The nitric oxide-producing neurons in the rat amygdala (Am) were studied, using reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. Almost all nuclei of the Am contained NADPHd-positive neurons and fibers, but the somatodendritic morphology and the intensity of staining of different subpopulations varied. The strongly stained neurons displayed labeling of the perikaryon and the dendritic tree with Golgi impregnation-like quality, whilst the dendrites of the lightly stained neurons were less successfully followed. Many strongly positive neurons were located in the external capsule and within the intraamygdaloid fiber bundles. A large number of small, strongly stained cells was present in the amygdalostriatal transition area. In the Am proper, a condensation of deeply stained cells occurred in the lateral amygdaloid nucleus. In the basolateral nucleus, the strongly NADPHd-positive neurons were few, and were located mainly along the lateral border of the nucleus. These cells clearly differed from the large, pyramidal, and efferent cells. The basomedial nucleus contained numerous positive cells but most of them were only lightly labeled. A moderate number of strongly stained neurons appeared in the medial division of the central nucleus, and a larger accumulation of strongly positive cells was present in the lateral and the capsular divisions. The medial amygdaloid nucleus contained numerous moderately stained neurons and displayed the strongest diffuse neuropil staining in Am. In the nucleus of the lateral olfactory tract, the first layer contained only NADPHd-stained axons, in the second layer, there were numerous moderately stained cells, and in the third layer, a few but deeply stained neurons. From the cortical nuclei, the most appreciable number of stained neurons was seen in the anterior cortical nucleus. The anterior amygdaloid area contained numerous NADPHd-positive neurons; in its dorsal part the majority of cells were only moderately stained, whereas in the ventral part the neurons were very strongly stained. The intercalated amygdaloid nucleus lacked NADPHd-positive neurons but an appreciable plexus of fine, tortuous axons was present. In the intra-amygdaloid part of the bed nucleus of the stria terminalis (st) some lightly stained cells were seen but along the entire course of st strongly stained neurons were observed. Some Am nuclei, and especially the central lateral nucleus and the intercalated nucleus, display considerable species differences when compared with the primate Am. The age-related changes of the nitrergic Am neurons, as well as their involvement in neurodegenerative diseases is discussed.

A recently revealed important function of the amygdala (Am) is that it acts as the brain’s “lighthouse”, which constantly monitors the environment for stimuli which signal a threat to the organism. The data from patients with extensive lesions of the striate cortex indicate that “unseen” fearful and fear-conditioned faces elicit increased Am responses. Thus, also extrageniculostriate pathways are involved. A multisynaptic pathway from the retina to the Am via the superior colliculus (SC) and the pulvinar was recently suggested. We here present data based on retrograde neuronal labeling following injection of the fluorescent tracer Fluoro-Gold in the rat Am that the parabigeminal nucleus (Pbg) emits a substantial, bilateral projection to the Am. This small cholinergic nucleus (Ch8 group) in the midbrain tegmentum is a subcortical relay visual center that is reciprocally connected with the SC. We suggest the existence of a second extrageniculo-striate multisynaptic connection to Am: retina–SC–Pbg–Am, that might be very effective since all tracts listed above are bilateral. In addition, we present hodological details on other brainstem afferent connections of the Am, some of which are only recently described, and some others that still remain equivocal. Following selective injections of Fluoro-Gold in the Am, retrogradely labeled neurons were observed in parasubthalamic nucleus, peripeduncular nucleus, periaqueductal gray, dopaminergic nuclear complex (substantia nigra pars lateralis and pars compacta, paranigral, parabrachial pigmented and interfascicular nuclei, rostral and caudal linear nuclei, retrorubral area), deep mesencephalic nucleus, serotoninergic structures (dorsal, median and pontine raphe nuclei), laterodorsal and pedunculopontine tegmental nuclei (Ch6 and Ch5 groups), parabrachial nuclear complex, locus coeruleus, nucleus incertus, ventrolateral pontine tegmentum (A5 group), dorsomedial medulla (nucleus of the solitary tract, A2 group), ventrolateral medulla (A1/C1 group), and pars caudalis of the spinal trigeminal nucleus. A bilateral labeling of the upper cervical spinal cord was also observed.

The dorsal column nuclei (DCN; gracile and cuneate nuclei) give rise to the medial lemniscus, the fibre system that provides an organised somatosensory input to the thalamus. Unlike the spinothalamic and trigeminothalamic tracts that project, also to the ipsilateral thalamus, the medial lemniscus system is believed to be entirely crossed. We demonstrate that DCN emit a small number of axons that reach the ipsilateral thalamus. As retrograde fluorescent neuronal tracer Fluoro-gold was stereotaxically injected in the ventrobasal thalamus of nine young adult Wistar rats. The injection foci were voluminous and encroached upon adjacent nuclei, but the periphery of the injection halo never spilled over to the contralateral thalamus. All sections of the contralateral gracile and cuneate nuclei and the midline nucleus of Bischoff contained abundant retrogradely labelled neurons. The comparison with the Nissl-stained parallel sections suggests that approximately 70–80% of the DCN neurons project to the contralateral thalamus. Counting of retrogradely labelled neurons in two cases revealed 4,809 and 4,222 neurons in the contralateral and 265 and 214 in the ipsilateral DCN, respectively. Thus, although less prominent than the ipsilateral spinothalamic tract, the lemniscal system also emits an ipsilateral projection that accounts for about 5% of the neuronal population in DCN that innervates the ventrobasal thalamus.

Glutamate was found to be an excitatory neurotransmitter in the enteric nervous system. Although several lines of evidence indicate a role of glutamate in the regulation of gut motility and secretion the physiological significance of glutamatergic transmission is not clear yet. We studied the effect of glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining in longitudinal muscle strips with attached myenteric plexus of guinea pig ileum. l-Glutamate (100 μM) significantly enhanced both the evoked [3H]acetylcholine release and the optical density of nicotinamide adenine dinucleotide phosphate-diaphorase positive neurones, i.e. the intensity of staining. The non-competitive N-methyl-d-aspartate receptor antagonist MK-801 (3 μM) abolished the stimulatory effect of l-glutamate on acetylcholine efflux. Similarly, the nitric oxide synthase inhibitor Nω-nitro-l-arginine (100 μM) significantly reduced the effect of l-glutamate on [3H]acetylcholine release and nicotinamide adenine dinucleotide phosphate-diaphorase staining. Our data suggest that endogenous nitric oxide seems to mediate the stimulatory effect of glutamate on acetylcholine release from guinea pig myenteric neurons.

Abstract  Muscarinic receptor activation by (4‐Hydroxy‐2‐butynyl)‐1‐trimethylammonium‐m‐chlorocarbanilate chloride (McN‐A‐343) was investigated both on NADPH‐d staining and on electrically induced responses in guinea‐pig gastric fundus. McN‐A‐343 (10 μmol L−1) significantly increased the optical density of NADPH‐d positive neurones, while blockade of nitric oxide synthase with Nω‐nitro‐L‐arginine (L‐NA) decreased it, suggesting facilitation of nitric oxide (NO) production. Electrical field stimulation (EFS; 2 Hz, 0.2 ms, supramaximal current intensity, 10 s train duration) elicited on‐contraction followed by off‐relaxation in the circular muscle strips. McN‐A‐343 (10 μmol L−1) transformed the EFS‐evoked response from on‐contraction into on‐relaxation, which was neurogenic, tetrodotoxin‐sensitive and hexamethonium‐resistant. L‐NA partly reduced the EFS‐evoked relaxation, revealing two components: a nitrergic and a non‐nitrergic one. The effect of McN‐A‐343 on the amplitude of the EFS‐evoked relaxation was not changed by the M3 receptor antagonist para‐fluoro‐hexahydro‐sila‐difenidol hydrochloride, but was significantly enhanced by M1 receptor blockade with telenzepine. In the presence of telenzepine, the L‐NA‐dependent nitrergic component of the EFS‐induced relaxation predominates. We suggest that cholinergic receptor activation has a dual effect on nitrergic neurotransmission: (i) stimulation of NOS by muscarinic receptor(s) different from M1 and M3 subtype, (ii) prejunctional inhibition of NO‐mediated relaxation via M1 receptors. In addition, M1 receptors may facilitate the non‐nitrergic relaxation.

The age-related differences in the synapses of the paraventricular hypothalamic nucleus (PVN) were studied with transmission electron microscopy in young (3-month-old) and senescent (25-month-old) Wistar rats. The changes in the axodendritic (ADS) and axospinous (ASS) synapses in the PVN neuropil were determined by morphometry. The majority of synapses were of the ADS type. In senescent rats the density of both ADS and ASS was diminished. The mean area of the ASS presynaptic boutons in aged rats was more enlarged than of the boutons in the ADS. The parameters characterising synaptic vesicles (SV) were diminished in aging rats. The number of SV per synaptic bouton decline was not significantly different. The changes in the length of synaptic contact zone (SCZ) were not significant with the exception of the total SCZ length per 1000 microm2 and the total area of the SCZ per 1000 microm3 in the ADS. The decrease of the number of dendritic spines (DS) per 1000 microm2 in the neuropil was significantly age-related diminished. Moreover, the mean area of the DS was significantly enlarged.

(2002). Neuromelanin in the Human Brain: A Review and Atlas of Pigmented Cells in the Substantia Nigra. Archives of Physiology and Biochemistry: Vol. 110, No. 4, pp. 257-369.

The effects of age on the dendritic tree of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons in the dorsolateral neuronal column of the periaqueductal gray (DLPAG) in the rat were examined, using NADPH-d histochemistry and computer-assisted analysis. In 26-month-old rats some of the neurons exhibited considerably branched dendrites, while others show amputation stumps, or local swellings in the initial dendritic portion. Quantitative analysis demonstrated that the number of dendritic segments in DLPAG increased with advancing age. The mean segment length and the mean length of the terminal segment significantly increased in 12-month-old rats compared to 3-month-old rats and decreased in 26-month-old rats in comparison to 12-month-old, as the reduction was significant for the terminal segments only. The total dendritic length of NADPH-d-stained neurons increased with aging. The increase was more pronounced in 12-month-old rats compared to 3-month-old rats and the difference was significant in 26-month-old rats. The number of the nodal vertices, Va and Vb, significantly increased between 12- and 26-month of age. The dendritic reorganization was considered a sign of compensatory mechanism counteracting the degenerative changes.

1. The effect of activation of nicotinic cholinoceptors in rat duodenal segments following electrical field stimulation (EFS) was investigated. 2. Electrical field stimulation elicited a two-component response: transient relaxation followed by contraction. The EFS-evoked response was tetrodotoxin (TTX; 1 mumol/L) sensitive. The relaxation component was NG-nitro-L-arginine (L-NNA; 100 mumol/L) sensitive, while the contractile response was atropine (1 mumol/L) sensitive. 3. 1,1-Dimethyl-4-phenyl-piperazinium iodide (DMPP; 20 mumol/L) induced relaxation of spontaneously active preparations that was L-NNA sensitive. L-Arginine (1 mmol/L) reversed the effects of L-NNA on DMPP-induced relaxation. 4. When EFS was applied, DMPP increased the amplitude of the relaxation component of the response and reduced the contractile component. 5. In the presence of L-NNA, the effect of DMPP on the relaxation component of the response to EFS was reduced, but the contractile response was not affected. L-Arginine partly reduced this effect of L-NNA. 6. Neither propranolol (1 mumol/L) nor yohimbine (1 mumol/L) had any effect on the actions of DMPP on EFS-evoked responses, but prazosin (1 mumol/L) strongly reduced the effect of DMPP on the contractile component of the response to EFS and slightly reduced the effect of DMPP on the relaxation response. 7. Histochemical studies demonstrated that, in the myenteric plexus of the rat duodenum, there are many reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons and that their number decreased after treatment with L-NNA. In the presence of L-arginine and L-NNA, the number of NADPH-d-positive neurons was similar to that found in control samples. 8. The data suggest that activation of nicotinic cholinoceptors modulates EFS-evoked responses in the rat duodenum as a result of the potentiation of nitrergic and adrenergic neurotransmission.

The differentiation of the argyrophil, argentaffin, 5-hydroxytryptamine (5-HT)-, somatostatin (SOM)-, cholecystokinin (CCK)-, substance P (SP)-, methionine-enkephalin (Met-Enk)- and vasoactive intestinal polypeptide (VIP)-immunoreactive entero-endocrine cells (EECs) was examined in human fetuses. A great increase in the frequency of EECs in the duodenum and the rectum was observed between the 7th and 12th gestation week. The differentiation of the EECs advanced distally in the small intestine and proximally in the large intestine. In 24-25-week-old fetuses the frequency of the EECs was also increased in the ileum and the colon. A different time-course of the appearance and differentiation of the types of EECs was observed. Met-Enk- and VIP-immunoreactive endocrine cells were not detected at any age. A regional difference in the frequency and morphology of the endocrine cell types was observed in the eldest fetuses.

The morphological appearance and quantitative parameters characterizing the dendrites of NADPH–diaphorase-stained neurons in the laterodorsal (LDT) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats were studied. All dendritic segments were classified according to the number of terminal and link segments they drain and the vertex analysis was used to quantify the dendritic tree and to determine its configuration. Morphological aberrations of the dendrites as local swelling, nodulation, thinning, shrinkage, folding and even the appearance of stumps were observed with advancing age. The quantitative analysis demonstrated a significant reduction (one-way ANOVA) of the total dendritic length, mean terminal path length, maximal segment length, total segment number and number of terminal segments at the rostral two thirds of the LDT and in the PPN. The mean vertex path length and the mean segment length significantly decreased only at the rostral level of the LDT. Plotting of the segment length against equivalent orders showed a decrease in all generations of the dendritic segments. The vertex ratios remained constant and indicated that the configuration of the dendritic tree remained unchanged during aging. The alterations in the dendrites mainly developed after 12 months of age. The age-related changes in the morphology and quantitative parameters of the dendrites in the rostral two thirds of the LDT and PPN were rather similar, which could be explained by the common anatomical, neurochemical and electrophysiological features. Thus, the present results suggest a mild, but continuous regression of the dendritic tree of the rat LDT and PPN in normal aging.

The aim of the present study was to compare the morphological pattern and the quantitative parameters of nitric oxide (NO)-containing neurons in the laterodorsal (LTD) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats. NADPH-diaphorase (NADPH-d) histochemical reaction, as a marker of the cholinergic neurons in the two mesopontine nuclei, and computer-assisted image analysis were used. The relationships between the neurons stained for NADPH-d and choline acetyltransferase (ChAT) were examined using a double-labelling procedure. The results demonstrated only occasional ChAT positive somata that did not exhibit NADPH-d staining. The volume of the LTD and PPN and the number of NADPH-d neurons remained unaltered with advancing age. However, ANOVA demonstrated a significant effect of age and level on the cross-sectional areas, maximum diameters and staining intensity of NADPH-d somata in the LTD and PPN. The three parameters were reduced in 26-month-old rats compared to 3-month-old rats. The changes in the morphological appearance of NADPH-d somata and processes as well as the quantitative analysis pointed to age-related neuronal atrophy. It was accompanied by hypertrophy of some neighbouring neurons, suggesting a compensatory mechanism which would counteract the degenerative changes. The age-dependant alterations in the LTD and PPN were rather similar.