Ultrastructural study of nitric oxide synthase-containing striatal neurons and their relationship with parvalbumin-containing neurons in rats

Single- and double-label electron microscopic immunocytochemistry was used to examine the ultrastructure of striatal neurons containing nitric oxide synthase (NOS⁺) and evaluate the synaptic relationship of NOS⁺ striatal neurons with those containing parvalbumin (PV⁺). In both the single-label and double-label studies, NOS⁺ perikarya were observed to possess polylobulated nuclei. In the single-label studies, NOS⁺ terminals were seen forming synaptic contacts with dendritic shafts and dendritic spines that did not contain NOS, but not with NOS⁺ perikarya or dendrites. In the double-label studies (using diaminobenzidine and silver intensified immunogold as markers), nitric oxide synthase and parvalbumin immunoreactions were found in two different populations of medium-sized aspiny striatal neurons. The PV⁺ axon terminals were seen forming symmetric synapses on the dendritic spines of neurons devoid of PV or NOS labeling, on PV⁺ dendrites, and on NOS⁺ soma and dendrites. In contrast, NOS⁺ terminals were not observed to form synaptic contacts with the dendrites or soma of either PV⁺ or NOS⁺ neurons. These findings suggest that NOS⁺ striatal interneurons form synaptic contact with the spines and presumably the dendrites of striatal projection neurons, but not with the dendrites or soma of PV⁺ or NOS⁺ striatal interneurons. NOS⁺ neurons do, however, receive synaptic input from PV⁺ neurons.     

The distribution of nitric oxide synthase-containing autonomic preganglionic terminals in the rat

Nitric oxide synthase (NOS)-immunoreactivity was co-localised with NADPH diaphorase activity in preganglionic sympathetic neurons and in their terminals in pre- and paravertebral sympathetic ganglia. The density of NOS-containing terminals varied between ganglia. Reactive terminals were densestin the superior cervical, stellate and inferior mesenteric ganglia, where the majority of the neurons were surrounded by reactive fibres, and the coeliac and superior mesenteric ganglia, where about half the postganglionic somata were sorrounded by reactive terminals. Fibres were least abundant in the pelvic ganglia and thoracic and lumbar sympathetic chain ganglia. NOS reactivity did not coincide with the distribution of calcitonin gene related peptide immunoreactivity, a marker for the terminals of NOS-containing sensory neurons in the rat. The distribution of nerve cells and terminals suggests that NOS is present in more than one functional subpopulation of sympathetic preganglionic neurons.     

Ultrastructural localization of tyrosine hydroxylase in rat nucleus accumbens

Immunocytochemical localization of tyrosine hydroxylase (TH) was used to determine the ultrastructural morphology and synaptic associations of catecholaminergic terminals in the nucleus accumbens of the rat. The brains were fixed by vascular perfusion with 4% paraformaldehyde and 0.2% glutaraldehyde. Coronal sections cut with a vibrating microtome were incubated with rabbit antiserum to TH then immunocytochemically labeled by the peroxidase-antiperoxidase method. Immunoreactivity for the enzyme was found within unmyelinated axons and axon terminals. These terminals contained either all small clear or combined small clear and large dense core vesicles. Approximately 40% of the labeled terminals formed symmetric synapses with unlabeled proximal or distal dendritic shafts. The dendrites showed a spare distribution of spines. Axosomatic synapses and axonal associations of the TH-containing terminals also were detected. The recipient perikarya were usually 10-20 micrometers in diameter and contained an indented nucleus and abundant cytoplasm. The content of large dense vesicles and synaptic associations with somata and proximal dendrites suggest that a certain proportion of the TH-containing terminals within the nucleus accumbens are morphologically distinct from catecholaminergic terminals within the dorsal striatum. These differences are discussed in relation to neuropeptides and functions of the dopaminergic mesolimbic and nigrostriatal pathways.     

Ultrastructural identification of synaptic terminals from the axon of type 3 interneurons in the cat lateral geniculate nucleus

Synaptic terminals from the axons of type 3 neurons in the A-laminae of the cat LGN impregnated with the Golgi gold-toning procedure were examined at light and electron microscopic levels. The axons were identified by their somatic origin, thin diameter, and, in one of these cells, by dense undercoating beneath the axolemma, which is a known characteristic of the axon initial segment. The axon of one of the analyzed cells was profusely branched and extended throughout most of lamina A within the dendritic domains of the cell, and both types of processes were oriented along projection lines in LGN. This suggests that the dendrites and axons of type 3 cells receive inputs and exert effects, of probably inhibitory nature, within restricted retinotopic regions of LGN. The vast majority of the axon terminals of these cells were distributed in series along axonal branches. In one of the type 3 cells, however, a dense cluster of terminals arising from a secondary axonal branch was observed. Ultrastructurally, the analyzed synaptic terminals of the type 3 cells contained flattened or pleomorphic synaptic vesicles, dark mitochondria, and established synapses that appeared to be of symmetrical type when the membranes were perpendicularly cut. On the basis of these characteristics these terminals are classified as F boutons, following Guillery's (Z. Zellforsch. 96:1-38, '69), nomenclature. The postsynaptic elements to the axon terminals were dendrites of small to medium size, which received "en passant" synaptic contacts in extraglomerular regions of the geniculate neuropil by the terminals distributed in series. The axon terminals located in clusters, however, made synapses with dendrites in glomerular regions of the neuropil, where they were not seen postsynaptic to retinal or other types of terminals. This is in contrast to the postsynaptic nature of F2 boutons in the same glomeruli, which have been identified as dendritic appendages of the GABA positive type 3 neurons in the cat LGN (Montero: J. Comp. Neurol. 254:228-245, '86). On the other hand, the axonal F terminals differ from F1 boutons in terms of synaptic relations and ultrastructure, since the latter have been shown to be presynaptic to F2s and somata and to contain crowded populations of flat synaptic vesicles which give them a characteristic dark appearance. Terminals equivalent to F1 boutons have been shown to originate from perigeniculate cells in the rat LGN. From these observations it is suggested that the geniculate GABAergic interneurons support two morphologically and functionally different type of inhibitory terminals synapsing the dendrites of relay cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dopaminergic modulation of nitric oxide synthase activity in subregions of the rat nucleus accumbens

Nitric oxide (NO) is a gaseous neurotransmitter synthesized in the nucleus accumbens (NAc) by aspiny interneurons containing neuronal NO synthase (nNOS). nNOS activity is readily assayed using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining and is believed to be regulated by activation of dopamine (DA) D1- and D2-like receptors. However, the role of DA transmission in the regulation of nNOS activity in identified subregions of the NAc remains unexplored. In this study, the impact of pharmacological manipulations of D1, D2, and NMDA receptors on nNOS activity was determined using optical density measures of NADPH-d staining preformed in multiple subdivisions (core, medial shell, intermediate shell, and lateral shell) of the NAc. Awake behaving rats received systemic administration of vehicle and/or the following drugs ~25 min prior to tissue harvesting: the nNOS inhibitor N(G) -propyl-L-arginine (NPA), the D1 receptor agonist SKF 81297, the D1 receptor antagonist SCH 23390, the D2 receptor agonist quinpirole (QNP), the D2 receptor antagonist eticlopride (ETI), or the NMDA receptor antagonist 3-((±)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). In vehicle-treated animals, a distinct medial-lateral histochemical gradient of NADPH-d staining was observed, which was characterized by moderate staining in the core and medial shell and more robust staining in the intermediate and lateral shell. Administration of NPA, SCH 23390, QNP, and CPP attenuated staining preferentially in the intermediate and lateral shell. SKF 81297 and ETI administration consistently increased staining in the medial shell in a manner, which was attenuated following pretreatment with SCH 23390, QNP, NPA, and CPP. These observations demonstrate that nNOS activity measured in distinct subregions of the NAc is differentially modulated by DA D1 and D2 receptor activation. Moreover, these findings demonstrate for the first time that DA D1 and D2 receptor activation regulates the facilitatory influence of glutamatergic transmission on nNOS activity in the NAc medial shell via facilitation (D1) or suppression (D2) of NMDA receptor function.     

Presynaptic muscarinic receptors on dopaminergic terminals in the nucleus accumbens

Levels of muscarinic receptors were measured in the nucleus accumbens of rat following 0.8 μg 6-hydroxydopamine or vehicle injections (0.2 μl) into the ventral tegmental area to investigate whether the dopaminergic terminals destroyed by this procedure bear muscarinic receptors. Dopamine levels in the nucleus accumbens ipsilateral to the injection of 6-hydroxydopamine were substantially reduced by 83% as compared to the unlesioned side after 7 days. Significant decreases in the specific binding of [³H]N-methylscopolamine of 9 and 15% were also seen in the nucleus accumbens ipsilateral to the lesion after 7 and 14 days respectively. The class of muscarinic receptor depleted by the lesion was further investigated using [³H]oxotremorine-M to label the ‘super high’ affinity binding sites. Thepercentage occupancy of total muscarinic receptors by [³H]oxotremorine-M was significantly decreased by lesion e.g. 23% after 7 days, indicating a selective loss of ‘super high’ affinity binding sites. The lesion caused no change in the affinity constant for the muscarinic antagonist, propylbenzilylcholine. Studies of the binding of the agonists carbachol and oxotremorine-M by competition with [³H]propylbenzilylcholine showed little change in the concentrations of affinity constants of the ‘high’ and ‘low’ affinity binding sites with the 6-hydroxydopamine lesion.     

Chemical and structural analysis of the relation between cortical inputs and tyrosine hydroxylase-containing terminals in rat neostriatum

gamma-Aminobutyric acid (GABA) increases the rate of 36Cl- efflux from preloaded rat hippocampal slices in a dose-dependent manner (EC50: 400 microM). This action has the pharmacological specificity expected of activation of GABA receptors in that it is mimicked by the agonists muscimol and 3-aminopropanesulfonic acid, and blocked by the antagonists bicuculline and picrotoxinin. GABA uptake inhibitors, nipecotic acid and 2,4-diaminobutyric acid, fail to increase 36Cl- flux. Pentobarbital produces a dose-dependent activation (EC50 = 1.5 mM) of 36Cl- efflux with maximal response greater than that of GABA. The effect of pentobarbital can be mimicked by 1,3-dimethylbutylbarbiturate, secobarbital, (+)hexobarbital but not (-)hexobarbital, and is blocked by bicuculline and picrotoxinin. Pentobarbital and the other active barbiturates also potentiate the action of GABA. Phenobarbital does not have any effect independently or in combination with GABA. It is suggested that GABA increases 36Cl- permeability by activation of a postsynaptic receptor which is in turn functionally coupled to a barbiturate receptor.     

Neurogenesis in the mammalian neostriatum and nucleus accumbens: Parvalbumin-immunoreactive GABAergic interneurons

We study the neurogenesis of a distinct subclass of rat striatum γ-aminobutyric acid (GABA)ergic interneurons marked by the calcium-binding protein parvalbumin (PV). Timed pregnant rats are given an intraperitoneal injection of bromodeoxyuridine (BrdU), a marker of cell proliferation, on designated days between embryonic day (E) 11 and E22. Birthdate of PV neurons is determined in the adult neostriatum and nucleus accumbens by using a BrdU-PV double-labeling immunohistochemical technique. PV-immunoreactive interneurons of the neostriatum show maximum birthrates (>10% double-labeling) between E14–E17, whereas PV-immunoreactive interneurons of the nucleus accumbens show maximum double-labeling between E16–E19. In the neostriatum, caudal PV-immunoreactive neurons are born before those at rostral levels, and lateral PV-immunoreactive neurons become postmitotic before medial neurons. In the postcommissural striatum, ventral PV-immunoreactive neurons become postmitotic before dorsal neurons. In the precommissural striatum, ventral neurons are born before dorsal neurons laterally, but a dorsoventral gradient is seen medially. At corresponding coronal levels, PV-immunoreactive neurons of the nucleus accumbens are born shortly after PV neurons of the neostriatum. Analysis of BrdU labeling intensity in the nucleus accumbens shows that medium spiny projection neurons of the shell become postmitotic before neurons of the core. Similarly, PV-immunoreactive interneurons of the nucleus accumbens shell are born before PV interneurons of the core. Compared with cholinergic interneurons of the neostriatum, PV-immunoreactive interneurons are born later, but neurogenetic gradients are similar. The period of striatum PV interneuron genesis encompasses the period for somatostatin interneurons, although the latter neurons do not show neurogenetic gradients, possibly due to heterogeneous subtypes. Consideration of basal telencephalon neurogenesis suggests that subpopulations of striatum interneurons may share common neurogenetic features with phenotypically similar populations in the basal forebrain, with final morphology and connectivity depending on local cues provided by the host environment. J. Comp. Neurol. 389:193–211, 1997. © 1997 Wiley-Liss, Inc.     

CGMP increases in satellite cells of nitric oxide synthase-containing sensory ganglia

cGMP and the enzymes, nitric oxide synthase (NOS) and heme oxygenase-1 (HO-1), the products of which stimulate soluble guanylyl cyclase activity, were investigated in cultured dorsal root ganglia (DRG), and nodose ganglia of adult rats. A dramatic increase of cGMP-positive satellite cells in ganglia cultured for 24 or 48 h was observed, particularly in Th8-L2 DRG and in nodose ganglia. These ganglia also contained most NOS-positive neurones, as reflected by NADPH-diaphorase histochemistry. HO-1 immunoreactivity increased in satellite cells, but in different cells to those in which cGMP increased. These results suggest that both NO and CO could be involved in signalling between neurones and satellite cells in sensory ganglia during regeneration.     

Ultrastructural analysis of synapses involving tyrosine hydroxylase-containing neurons in the ventral periventricular hypothalamus of the macaque

Immunocytochemical staining for tyrosine hydroxylase (TH) in the adult macaque brain revealed a network of catecholaminergic (CA) cell bodies and fibers in the arcuate (ARC), anterior ventral periventricular (APV) and lateral suprachiasmatic nuclei (SCN). Coronal Vibratome sections immunostained with PAP or colloidal gold (15 nm) were thin sectioned and examined by electron microscopy. We examined 280 TH-immunopositive processes in individual or in serial thin sections. Of these, 190 engaged in a total of 270 synapses identified as Gray Type I asymmetrical synapses (AS) with distinct postsynaptic densities or Gray Type II symmetrical synapses (SS) without such specializations. The majority (80%) of all synapses were axodendritic, 63% of which exhibited SS and 37% AS, representing almost all of the AS observed. In nearly every case, unlabeled axon terminals containing round, 45 nm, clear vesicles and occasional small dense core vesicles contacted TH-labeled dendrites. About 15% of the synapses were dendrodendritic, all of which were symmetrical. Rare contacts involving other elements (axosomatic, dendrosomatic) constituted only 5% of the total, and occurred predominantly as SS. The predominance of AS and the prevalence of SS almost exclusively on TH-containing dendrites indicates that these CA neurons receive extensive afferent input from other neurotransmitters. TH-labeling of both neural elements in most dendrodendritic, and in some axodendritic SS, also suggests that they modulate one another within the ARC, APV and SCN. The results suggest that these CA neurons perform an important role in local integration, and may act elsewhere to affect the common final pathway of the neuroendocrine system in primates.     

Y retinal terminals contact interneurons in the cat dorsal lateral geniculate nucleus

One of the largest influences on dorsal lateral geniculate nucleus (dLGN) activity comes from interneurons, which use the neurotransmitter gamma-aminobutyric acid (GABA). It is well established that X retinogeniculate terminals contact interneurons and thalamocortical cells in complex synaptic arrangements known as glomeruli. However, there is little anatomical evidence for the involvement of dLGN interneurons in the Y pathway. To determine whether Y retinogeniculate axons contact interneurons, we injected the superior colliculus (SC) with biotinylated dextran amine (BDA) to backfill retinal axons, which also project to the SC. Within the A lamina of the dLGN, this BDA labeling allowed us to distinguish Y retinogeniculate axons from X retinogeniculate axons, which do not project to the SC. In BDA-labeled tissue prepared for electron microscopic analysis, we subsequently used postembedding immunocytochemical staining for GABA to distinguish interneurons from thalamocortical cells. We found that the majority of profiles postsynaptic to Y retinal axons were GABA-negative dendrites of thalamocortical cells (117/200 or 58.5%). The remainder (83/200 or 41.5%) were GABA-positive dendrites, many of which contained vesicles (59/200 or 29.5%). Thus, Y retinogeniculate axons do contact interneurons. However, these contacts differed from X retinogeniculate axons, in that triadic arrangements were rare. This indicates that the X and Y pathways participate in unique circuitries but that interneurons are involved in the modulation of both pathways.     

Substance P in the rat nucleus accumbens: ultrastructural localization in axon terminals and their relation to dopaminergic afferents

Dual labeling electron microscopic immunocytochemistry was used to investigate the cellular substrate for functional interactions between substance P (SP) and dopamine in the rat nucleus accumbens. Coronal vibratome sections from acrolein-fixed brains were sequentially processed for the localization of: (1) a rat monoclonal antiserum against SP identified by the peroxidase--anti-peroxidase immunocytochemical method, and (2) a rabbit polyclonal antiserum against tyrosine hydroxylase (TH) identified by immunoautoradiography. The monoclonal rat antiserum recognized principally SP, but also exhibited cross-reactivity with certain other tachykinins such as substance K. Terminals showing SP-like immunoreactivity (SPLI) were 0.2-1.5 microns in diameter and contained numerous small (30-40 nm), round vesicles; one or more large (80-150 nm), dense-core vesicles; and an occasional membrane-bound multivesicular body. From a total of 114 SP-labeled terminals that were quantitatively analyzed, 30.1% formed symmetric synapses with dendrites; whereas only 8% formed asymmetric junctions with dendritic spines. Terminals showing SPLI also occasionally formed junctions with dendrites receiving synaptic input from other terminals that were similarly labeled for the peptide or from terminals immunoautoradiographically labeled for TH. In contrast to the low frequency of postsynaptic relationships, 39.8% of the terminals containing SPLI showed close associations with other unlabeled or TH-labeled terminal or preterminal axons. The axonic contacts were characterized by equally spaced membranes that were not separated by glial processes. Within the terminals containing SPLI, vesicles were located near the axonic contacts; whereas vesicles in unlabeled terminals were located more distally with respect to these appositions. We conclude that in the rat nucleus accumbens SP or a closely related tachykinin subserves principally inhibitory functions at postsynaptic sites as indicated by the prominence of symmetric junctions. The abundance of axonic associations and sparsity of convergent input from TH- and SP-labeled terminals at closely spaced sites on dendrites supports the concepts that a SP-like tachykinin also may modulate the release of dopamine through direct or indirect presynaptic mechanisms. The possibility that there may be more extensive postsynaptic associations through convergence at widely spaced sites on common neurons is discussed.     

Synaptic microcircuitry of tyrosine hydroxylase-containing neurons and terminals in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys

A population of tyrosine hydroxylase (TH)-containing neurons that is up-regulated after lesion of the nigrostriatal dopaminergic pathway has been described in the primate striatum. The goal of this study was to examine the morphology, synaptology, and chemical phenotype of these neurons and TH-immunoreactive (-ir) terminals in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys. TH-ir perikarya were small (10-12 microm), displayed nuclear invaginations, and received very few synaptic inputs. On the other hand, TH-containing dendrites were typically large in diameter (>1.0 microm) and received scarce synaptic innervation from putative excitatory and inhibitory terminals forming asymmetric and symmetric synapses, respectively. More than 70% of TH-positive intrastriatal cell bodies were found in the caudate nucleus and the precommissural putamen, considered as the associative functional territories of the primate striatum. Under 10% of these cells displayed calretinin immunoreactivity. TH-ir terminals rarely formed clear synaptic contacts, except for a few that established asymmetric axodendritic synapses. Almost two-thirds of TH-containing boutons displayed gamma-aminobutyric acid (GABA) immunoreactivity in the striatum of parkinsonian monkeys, whereas under 5% did so in the normal striatum. These findings provide strong support for the existence of a population of putative catecholaminergic interneurons in the associative territory of the striatum in parkinsonian monkeys. Their sparse synaptic innervation raises interesting issues regarding synaptic and nonsynaptic mechanisms involved in the regulation and integration of these neurons in the striatal microcircuitry. Finally, the coexpression of GABA in TH-positive terminals in the striatum of dopamine-depleted monkeys suggests dramatic neurochemical changes in the catecholaminergic modulation of striatal activity in Parkinson's disease.     

Distribution of nitric oxide synthase-containing ganglionic neuronal somata and postganglionic fibers in the rat kidney

Nitric oxide synthase (NOS)-immunoreactive neurons were identified in the rat kidney by using an antibody against type Ia NOS and the avidin-biotin complex immunoperoxidase method in whole kidneys examined in 100 μm serial sections. The histochemical method for demonstration of the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was also used to characterize NOS-containing neurons. All somata showing NOS immunoreactivity also displayed NADPH-d activity. The greatest number of neuronal somata were observed in groups at the wall of the renal pelvis and in the angular space formed by the pole of the renal parenchyma and renal pelvic wall. They were also seen at the renal hilus close to the renal artery and along the interlobar vasculature. The size of the neuronal somata in the 35-day-old rat ranged from 13.6 to 34.8 μm, with a mean size of 21.52 ± 4.81 μm. Seventy percent, however, ranged in size from 17.8 to 26.8 μm. The shape of the neuronal somata also varied, with the majority having an ovoid or round shape. The distribution of the postganglionic fibers was investigated by means of the camera lucida. Postganglionic fibers projected into the wall of the renal pelvis and/or to the interlobar arteries extending to the arcuate arteries and to the beginning of the afferent arterioles. The NOS-immunoreactive neurons may have a vasodilator and relaxing function on the renal pelvic wall and vasculature. In addition, the presence of NOS-containing nerve fibers in nerve bundles, which are known to have predominantly vasomotor and sensory fibers, suggest that they may have a possible modulatory role on renal neural function. © 1996 Wiley-Liss, Inc.     

Y1 receptors in the nucleus accumbens: Ultrastructural localization and association with neuropeptide Y

Neuropeptide Y (NPY) is present in aspiny neurons in the nucleus accumbens (NAc), which also contains moderate levels of ligand binding and mRNA for the Y1 receptor. To determine the potential functional sites for receptor activation, we examined the electron microscopic immunocytochemical localization of antipeptide antisera against the Y1 receptor in the rat NAc. We also combined immunogold and immunoperoxidase labeling to show that, in this region, Y1 receptors are present in certain somatodendritic and axonal profiles that contain NPY or that appose NPY containing neurons. The Y1-like immunoreactivity (Y1-LI) was seen occasionally along plasma membranes but was associated more commonly with smooth endoplasmic reticulum (SER) and tubulovesicular organelles in somata and dendrites of spiny and aspiny neurons. The mean density of immunoreactive dendrites and spines per unit volume was greater in the “motor-associated” core than in the shell of the NAc. Y1-LI was also seen in morphologically heterogenous axon terminals, including those forming asymmetric excitatory-type synapses, and in selective astrocytic processes near this type of junction. We conclude that Y1 receptors play a role in autoregulation of NPY-containing neurons but are also likely to be internalized along with endogenous NPY in NAc. Our results also implicate Y1 receptors in the NAc in post- and presynaptic effects of NPY and in glial functions involving excitatory neurotransmission. In addition, they suggest involvement of Y1 receptors in determining the output of a select population of neurons associated with motor control in the NAc core. J. Neurosci. Res. 52:54–68, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural characteristics of enkephalin-immunoreactive boutons and their postsynaptic targets in the shell and core of the nucleus accumbens of the rat

The present study compared the ultrastructural morphology of enkephalin-immunoreactive boutons and their postsynaptic targets in different territories of the nucleus accumbens in the rat. The synaptic bouton profiles were identified by antibodies directed against [leu⁵]enkephalin. Ninety-five percent of the synaptic contacts were symmetric in configuration and the remaining 5% were asymmetric. Axosomatic contacts comprised 6% of all enkephalin-immunoreactive junctions and were distributed equally in all parts of the nucleus. Most (76%) synaptic terminals contacted dendrites but they contacted proportionally fewer dendrites in the shell (71%) than in the core (78%). Moreover, enkephalin-immunoreactive synaptic boutons in the shell (19%) and caudal enkephalin-rich areas (17%) of the core contacted twice as many spines than in the remaining parts of the core (8.5%). In the core, long pallidum-like dendrites were occasionally found ensheathed in enkephalin-immunoreactive terminal boutons. We conclude that the differential arrangement of enkephalinergic contacts in the shell and core could have important functional consequences, especially when considered in relation to other known morphological and neurochemical differences between these regions. © 1993 Wiley-Liss, Inc.     

Ultrastructural and immunocytochemical characterization of primary afferent terminals in the rat cuneate nucleus

The cuneate nucleus is a relay center for somatosensory information by receiving tactile and proprioceptive inputs from primary afferent fibers that ascend in the dorsal funiculus. The morphology, synaptic contacts, and neurochemical content of primary afferent terminals in the cuneate nucleus of rats were investigated by combining anterograde transport of horseradish peroxidase conjugated to wheat-germ agglutinin or to cholera toxin (injected in cervical dorsal root ganglia) with postembedding immunogold labeling for glutamate and GABA. Both tracers gave similar results. Two types of terminals were labeled: type I terminals were irregularly shaped, had a mean area of 4.0 μm², synapsed on several dendrites, and were contacted by other terminals, some of which were GABA positive. Type II terminals were dome-shaped, had a mean area of 2.18 μm², and made synaptic contact on a single dendrite. All the anterogradely labeled terminals (interpreted as endings of primary afferents) were enriched in glutamate but not in GABA. The finding that identified primary afferent terminals are enriched in glutamate with respect to other tissue profiles strongly suggests a neurotransmitter role for glutamate in this afferent pathway to the rat cuneate nucleus. © 1994 Wiley-Liss, Inc.     

Relative sparing of nitric oxide synthase-containing neurons in the hippocampal formation in Alzheimer's disease.

Nitric oxide (NO) is an endogenous neuromodulator that may mediate neurotoxic effects of glutamate. NO-synthesizing neurons are, however, resistant to NO- and glutamate-induced neurotoxicity. We now show that NO synthase neurons are selectively spared in patients with Alzheimer's disease, even in a severely affected region of the brain such as the hippocampal formation.     

Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after local cerebral ischemia in rat

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2–48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24–48 h after ischemia, neurons in the ischemia lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.