi-square analysis confirmed that no significant difference existed between the density of type 1 cells in blob and interblob compartments (P = 0.20). When type 2 cells were qualitatively analyzed using a similar plot against an adjacent section stained for CO, no relationship between blob compartments and cell distribution was found (see Fig. 10Go).



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Figure 10.  Type 2 cells are evenly distributed in relationship to the blob and interblob compartments. Fifteen thousand type 2 cells (indicated by dots) in cortical layer III are plotted in relationship to CO blobs on an adjacent section. At this magnification, all dots cannot be resolved because of overlap. The scale bar is 1 mm.

 
NADPH Diaphorase and bNOS Labeling in the LGN

Closer examination of the cortical neuropil revealed that a large number of bNOS and NADPH diaphorase labeled fibers coursed through the cortex, and some of these exhibited varicosities. To decide whether the K cells in the LGN are responsible for the NADPH diaphorase dense activity in the blobs of V1 (the cortical target of these cells), we examined the distribution of NADPH diaphorase and bNOS in the LGN. No darkly labeled cells were observed in the LGN of any of the three primate species. Some lightly labeled cells were present, but these were distributed in all layers of the LGN and were very small when compared with the size distribution of cells in an adjacent section stained for Nissl substance (see Fig. 11Go). The homogeneous distribution and small size of the lightly stained NOS positive cells suggest that they are interneurons in the LGN. In the rat, NADPH diaphorase and GABA colocalize in the LGN (Mitrofanis, 1992Go; Gabbott and Bacon, 1994Go). The neuropil labeling was darkest in the M layers of the LGN. The K layers contained the lightest neuropil labeling. In the cat, the LGN neuropil labeling originates in the parabrachial region of the brainstem (Bickford et al., 1993Go).



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Figure 11.  Relay cells in the LGN are not stained with NADPH diaphorase. Adjacent coronal sections through the LGN of the squirrel monkey are stained for Nissl substance (A) and NADPH diaphorase (B). Letters indicate the M, P and K layers. Some small presumed interneurons are stained with NADPH diaphorase. At higher magnification in an M layer, NADPH diaphorase positive cells (representative cell indicated by arrowhead) are clearly not large enough to be M relay cells (representative M relay cell indicated by arrow). The scale bar is 150 µm in (A, B), 30 µm in (C).

 
From these data, we can conclude either that LGN cells are not responsible for the compartmental neuropil staining with NADPH diaphorase in V1, or that only the axons of those cells contain NOS.


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Our chief finding was that dense neuropil staining for NADPH diaphorase mirrors the pattern of high metabolic activity shown with CO in V1 and V2 of all three primate species, suggesting that the distribution of this enzyme relates more to metabolic demand than to functions associated with specific visual pathways. A second finding was that the distribution of cells stained for NADPH diaphorase or bNOS does not correlate with the distribution of NADPH diaphorase neuropil or CO staining. Darkly stained type 1 cells are sparse in all layers of V1 and V2 and are most dense in the white matter; type 2 cells are most dense in the supragranular layers of cortex. Finally, our results indicate that the somata of LGN cells projecting to V1, including cells in the K, M and P layers, are negative for bNOS and NADPH diaphorase, indicating that these V1 inputs are not likely to be the source of NOS neuropil staining in V1 of cortex. In the following discussion, we consider the interpretation of these results in light of previous findings and the proposed functions of NO in visual cortex.

The Search for a Visual Source of the NADPH Diaphorase Positive Cortical Neuropil

We examined presumed LGN relay cells for the presence of bNOS positive somata and did not find any to be present. It is possible that NOS is only located in the axons and not the somata of these cells. The major functional splice variant of bNOS, bNOS{alpha}, was shown to be associated with a protein which is tethered at the postsynaptic density (Brenman et al., 1996Go). Also, in macaque V1 at the EM level, bNOS is located mainly in spines and axon terminals, and is concentrated over membranes (Aoki et al., 1993Go, 1997Go; Wong-Riley et al., 1998Go). One study has shown that cells in the dLGN of the rat contain bNOS mRNA; however, these cells are likely to be interneurons since GABA and NADPH diaphorase colocalize in the LGN of the rat (Mitrofanis, 1992Go; Gabbott and Bacon, 1994Go; Iwase et al., 1998Go). It is possible that bNOS positive cells in non-visual subcortical nuclei, such as the claustrum, raphe nucleus, locus coeruleus or intralaminar nuclei of the thalamus, contribute to the cortical neuropil staining, although none of these sources of input to V1 have been shown to project specifically to the blobs.

In agreement with previous reports in the squirrel monkey and macaque monkey (Sandell, 1986Go; Franca et al., 1997Go; Wong-Riley et al., 1998Go), we did not find any correlation between the distribution of type 1 cell somas and the CO blob and interblob compartments. The data suggest that the darkly labeled cells have long dendrites that reach into the blob compartments but are not concentrated exclusively in these compartments. Our data do not rule out the possibility that irregularly shaped dendritic fields are responsible for the compartmental neuropil staining in V1. Some support for this theory comes from studies in rat somatosensory cortex where NOS cells are present in barrel septa, but their axons enter the barrels (Valtschanoff et al., 1993Go). Although, we did not include white matter cells in our statistical analysis of the compartmental distribution of type 1 bNOS positive cells, type 1 cells are most numerous in the white matter and often have long, horizontally oriented processes that sometimes course into the gray matter (Aoki et al., 1993Go). Therefore, processes of these white matter cells could contribute to the neuropil staining in the overlying layers and compartments of V1 and V2.

We and others find that the second class of NOS positive cells, type 2 cells, are much more lightly stained than type 1 cells, with only the most proximal portion of the dendrites discernible (Sandell, 1986Go; Aoki et al., 1993Go; Luth et al., 1994Go; Tomimoto et al., 1994Go; Yan et al., 1996Go; Franca et al., 1997Go; Wong-Riley et al., 1998Go). When we examined the distribution of type 2 cells in relationship to the blobs, no correlative pattern emerged. However, type 2 cells in extrastriate visual areas may project back to V1, forming the blob-like pattern of NADPH diaphorase positive neuropil staining in V1. Unlike type 1 cells, it is not clear that type 2 cells are interneurons. Although they do not have an obvious pyramidal morphology, their dendrites are hard to see. If extrastriate type 2 cells project to V1, then they would be good candidates for the source of the NOS positive neuropil staining in V1.

A Puzzling Finding

The neuropil staining pattern revealed with NADPH diaphorase histochemistry is different from that seen with bNOS immunocytochemistry. The immunocytochemistry did not reveal the dense compartmental neuropil staining seen with histochemistry. In a set of experiments using a polyclonal bNOS antibody not used here, Aoki et al. (Aoki et al., 1993Go) also did not see blob staining with immunocytochemistry (see Fig. 3BGo). The latter antibody did reveal darker neuropil in layer IV, but there was no evidence of patchy staining in layer III. Because a second lot of our original bNOS antibody from Transduction Labs was inferior to the first, we used a third polyclonal antibody from another source (Incstar) for some experiments. We incubated sections of owl monkey tissue containing V1 with this third antibody, and it also did not reveal any compartmental staining in layer III. Wong-Riley et al. did note patchy staining in the supragranular layers of V1 in the macaque monkey, although they admit that this pattern was faint (Wong-Riley et al., 1998Go). Our inability to see this patchy staining with immunocytochemistry may be due to the presence of a slightly different bNOS epitope in the species of primate which we examined.

Another explanation for the discrepancy we see in neuropil staining with histochemical and immunocytochemical methods is that the histochemical staining is revealing another enzyme as well as NOS, such as an oxidoreductase not detected with the antibodies. It is possible that the amorphous type of neuropil staining that could not be attributed to any neural structure in NADPH diaphorase stained tissue might represent mitochondrial staining in otherwise NOS negative cells, as has been seen in the rat at the EM level (Wolf et al., 1992Go). Moreover, this type of mitochondrial staining was not noted in tissue stained with immunocytochemical methods (Mizukawa et al., 1988Go). However, another group of authors looking in the cat with NADPH diaphorase at the EM level described a lack of mitochondrial staining (Aoki et al., 1993Go). At any rate, oxido-reductases are likely to be found in and around mitochondria. It would be interesting to compare directly the prevalence of the mitochondrial NADPH diaphorase staining in blob and interblob compartments at the EM level.

A third possibility for the discrepancy between the two staining methods concerns the presence of several splice variants of bNOS. Splice variants may have differing immunoreactivity depending on whether the epitope is present in the protein or spliced out. Each variant also can have very different catalytic activities. Several groups have suggested that bNOS is translationally modified (Ogura et al., 1993Go; Hall et al., 1994Go; Eliasson et al., 1997Go). The second exon is spliced out in at least one variant, called bNOSß, and this protein is responsible for the residual NOS activity in transgenic mice that lack exon 2 of bNOS. This alternative form of bNOS also is present in normal mice and is catalytically functional in vivo (Eliasson et al., 1997Go). The bNOS antibody that we used was raised against a peptide in the carboxyl terminal region of the protein, and so we would expect it to detect both bNOS{alpha} and bNOSß. We did see two major bands in our Western blots corresponding to the expected molecular weight of the two splice variants. Other splice variants, possibly involving the carboxyl terminal, may exist and cannot be ruled out as a possible explanation for the discrepancy that we see between the two detection methods.

Finally, it could be that the NADPH diaphorase histochemistry did not reveal the brain isoform of NOS, but instead was indicative of another isoform. It did seem odd to us that the two methods we used for NADPH diaphorase histochemistry gave us slightly different staining patterns. The NADP–malic acid method always revealed the neuropil staining more clearly. The latter also did not stain the blood vessels, presumably containing the endothelial isoform of NOS, as darkly (see Fig. 2Go). Sometimes, the NADP–malic acid method did not stain any type 2 cells, and only stained a few type 1 cells (we could see this by comparing the number of stained neurons from different brains of the same species reacted on different occasions). These differences may be attributed to different fixation conditions or the age of the tissue, but we could not be sure since these parameters were not systematically varied. In contrast to the NADP–malic acid method, the NADPH method always revealed stained cells and darkly stained blood vessels, but only occasionally stained the neuropil darkly (see Figs 3, 5GoGo). Clearly, the two methods have different sensitivities to the presumptive endothelial NOS activity in the blood vessels. We did examine the staining pattern with two polyclonal antibodies (Transduction Labs and Santa Cruz) against the endothelial form of NOS and saw no evidence of compartmental staining with this antibody (Wiencken and Casagrande, 1999Go).

Functional Considerations

Nitric oxide has been proposed to play a variety of functional roles in the brain. One suggestion is that NO, a vasodilator, controls local blood flow in the cortex. Nitric oxide is the endothelium derived relaxing factor which affects smooth muscle relaxation (Palmer et al., 1987Go; Garthwaite, 1991Go). While considerable evidence exists for an effect of NO on cortical blood flow, NO apparently is not necessary for cortical blood flow changes to occur (Goadsby et al., 1992Go; Iadecola, 1993Go; Kobari et al., 1993Go; Adachi et al., 1994Go; Faraci and Brian, 1994Go; Irikura et al., 1995Go). Several authors have shown that NADPH diaphorase positive fibers are near blood vessels in the brain (Iadecola et al., 1993Go; Nozaki et al., 1993Go; Luth et al., 1994Go; Moro et al., 1995Go; Yan et al., 1996Go). We also saw type 1 cell processes intimately entwined with blood vessels. Therefore, NOS positive cells are in a very good position to link local blood flow to neural activity. This possibility seems reasonable when one considers that a marker for high metabolic activity, CO, and markers for NOS colocalize in the neuropil of V1 and V2. Areas of high metabolic activity would be expected to require greater amounts of oxygen than surrounding areas with less activity. Direct examination of this issue showed that NOS immunoreactive cells in the blob regions of macaque V1 have greater CO activity than those in the interblob compartments, suggesting that NOS positive cells in the blobs are more metabolically active than those in the interblobs (Wong-Riley et al., 1998Go).

A second suggestion is that NO is involved in cortical plasticity. The second exon of bNOS, which is spliced out in bNOSß, contains a special domain that interacts with postsynaptic density-95 protein (PSD-95). An NMDA receptor type 2B peptide that also binds PSD-95 potently blocks this association (Brenman et al., 1996Go). This relationship suggests that bNOS and the NMDA receptor are tethered together in a complex at some locations. Several recent findings in macaque V1 support this conclusion, showing that bNOS and the NMDAR1 subunit are colocalized in dendritic shafts, spines and axon terminals in the cortex (Aoki et al., 1997Go), and that ~50% of NOS immunoreactive cells also contain the NMDAR1 subunit (Wong-Riley et al., 1998Go). The proximity of bNOS to the NMDA receptor means that bNOS would be exposed to higher local calcium concentrations when the NMDA receptor was activated and so could produce NO at these times. It has been shown that NMDA activation, but not the activation of AMPA or kainate receptors, results in NO release (Alagarsamy et al., 1994Go; Liu et al., 1997Go). A form of transient synaptic potentiation has been noted in the supragranular layers of the visual cortex. This potentiation is due to up-regulation of excitatory synaptic transmission and requires NMDA activation. Nitric oxide can modulate this form of transient potentiation positively or negatively (Montague et al., 1994Go; Friedlander et al., 1996Go; Harsanyi and Friedlander, 1997Go). Nitric oxide might be involved in modulating visual signals in a plastic way during visual processing.

In summary, the zones of primary terminations of the LGN in the cortex are clearly zones that are both high in neural activity (as revealed by CO staining) and high in NADPH diaphorase activity (possibly indicating NOS activity). A simple explanation for the presence of NOS in these areas is that areas of high activity are areas that need exquisite blood flow control. However, NOS is also closely associated with the NMDA receptor, which has been implicated in plastic changes in the brain. The tethering of NOS to the NMDA receptor suggests that the functional modules in V1 and V2 may be areas of exceptional cortical plasticity and that NO may contribute to that plasticity.


    Notes
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
We thank John Allison, Julie Mavity-Hudson, Marta Kamburowski, Jan Rosemergy and Jennifer Ichida for comments on the manuscript. We also thank Annette Filiatro for her work on the project and Dr Julie Sandell for her suggestions at the beginning of the research. Supported by NEI grant EY01778 (to V.A.C.), core grants EY08126 and HD15052, and training grant EY07135.

Address correspondence to V.A. Casagrande, Department of Cell Biology, Vanderbilt Medical School, Medical Center North, Rm C2310, Nashville, TN 37232-2175, USA. Email: vivien.casagrande{at}mcmail.vanderbilt.edu.


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