NADPH-diaphorase reactive pyramidal neurons in Ammon's horn and the subiculum of the rat hippocampal formation

doi:10.1016/0006-8993(96)00530-6

Brain Research

Volume 733, Issue 1, 9 September 1996, Pages 31-40

https://doi.org/10.1016/0006-8993(96)00530-6 Get rights and content

Abstract

NADPH-diaphorase histochemistry has been shown to stain cells which contain nitric oxide synthase, an enzyme responsible for the biosynthesis of the freely diffusable gas nitric oxide. A number of studies have mapped the distribution of NADPH-diaphorase-reactive neurons in the hippocampal formation but they have failed to yield consistent data. The major point of controversy concerns the presence of NADPH-diaphorase-reactive pyramidal cells in the CA1 subfield of the rat hippocampal formation. The present results show that CA1 pyramidal neurons do contain nitric oxide synthase (NOS) which can be reliably demonstrated with the appropriate histochemical procedure. One of the critical determinants of CA1 pyramidal cell NADPH-diaphorase activity is shown to be incubation of brains in sucrose solution prior to histochemical processing. Subicular pyramidal cells were also found to contain NOS and to possess NADPH-diaphorase activity. These results explain a number of contradictory reports in the literature relating to the presence of NADPH-diaphorase activity in hippocampal principal cells. Additionally, densitometric analysis carried out on 20 μm thick sections, from brains incubated in sucrose solution, indicated that there were characteristic gradients. The intensity of NADPH-diaphorase activity in pyramidal cells located in the ventral subiculum was found to be greater than those in the dorsal subiculum. A similar, yet marginal, trend was apparent for pyramidal cells in CA1 and CA3, as well as nonpyramidal cells in CA1. At both dorsal and ventral levels, NADPH-diaphorase-positive subicular pyramidal cells and CA1 nonpyramidal cells also show a greater intensity than CA1 or CA3 reactive pyramidal neurons. This study also showed that tissue incubation in sucrose solution prior to immunocytochemistry, enhanced immunoreactivity of the endothelial isoform of NOS whilst having little effect on neuronal NOS reactivity.

References (34)

BöhmeG.A. et al.
Possible involvement of nitric oxide in long-term potentiation
Eur. J. Pharmacol.
(1991)
BredtD.S. et al.
Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase
Neuron
(1991)
BusconiL. et al.
Endothelial nitric oxide synthase. N-terminal myristoylation determines subcellular localization
J. Biol. Chem.
(1993)
ChiangL.W. et al.
Nitric oxide synthase expression in single hippocampal neurons
Mol. Brain Res.
(1994)
EndohM. et al.
Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons
Neuroscience
(1994)
GarthwaiteJ.
Glutamate nitric oxide and cell-cell signalling in the nervous system
Trends Neurosci.
(1991)
FörstermannU. et al.
Isoforms of nitric oxide synthase. Characterization and purification from different cell types
Biochem. Pharmacol.
(1991)
HaleyJ.E. et al.
The role of nitric oxide in hippocampal long-term potentiation
Neuron
(1992)
LeighP.N. et al.
Distribution of NADPH-diaphorase positive cells in the rat brain
Comp. Biochem Physiol.
(1990)
MacklerS.A. et al.
Stimulus-induced coordinate changes in mRNA abundance in single postsynaptic hippocampal CAI neurons
Neuron
(1992)

VincentS.R. et al.

Brain heme isoenzymes and nitric oxide synthase are co-localized in select neurons

Neuroscience

(1994)

VincentS.R. et al.

Neurons that say NO

Trends Neurosci.

(1992)

VincentS.R. et al.

Histochemical mapping of nitric oxide synthase in the rat brain

Neuroscience

(1992)

BaimbridgeK.G. et al.

Calcium-binding protein distribution in the rat brain

Brain Res.

(1982)

BredtD.S. et al.

Cloned and expressed nitric oxide synthase protein structurally resembles cytochrome P450 reductase

Nature

(1990)

DinermanJ.L. et al.

Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity

EndohM. et al.

Reactive astrocytes express NADPH diaphorase in vivo after transient ischemia

Neurosci. Lett.

(1990)

Cited by (33)

Alterations in the intrinsic discharge activity of CA1 pyramidal neurons associated with possible changes in the NADPH diaphorase activity in a rat model of autism induced by prenatal exposure to valproic acid
2022, Brain Research
Citation Excerpt :
The aberrant NO signaling could therefore contribute to a variety of pathophysiological conditions, such as intellectual disabilities, excitotoxicity, Alzheimer’s disease, multiple sclerosis, neuropathological pain, and Parkinson’s disease (Steinert et al., 2010; Liu et al., 2015). Previous studies have widely reported that the CA1 pyramidal neurons contain NADPH-d (Nicotinamide adenine dinucleotide phosphate-diaphorase) activity (Vaid et al., 1996; Valtschanoff et al., 1993; Bredt et al., 1991). Histological reports have indicated an increase in the number of NADPH-d positive neurons in the CA1 region of the hippocampus as a consequence of epilepsy that is comorbid with ASD (Talavera et al., 1997; Lerner-Natoli et al., 1994).
Autism spectrum disorder is a neurodevelopmental disorder characterized by sensory abnormalities, social skills impairment and cognitive deficits. Although recent evidence indicated that induction of autism-like behavior in animal models causes abnormal neuronal excitability, the impact of autism on neuronal properties is still an important issue. Thus, new findings at the cellular level may shed light on the pathophysiology of autism and may help to find effective treatment strategies. Here, we investigated the behavioral, electrophysiological and histochemical impacts of prenatal exposure to valproic acid (VPA) in rats. Findings revealed that VPA exposure caused a significant increase in the hot plate response latency. The novel object recognition ability was also impaired in VPA-exposed rats. Along with these behavioral alterations, neurons from VPA-exposed animals exhibited altered excitability features in response to depolarizing current injections relative to control neurons. In the VPA-exposed group, these changes consisted of a significant increase in the amplitude, evoked firing frequency and the steady-state standard deviation of spike timing of action potentials (APs). Moreover, the half-width, the AHP amplitude and the decay time constant of APs were significantly decreased in this group. These changes in the evoked electrophysiological properties were accompanied by intrinsic hyperexcitability and lower spike-frequency adaptation and also a significant increase in the number of NADPH-diaphorase stained neurons in the hippocampal CA1 area of the VPA-exposed rats. Taken together, findings demonstrate that abnormal nociception and recognition memory is associated with alterations in the neuronal responsiveness and nitrergic system in a rat model of autism-like.
Adult brain nitrergic activity after concomitant prenatal exposure to ethanol and methyl mercury
2010, Acta Histochemica
Pregnant rats were exposed to ethanol (EtOH) and/or methyl mercury (MeHg) during fetal brain development. Nitrergic activity was quantified by densitometric measurement of formazan deposits in the hippocampus, cerebellum and striatum of two-month-old offspring following histochemical assay for NADPH-diaphorase (NADPH-d) activity. Compared to control subjects, an increase in nitrergic activity was found in the molecular layer of dentate gyrus and in the lacunosum molecular and stratum radiatum of CA1 (cornus amoni 1) in the EtOH+MeHg group, whereas a single administration of EtOH increased the activity in all striatal segments. The cerebellum seems to be less sensitive at this time-point to intoxication, and presented an increase only at the molecular layer of EtOH-exposed animals when compared to the MeHg and EtOH+MeHg groups (ANOVA, one-way followed by Tukey's test, p<0.05 or p<0.01). Taken together, results suggest that developmental exposure to EtOH and MeHg, singularly or in combination, alters nitrergic activity in adult rat in different ways depending on the region and layer of the central nervous system (CNS), and that these alterations might be related to different local metabolic properties.
Oxytocin induces penile erection when injected into the ventral subiculum: Role of nitric oxide and glutamic acid
2010, Neuropharmacology
Citation Excerpt :
First, the ventral subiculum contains oxytocinergic fibres (Buijs, 1980; Sofroniew, 1980; Hawthorn et al., 1985; Yoshimura et al., 1993), oxytocin receptors and oxytocin receptor messenger RNA (Vaccari et al., 1998; Yoshimura et al., 1993; Adan et al., 1995; for a review see Lee et al., 2009). Second, pyramidal NO-containing neurons of the projection-type not GABAergic-type are present in the ventral subiculum (Valtschanoff et al., 1993; Vaid et al., 1996; Seress et al., 2002). As oxytocin was found to be ineffective on spontaneous inhibitory post-synaptic currents (IPSCs) of ventral subiculum pyramidal neurons (Ogier et al., 2008), it is likely that oxytocin receptors in the ventral subilculum, whose stimulation leads to penile erection, are located in these pyramidal NO-containing neurons of the projection type.
Oxytocin (100 ng) induces penile erection when injected unilaterally into the ventral subiculum of the hippocampus of male rats. The pro-erectile effect started mostly 30 min after treatment and occurred 15 min after an increase in both nitric oxide (NO) production, measured by the concentration of NO₂⁻ and NO₃⁻, the main metabolites of newly formed NO, and extra-cellular glutamic acid concentration in the dialysate obtained from the ventral subiculum by intracerebral microdialysis. These responses were abolished by d(CH₂)₅Tyr(Me)²-Orn⁸-vasotocin (2 μg), an oxytocin receptor antagonist, S-methyl-l-thiocitrulline (SMTC), a selective inhibitor of neuronal NO-synthase (25 μg), and haemoglobin, a NO scavenger (25 μg), given into the ventral subiculum before oxytocin. Unlike d(CH₂)₅Tyr(Me)²-Orn⁸-vasotocin, SMTC and haemoglobin, (+)MK-801 (5 μg), a noncompetitive antagonist of NMDA receptors abolished oxytocin-induced penile erection, but reduced only partially the increase in NO production and extra-cellular glutamic acid. As NMDA (0.25–1 μg) injected into the ventral subiculum induces penile erection episodes, which also occurred with an increase of NO production and extra-cellular glutamic acid, and NMDA responses were abolished by (+)MK-801 (5 μg), but not by SMTC (25 μg) or haemoglobin (25 μg), injected into the ventral subiculum, these results show that oxytocin injected into the ventral subiculum increases NO production by activating its own receptors. NO in turn increases glutamic acid neurotransmission, leading to penile erection, possibly through neural (glutamatergic) efferent projections from the ventral subiculum to extra-hippocampal brain areas (e.g., prefrontal cortex) modulating the activity of mesolimbic dopaminergic neurons.
A simple and fast densitometric method for the analysis of tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta and in the ventral tegmental area
2005, Brain Research Protocols
Parkinson's disease is a progressive dyskinetic disorder caused by degeneration of mesencephalic dopaminergic neurons in the substantia nigra pars compacta (SNpc) and, to a lesser extent, in the ventral tegmental area (VTA). Tyrosine hydroxylase (TH) is a rate-limiting enzyme for dopamine synthesis, therefore immunohistochemistry for TH can be used as an important marker of dopaminergic cell loss in these regions. Traditionally, immunohistochemical experiments are analyzed qualitatively by optical microscopic observation or more rarely semi-quantitatively evaluated by densitometry. A common problem with such papers is the lack of a clear explanation of the algorithms and macros employed in the semi-quantitative approaches. In this paper, we describe, in detail, an easy, fast and precise protocol for the analysis of TH immunoreactivity in SNpc and VTA using one of the most popular image analysis software packages (Image Pro-Plus). We believe that this protocol will facilitate the evaluation of mesencephalic TH immunoreactivity in various available animal models of Parkinson's disease.
Structural and functional abnormalities of the hippocampal formation in rats with environmentally induced reductions in prepulse inhibition of acoustic startle
2001, Neuroscience
The effects of social isolation on prepulse inhibition of acoustic startle (PPI), electrophysiology and morphology of subicular pyramidal neurons and the densities of interneuronal sub-types in the hippocampal formation were examined. Wistar rats (male weanlings) were housed socially (socials, n=8) or individually (isolates, n=7). When tested eight weeks later, PPI was lower in isolates. Rats then received terminal anaesthesia before slices of hippocampal formation were made in which the electrophysiological properties of a total of 108 subicular neurons were characterized. There were no differences in neuronal sub-types recorded in socials compared with isolates. Intrinsically burst-firing and regular spiking pyramidal neurons were examined in detail. There were no differences in resting membrane potential or input resistance in isolates compared with socials but action potential height was reduced and action potential threshold raised in isolates. A limited morphological examination of Neurobiotin-filled intrinsically burst-firing neurons did not reveal differences in cell-body area or in number of primary dendrites. Sections from the contralateral hemispheres of the same rats were stained with antibodies to calretinin, parvalbumin and the neuronal isoform of nitric oxide synthase (nNOS). In isolates, the density of calretinin positive neurons was increased in the dentate gyrus but unchanged in areas CA3, CA1 and subiculum. Parvalbumin and nNOS positive neuronal densities were unchanged.
Hence in rats with environmentally induced reductions in PPI there are structural and functional abnormalities in the hippocampal formation. If the reduction in PPI stems from these abnormalities, and reduced PPI in rats is relevant to schizophrenia, then drugs that correct the reported electrophysiological changes might have antipsychotic effects.
Chapter III Comparative and developmental neuroanatomical aspects of the NO system
2000, Handbook of Chemical Neuroanatomy
This chapter discusses the anatomical aspects of the distribution of nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in the nervous system of all major groups of vertebrates and during the development of the mammalian brain. In the central nervous system, three NOS isoforms—namely, neuronal, endothelial, and immunological—have been detected; the neuronal isoform being the most widely expressed but not exclusive in neural elements. Although the NOS-positive neurons were originally described as “solitary active cells,” there are brain regions where neuronal NOS is expressed by dense populations of neurons, such as the cerebellum, the olfactory bulb, or the hypothalamic supraoptic nucleus, and not by isolated, scattered cells. Endothelial NOS is constitutively expressed not only in blood vessels but also in some neural cells, occasionally in the same population as neuronal NOS. While both neuronal and endothelial isoforms are constitutively expressed in the nervous system, immunological NOS can be detected in neural elements only after the induction of its expression in experimental or pathological situations.

View all citing articles on Scopus

View full text

NADPH-diaphorase reactive pyramidal neurons in Ammon's horn and the subiculum of the rat hippocampal formation

Abstract

Eur. J. Pharmacol.

Neuron

J. Biol. Chem.

Mol. Brain Res.

Neuroscience

Trends Neurosci.

Biochem. Pharmacol.

Neuron

Comp. Biochem Physiol.

Neuron

Neuroscience

Trends Neurosci.

Neuroscience

Calcium-binding protein distribution in the rat brain

Brain Res.

Cloned and expressed nitric oxide synthase protein structurally resembles cytochrome P450 reductase

Nature

Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity

Reactive astrocytes express NADPH diaphorase in vivo after transient ischemia

Neurosci. Lett.