Neurochemical and Structural Organization of the Principal Nucleus of the Inferior Olive in the Human

The inferior olive (IO) is the sole source of the climbing fibers that innervate the Purkinje cells of the cerebellar cortex. The IO comprises several subdivisions, the dorsal accessory olive (DAO), medial accessory olive (MAO), and principal nuclei of the IO (IOpr); the relative sizes of these subnuclei vary among species. In human, there is an expansion of the cerebellar hemispheres and a corresponding expansion of the IOpr. We have examined the structural and neurochemical organization of the human IOpr, using sections stained with cresyl violet (CV) or immunostained for the calcium‐binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV), the synthetic enzyme for nitric oxide (nNOS), and nonphosphorylated neurofilament protein (NPNFP). We found qualitative differences in the folding patterns of the IOpr among individuals and between the two sides of the brainstem. Quantification of IOpr volumes and indices of folding complexity, however, did not reveal consistent left–right differences in either parameter. Single‐label immunohistochemistry showed that populations of neurons in the IOpr express CB, CR, NPNFP, and nNOS. Individual fibers labeled for PV, CB, CR, NPNFP, and nNOS were also found. There was individual variability in the numbers and density of stained neurons in the human IOpr; such variability was not seen in other brainstem nuclei. These data are consistent with, and complement, earlier studies showing a dramatic age‐related increase in lipofuscin and decrease in RNA in the human IOpr. The impact of these changes in the IOpr on cerebellar function is, however, not known. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

Visually-induced NGFI-A protein expression in the calbindin-, parvalbumin- and nitric oxide synthase-neuronal populations of the rat superior colliculus

The expression of the immediate early gene NGFI-A in the nervous system is induced by sensory stimulation and seems to be related to long-term synaptic plasticity. We have used double-labeling immunohistochemistry to identify calbindin (CB)(+), parvalbumin (PV)(+) and neuronal nitric oxide synthase (nNOS)(+) neurons that also expressed the protein encoded by this immediate early gene after light-exposure on in the superficial layers of the rat superior colliculus (sSC). The majority of the NGFI-A(+) cells were not double-labeled for the tested markers. In the stratum zonale+stratum griseum superficiale (SZ/SGS), only 17.8%, 8.0% and 12.1% of NGFI-A(+) cells were also labeled for CB, PV or nNOS, respectively. In the stratum opticum (SO), only 10.5% of the NGFI-A(+) cells were also CB(+). Furthermore, only a small subset of each population expressed the NGFI-A protein after light-exposure. In the SZ/SGS, 35.7% of the CB(+), 32.1% of the PV(+) and 26.6% of the nNOS(+) neurons also expressed the NGFI-A. In the SO, 31.7% of the CB(+) neurons also expressed the NGFI-A. The proportional distribution of the nNOS(+)/NGFI-A(+) neurons throughout the SZ/SGS layers showed a slight but significant rostro-caudal gradient. No significant difference was observed for the other markers, indicating homogeneous activation of these populations throughout the retinotopic map. Our results suggest that the visually-driven NGFI-A expression is not restricted to a specific population of the sSC and that visual processing in this structure, as assessed by the expression of this candidate-plasticity protein, involves the activation of subsets of ascending and non-ascending projection neurons.     

Colocalization of parvalbumin, calretinin and calbindin D-28k in human cortical and subcortical visual structures

Several studies have demonstrated that three calcium-binding proteins parvalbumin (PV), calbindin D-28k (CB) and calretinin (CR) mark distinct subsets of cortical interneurons. This study demonstrates, in cortical and subcortical visual structures, the coexistence of two calcium-binding proteins in some neuronal subpopulations. The human visual cortex (VC), lateral geniculate nucleus (LGN), lateral inferior pulvinar (LIP) and superior colliculus (SC) were examined by a double-labelling immunocytochemical technique. The VC showed mostly separate populations of PV, CB and CR immunoreactive (-ir) interneurons, but also small populations of double-stained PV+CR and CR+CB neurons, while PV+CB neurons were less frequent. An average of 2.5% of the immunoreactive neurons were double-stained for PV+CR and 7.1% for CR+CB in area 17, while this percentage was slightly higher in association area 18 (3.3 and 7.4%, respectively). In the LGN and LIP, double-stained neurons were scarce, but in the fibre capsule of these nuclei, as well as in the optic radiation (OR) and white matter underlying area 17, both double-stained PV+CR or CR+CB and separate populations of PV-ir, CB-ir and CR-ir neurons and fibres were observed. Unlike the thalamic regions, the SC showed some double-stained PV+CR and CR+CB neurons, scattered both in the superficial and deep layers. These findings are discussed in the light of similar observations recently reported from other regions of the human brain.     

The claustrum of the sheep and its connections to the visual cortex

The present study analyses the organization and selected neurochemical features of the claustrum and visual cortex of the sheep, based on the patterns of calcium-binding proteins expression. Connections of the claustrum with the visual cortex have been studied by tractography. Parvalbumin-immunoreactive (PV-ir) and Calbindin-immunoreactive (CB-ir) cell bodies increased along the rostro-caudal axis of the nucleus. Calretinin (CR)-labeled somata were few and evenly distributed along the rostro-caudal axis. PV and CB distribution in the visual cortex was characterized by larger round and multipolar cells for PV, and more bitufted neurons for CB. The staining pattern for PV was the opposite of that of CR, which showed densely stained but rare cell bodies. Tractography shows the existence of connections with the caudal visual cortex. However, we detected no contralateral projection in the visuo-claustral interconnections. Since sheep and goats have laterally placed eyes and a limited binocular vision, the absence of contralateral projections could be of prime importance if confirmed by other studies, to rule out the role of the claustrum in stereopsis.     

Altered expression of parvalbumin and calbindin in interneurons within the primary visual cortex of neonatal enucleated hamsters

In the present study, we tested the hypothesis that the expression of calcium binding proteins (CaBPs), parvalbumin (PV), calretinin (CR) and calbindin (CB), is dependent upon sensory experience as emphasized in visual deprivation and deafferentation studies. The expression of CaBPs was studied in interneurons within the primary and extrastriate visual cortices (V1, V2M, V2L) and auditory cortex (AC) of adult hamsters enucleated at birth. The effects of enucleation were mainly confined to area V1 where there was a significant volume reduction (26%) and changes in the laminar distribution of PV and CB immunoreactive (IR) cells. The density of PV-IR cell bodies was significantly increased in layer IV and reduced in layer V. Moreover, the density of CB-IR neurons was inferior in layer V of V1 in enucleated hamsters (EH) compared to controls. These results suggest that some features of the laminar distribution of specific CaBPs, in primary sensory cortices, are dependent upon or modulated by sensory input.     

胸腺及其投射神经元一氧化氮的分布

本实验用 NADPH-d组织化学方法及 n NOS免疫细胞化学方法 ,对大鼠胸腺内一氧化氮 (NO)阳性细胞的分布进行了研究。采用 CB-HRP逆行追踪结合 n NOS免疫细胞化学双重反应技术 ,观察了大鼠胸腺投射神经元 n NOS的分布。结果显示 :(1)在脑干的疑核、面后核内有 CB-HRP与 n NOS双重阳性细胞 ;(2 )在胸腺内有多种 NADPH-d和 n NOS阳性细胞 ,按其形态可分为 :髓质上皮样细胞、胸腺树突样细胞、神经元样细胞、胸腺细胞样细胞及胸腺小体 ;(3 )在胸腺的被膜下、小梁内、皮髓质交界处、小血管的周围有丰富的 n NOS阳性纤维。提示 ,胸腺内 NO的来源不同 ,其在调节胸腺的各种活动中可能发挥的作用也不同     

Expression and regulation of NMDA receptor subunit R1 and neuronal nitric oxide synthase in cortical neuronal cultures: correlation with cytochrome oxidase

Our previous studies showed a differential distribution of the glutamatergic terminals in cytochrome oxidase-rich and -poor regions of the visual cortex. The NMDA type of glutamate receptors have been proposed to be involved in the activation of nitric oxide synthase to produce nitric oxide, the neurotransmitter. In the present study, we hypothesized that the expressions of glutamate receptor, NMDA receptors (NMDAR1) and neuronal nitric oxide synthase (nNOS) were colocalized and were also correlated with that of cytochrome oxidase (CO) in a subset of neurons. We used primary cultures of postnatal rat visual cortical neurons as a model system, so that we could examine both the somatic and dendritic expressions of these neurochemicals in individual neurons. We found a difference in the sequence of developmental expressions of NMDAR1, nNOS, CO, and Na⁺/K⁺ ATPase. Triple labeling showed that all nNOS-positive neurons were immunoreactive for NMDAR1, and a subpopulation of them had high CO activity. The expression of NMDAR1 was positively correlated with CO activity. This is consistent with our previous finding that CO activity is strongly governed by excitatory glutamatergic synapses. After 40 hours of depolarizing potassium chloride treatment, CO activity was increased, and NMDAR1and nNOS levels were up-regulated in parallel. One week of tetrodotoxin significantly decreased the expression of NMDAR1, nNOS, and CO activity. Our results demonstrate that NMDA receptors and nNOS do co-exist in a subset of neurons that have high CO activity and their expressions are under the control of neuronal activity.     

NADPH-diaphorase distribution in the rabbit superior colliculus and co-localization with calcium-binding proteins

Nitric oxide (NO) and calcium‐binding proteins (CaBP) are important neuromodulators implicated in brain plasticity and brain disease. In addition, the mammalian superior colliculus (SC) has one of the highest concentrations of NO within the brain. The present study was designed to determine the distribution of nitric oxide‐synthesizing neurons in the SC of the rabbit by enzyme histochemistry for reduced nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d), and its degree of co‐localization with CaBP, parvalbumin (PV) and calbindin (CB). NADPH‐d‐labelled fibres formed dense patches of terminal buttons within the intermediate grey layer and streams of fibres within the deepest layers of SC. Cells expressing NOS constitute a subpopulation of neurons in which practically all cell types are represented. Combined PV/NADPH‐d experiments showed a complete lack of co‐localization within individual neurons and fibres. On the contrary, double‐labelled neurons appeared in CB/NADPH‐d‐stained sections, only in the superficial layers, and mostly in the SGS and SO. These cells, which were intermingled with other neurons containing either NADPH‐d or CB, appear to be a subtype of narrow‐field and wide‐field vertical cells, and display an anterior–posterior gradient of density. Owing to the involvement of the superficial layers of the SC in the organization and integration of the visual information, it is suggested that these neurons may play a concrete role within the visual circuits. Our data indicate a clear selectivity in the expression of NADPH‐d, PV and CB in the SC, and that NO and CB probably serve as co‐modulators and/or co‐transmitters in the connectivity of the superficial layers of this midbrain structure.     

The calcium binding proteins calbindin, parvalbumin, and calretinin have specific patterns of expression in the gray matter of cat spinal cord

Calcium binding proteins (CBPs) regulate intracellular levels of calcium (Ca²⁺) ions. CBPs are particularly interesting from a morphological standpoint, because they are differentially expressed in certain sub-populations of cells in the nervous system of various species of vertebrate animals. However, knowledge on the cellular regulation governing such cell-specific CBP expression is still incomplete. In this work on the L7 segment of the cat spinal cord, we analyzed the localization and morphology of neurons expressing the CBPs calbindin-28 KD (CB), parvalbumin (PV), and calretinin (CR), and co-expressing CB and PV, CB and CR, and PV and CR. Single CBP-positive (⁺) neurons showed specific distributions: (1) CB was present in small neurons localized in laminae I, II, III and X, in small to medium size neurons in laminae III–VI, and in medium to large neurons in laminae VI–VIII; (2) PV was present in small size neurons in laminae III and IV and in medial portions of laminae V and VI, medium neurons and in lamina X at the border with lamina VII, in medium to large neurons in laminae VII and VIII; (3) CR labeling was detected in small size neurons in laminae I, II, III and VIII, in medium to large size neurons in laminae I and III–VII, and in small to medium size neurons in lamina X. Double labeled neurons were a small minority of the CBP⁺ cells. Co-expression of CB and PV was seen in 1 to 2% of the CBP⁺ cells, and they were detected in the ventral and intermediate portions of lamina VII and in lamina X. Co-localization of CB and CR was present in 0.3% of the cells and these cells were localized in lamina II. Double labeling for PV and CR occurred in 6% of the cells, and the cells were localized in ventral part of lamina VII and in lamina VIII. Overall, these results revealed distinct and reproducible patterns of localization of the neurons expressing single CBPs and co-expressing two of them. Distinct differences of CBP expression between cat and other species are discussed. Possible relations between the cat L7 neurons expressing different CBPs with the neurons previously analyzed in cat and other animals are suggested.     

Immunocytochemical localization of the AMPA glutamate receptor subtype GluR2/3 in the squid optic lobe

As a major excitatory neurotransmitter in the cephalopod visual system, glutamate signaling is facilitated by ionotropic receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR). In cephalopods with large and well-developed brains, the optic lobes (OL) mainly process visual inputs and are involved in learning and memory. Although the presence of AMPAR in squid OL has been reported, the organization of specific AMPAR-containing neurons remains unknown. This study aimed to investigate the immunocytochemical localization of the AMPA glutamate receptor subtype 2/3-immunoreactive (GluR2/3-IR) neurons in the OL of Pacific flying squid (Tordarodes pacificus). Morphologically diverse GluR2/3-IR neurons were predominantly located in the tangential zone of the medulla. Medium-to-large GluR2/3-IR neurons were also detected. The distribution patterns and cell morphologies of calcium-binding protein (CBP)-IR neurons, specifically calbindin-D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-IR neurons, were similar to those of GluR2/3-IR neurons. However, two-color immunofluorescence revealed that GluR2/3-IR neurons did not colocalize with the CBP-IR neurons. Furthermore, the specific localizations and diverse types of GluR2/3-IR neurons that do not express CB, CR, or PV in squid OL were determined. These findings further contribute to the existing data on glutamatergic visual systems and provide new insights for understanding the visual processing mechanisms in cephalopods.     

Distribution of calbindin-D28k, neuronal nitric oxide synthase, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the lateral nucleus of the sheep amygdaloid complex

This study describes calbindin-D28k (CB), neuronal nitric oxide synthase (nNOS), and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) expression in the lateral nucleus of the sheep amygdaloid complex. Double immunofluorescence protocol was used in order to determine whether there is colocalization of CB and nNOS. The CB-immunoreactive (IR) neuronal population was composed especially of non-pyramidal neurons, but a few pyramidal cells were also present. The non-pyramidal neurons showed a multipolar and, occasionally, a fusiform morphology. The comparison between single-labeled CB-IR non-pyramidal neurons and cells belonging to CB-IR neuronal population showed they were identical for morphology, mean size, and distribution. The single-labeled CB-IR non-pyramidal neurons were only the 17.8% of the total non-pyramidal neurons counted. The nNOS-IR neuronal population was represented by non-pyramidal multipolar and fusiform neurons. Single-labeled nNOS-IR non-pyramidal neurons had the same morphology, mean area, and distribution as cells belonging to nNOS-IR neuronal population. Single-labeled nNOS-IR non-pyramidal neurons were more numerous than single-labeled CB-IR, and represented the 73.7% of total non-pyramidal neurons counted. NADPH-d-positive cells had the same morphology and distribution as the nNOS-IR neurons. Double immunolabeling (CB/nNOS) was found mostly in non-pyramidal multipolar neurons and only in a few non-pyramidal fusiform cells. These neurons had a mean perikaryal area significantly higher and significantly smaller than that of single-labeled nNOS and single-labeled CB-IR non-pyramidal neurons, respectively. CB and nNOS coexist only in a minority of non-pyramidal neurons (8.5%). The 32.4% of all CB-IR non-pyramidal neurons were nNOS-positive; only 10.4% of nNOS-IR non-pyramidal neurons were CB-positive. These results indicate that CB and nNOS are expressed by selective neurons and that the majority of nNOS-IR non-pyramidal neurons are lacking in CB.     

Upregulation of NMDA receptor and neuronal NADPH-d/NOS expression in the nodose ganglion of acute hypoxic rats

Nitric oxide may serve as a neuronal messenger in the regulation of cardiorespiratory function via the N-methyl-D-aspartate (NMDA) receptor-mediated neuronal nitric oxide synthase (nNOS) activation. Since hypoxic stress would drastically influence the cardiorespiratory function, the present study aimed to examine if the expression of nNOS and NMDA receptor subunit 1 (NMDAR1) in the nodose ganglion (NG) would alter under different extents of hypoxia treatment. The nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, nNOS and NMDAR1 immunofluorescence were used to examine nNOS and NMDAR1 expression in the NG following exposing of adult rats in the altitude chamber (0.27 atm, PO(2)=43 torr) for 2 and 4 h. The present results showed that NADPH-d, nNOS and NMDAR1 reactivities were co-localized in the NG under normoxic and hypoxic environment. Quantitative evaluation revealed that about 43% of neurons in the NG showed positive response for NADPH-d/nNOS and NMDAR1 reactivities. However, in animals subjected to hypoxia, both the percentage and the staining intensity of NADPH-d/nNOS and NMDAR1 labeled neurons were drastically increased. The percentage of NADPH-d/nNOS and NMDAR1-immunoreactive neurons in the NG was raised to 68% as well as 77%, respectively, following 2 and 4 h of hypoxic exposure. The magnitude of up-regulation was positively correlated with the duration of hypoxic periods. No significant cell loss was observed under this experimental paradigm. These findings suggest that different extents of hypoxia might induce the higher expression of nNOS and NMDAR1 in the NG, which could contribute to the neuronal integration as responding to the different physiological demands under hypoxic stress.     

Quantitative estimation and chemical coding of spiny type I neurons in human intestines

Previous studies have shown that most human myenteric neurons co-staining for vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and neurofilaments (NF) display the morphology of spiny type I neurons displaying a descending projection pattern. Here, we estimated the proportions of spiny neurons in human intestines, the amount of congruence of VIP/nNOS-immunoreactive with spiny neurons and whether galanin (GAL) is co-localized with VIP. Three sets of colchicine-pretreated and fixed whole mounts of 21 patients or body donors (median age 65 years; 10 female, 11 male) were stained for VIP, nNOS and NF, for VIP, nNOS and the human neuronal protein Hu C/D (HU) as well as for VIP, nNOS and GAL. The majority of VIP/nNOS-co-reactive neurons were spiny neurons (79/80% in small/large intestine, respectively) and the majority of spiny neurons co-stained for VIP and nNOS (82/69%). Neurons co-immunoreactive for VIP/nNOS/HU amounted to 7 and 4%, respectively. GAL/VIP-co-immunoreactivity was demonstrated in 69 and 27% of spiny neurons, respectively. We conclude that the number of neurons displaying co-reactivity for VIP and nNOS is a quantitative indicator of spiny neurons in both small and large intestine and that the proportion of spiny neurons is about 7% in small and 4% in large intestines. Since nerve fibres co-staining for NF/VIP/nNOS were found mainly in the circular muscle layer but not the surrounding perikarya of spiny neurons, we suggest that they may represent inhibitory motor neurons rather than descending interneurons.     

大鼠中缝背核参与NO介导的脊髓对吗啡依赖和戒断的调节

目的:探索大鼠脑干内神经核团中缝背核(dorsal raphe nucleus,DRN)在吗啡依赖和戒断形成过程中的作用及其机制。方法:雄性成年SD大鼠、(260±20)g,随机分为戒断组,依赖组,生理盐水组,纳洛酮组和抑制剂组。建立吗啡依赖与戒断并进行行为学观测评分后取材相关部位,连续冠状冰冻切片,神经元型一氧化氮合酶(neuron nitric oxide synthase,nNOS)免疫组织化学标记。计数各组动物相同层面脑片和脊髓背角nNOS标记细胞的表达情况。结果:戒断组大鼠,戒断症状及总评分较对照组和依赖组大鼠差异显著(P<0.01);给NOS抑制剂组戒断症状评分较戒断组明显降低(P<0.05)。生理盐水组和纳洛酮组于中缝背核相应区域计数到部分nNOS标记神经元,但两但间无显著性差异(P<0.05);依赖组与戒断组大鼠nNOS标记神经元计数明显增加(P<0.05);而NOS抑制剂组大鼠nNOS标记神经元数量较戒断组明显减少(P<0.05)。脊髓背角切片显示,依赖组与戒断组大鼠nNOS标记神经元计数均较各对照组明显增加(P<0.05);而NOS抑制剂组大鼠nNOS标记神经元数量较戒断组减少显著(P<0.05),其变化与中缝背核结果一致。结论:脑内中缝背核可能参与通过一氧化氮(nitric oxide,NO)信号通路介导的脊髓对吗啡依赖和戒断形成的调节。     

Parvalbumin and calbindin D-28K immunoreactive neurons in area MT of rhesus monkey

The chemical characteristics of the neurons of the motion sensitive visual area, area MT, remain to be established. We studied the distribution pattern of two calcium binding proteins, parvalbumin (PV) and calbindin D28K (CB) in this area, using specific monoclonal antibodies and the peroxidase-antiperoxidase (PAP) immunohistochemical technique. Aldehyde fixed 30-micron-thick cryostat sections from area MT of five animals were processed free floating for immunohistochemical staining. Besides studying the morphological characteristics of PV and CB positive neurons, quantitative analysis was carried out to determine their (1) perikaryal area (Pa) and diameter, (2) numerical densities (NV)/mm3 cortical tissue, (3) absolute number (NC) in a column of cortex under 1 mm2 cortical surface along with (4) layerwise absolute number (NL) under 1 mm2 cortical surface and (5) laminar percentage distribution of immunoreactive (IR) neurons. Quantitative analysis was carried out using a Leica QMC 500 image analysis system connected to a DMRE microscope. The results showed that both types of IR neurons were localized to all cortical layers except layer I. The PV +ve neurons were equidistributed between the supra- and infragranular layers, with the highest percentage being present in layer III (45%) followed by layer V (21%). The CB +ve neurons, on the other hand, were predominantly localized in supragranular layers, with the highest percentage being in layer III (54%) and the next highest percentage in layer II (18%). The average Pa and diameter of PV +ve neurons were found to be 96.90 +/- 28.43 micron 2 and 11.01 +/- 1.61 microns respectively. The CB +ve neurons were significantly smaller in size than the PV +ve neurons, with average Pa and diameter of the former being 92.23 +/- 26.18 micron 2 and 10.39 +/- 1.23 microns respectively. The NV for PV and CB +ve neurons showed ranges of 3157-3894 and 2303-2585, with means of 3347 +/- 285 (+/- SD) and 3436 +/- 100 respectively. The values for NC showed ranges of 5230-5444 and 4020-4268 with means of 5378 +/- 85 and 4167 +/- 95 for PV and CB neurons respectively. Variations in size together with the differential distribution of these neurons in the cortical layers may indicate their involvement in different functional circuitaries.     

Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.     

Roles of nitric oxide in the homeostatic control of the excitation–inhibition balance in rat visual cortical networks

The level of excitability of cortical neurons depends on the balance between their excitatory and inhibitory inputs (excitation/inhibition [E/I] balance). In the cortex, the E/I balance received by a neuron is dynamically maintained through a coordinated regulation of the strength of these inputs, described in term of homeostatic plasticity. Using a method allowing the determination of the E/I balance in rat cortical layer 5 pyramidal neurons (L5-PNs, the main output stage of the cortex), while keeping the interactions between excitatory and inhibitory networks functional, we examined the effects of high or low frequency of stimulation (HFS or LFS) protocols in layer 4 (in order to mimic thalamo–cortical entries) on the E–I level of the neuronal network. We previously showed that the E/I balance of L5-PNs remains stable due to a dual potentiation or dual depression of E and I after HFS or LFS protocols. Here, using a specific neuronal nitric oxide synthase (nNOS) inhibitor, we show that the related potentiation or depression of E and I (underlying homeostatic plasticity processes) required nNOS activation. We also show that application of an unspecific blocker of nitric oxide synthase (NOS) or a nitric oxide (NO) scavenger induces an increase of the E/I balance suggesting a role for a tonic NO synthesis in the regulation of the network activity. It is concluded that, in the cortex, a phasic NO effect (due to activation of nNOS) is required for the induction of homeostatic plasticity processes whereas a tonic NO signal is involved in the regulation of a set-point value for the E/I balance.     

Spinal cord transection modifies neuronal nitric oxide synthase expression in medullar reticular nuclei and in the spinal cord and increases parvalbumin immunopositivity in motoneurons below the site of injury in experimental rabbits

Using immunohistochemistry, we detected the expression of neuronal nitric oxide synthase (nNOS) in ventral medullary gigantocellular reticular nuclei and in the lumbosacral spinal cord 10 days after thoracic transection in experimental rabbits. We tried to determine whether neurons located below the site of injury are protected by the calcium binding protein parvalbumin (PV). Changes of nNOS immunoreactivity (IR) in spinal cord were correlated with the level of nNOS protein in dorsal and ventral horns. Ten days after transection, nNOS was upregulated predominantly in lateral gigantocellular nuclei. In the spinal cord, we revealed a significant increase of nNOS protein in the dorsal horn. This is consistent with a higher density of punctate and fiber-like immunostaining for nNOS in laminae III-IV and the up-regulation of nNOS-IR in neurons of the deep dorsal horn. After surgery, the perikarya of motoneurons remained nNOS immunonegative. Contrary to nNOS, the PV-IR was upregulated in α-motoneurons and small-sized neurons of the ventral horn. However, its expression was considerably reduced in neurons of the deep dorsal horn. The findings indicate that spinal transection affects nNOS and PV in different neuronal circuits.     

Immunohistochemical characterization of parvalbumin-containing interneurons in the monkey basolateral amygdala

Interneurons expressing the calcium-binding protein parvalbumin (PV) are a critical component of the inhibitory circuitry of the basolateral nuclear complex (BLC) of the mammalian amygdala. These neurons form interneuronal networks interconnected by chemical and electrical synapses, and provide a strong perisomatic inhibition of local pyramidal projection neurons. Immunohistochemical studies in rodents have shown that most parvalbumin-positive (PV+) cells are GABAergic interneurons that co-express the calcium-binding protein calbindin (CB), but exhibit no overlap with interneuronal subpopulations containing the calcium-binding protein calretinin (CR) or neuropeptides. Despite the importance of identifying interneuronal subpopulations for clarifying the major players in the inhibitory circuitry of the BLC, very little is known about these subpopulations in primates. Therefore, in the present investigation dual-labeling immunofluorescence histochemical techniques were used to characterize PV+ interneurons in the basal and lateral nuclei of the monkey amygdala. These studies revealed that 90–94% of PV+ neurons were GABA+, depending on the nucleus, and that these neurons constituted 29–38% of the total GABAergic population. CB+ and CR+ interneurons constituted 31–46% and 23–27%, respectively, of GABAergic neurons. Approximately one quarter of PV+ neurons contained CB, and these cells constituted one third of the CB+ interneuronal population. There was no colocalization of PV with the neuropeptides somatostatin or cholecystokinin, and virtually no colocalization with CR. These data indicate that the neurochemical characteristics of the PV+ interneuronal subpopulation in the monkey BLC are fairly similar to those seen in the rat, but there is far less colocalization of PV and CB in the monkey. These findings suggest that PV+ neurons are a discrete interneuronal subpopulation in the monkey BLC and undoubtedly play a unique functional role in the inhibitory circuitry of this brain region.     

Subcellular localization of neuronal nitric oxide synthase in the superficial gray layer of the rat superior colliculus

The superficial layers of the rat superior colliculus (sSC) receive innervation from retina and include nitric oxide synthase (NOS)-immunoreactive neurons. We used electron microscopic immunocytochemistry to assess the subcellular localization of neuronal NOS (nNOS) in the sSC. nNOS immunoreactivity was detected on the external membrane of mitochondria, endoplasmic reticulum, in pre- and postsynaptic profiles and also diffusely distributed in the cytosol. Postsynaptic labeled regions were often associated with presumptive retinal unlabeled terminals. Microtubules also appeared intensely labeled. These results show that NOS immunoreactive neurons may be innervated by retinal terminals and suggest an association of nNOS with cytoskeletal elements.