Hilar neuropeptide Y interneuron loss in the aged rat hippocampal formation

Neuropeptide Y-immunoreactive (NPY-I) interneurons in the dentate gyrus are vulnerable to various insults, including septohippocampal cholinergic deafferentation. The present study examined whether a loss of NPY-I neurons occurs during aging, when the functional integrity of the septohippocampal pathway is thought to be compromised. Sets of male Long Evans rats (consisting of young and aged rats, with and without spatial learning impairments assessed by the Morris water maze) were examined. Light microscopic analysis revealed that hilar NPY-I neuronal number in matched dorsal sections was significantly decreased in aged compared to young rats. Ultrastructural analysis disclosed that the microenvironment (the types of processes apposed to the plasmalemmal surface) of NPY-I neurons also differed significantly between young and aged rats. In particular, a subgroup of NPY-I neurons, distinguished by a higher percentage of unmyelinated axon coverage of the plasmalemmal surface, was present in young, but not aged, rats. Neither the number nor the microenvironment of NPY-I neurons significantly differed between aged animals that were impaired versus unimpaired in spatial learning performance. To our knowledge these findings represent the first report of an age-associated decline in the number of a specific, neurochemically identified neuronal subpopulation within the hippocampal formation. Additionally, they closely parallel observations in 192 IgG-saporin-lesioned animals, suggesting that a distinct subgroup of NPY-I interneurons is particularly dependent on the viability of septohippocampal cholinergic innervation for its survival. Since neuronal loss was not correlated with performance, this alteration by itself does not appear to be sufficient to produce learning impairment.     

Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.     

Glutamate decarboxylase-like immunoreactive neurons in the rat caudate putamen

GAD-IR neurons were roughly divided into those with medium sized perikarya and large perikarya. The medium-sized GAD-IR neurons accounted for about 85% of the GAD-IR neurons. The medium-sized perikarya were further divided into two, those with a smooth nuclear membrane and those with an indented nucleus. The former were very similar to medium-sized spiny neurons and the latter corresponded to medium-sized aspiny neurons. The GAD-IR large cells that were identified by light microscopy, had nuclear indentations and were divided into two classes based on their ultrastructural features, type 1 large cells received few synaptic inputs and type 2 large cells received many synaptic contacts from non-immunoreactive or immunoreactive boutons. The former resembles Type I large cells and the latter Type II large cells identified recently by Chang and Kitai; the latter are also similar to the second type of projecting neurons identified by Bolam et al.     

An ultrastructural study of dynorphin-immunoreactive nerve fibers and terminals in the celiac-superior mesenteric ganglion of the guinea pig

Dynorphin-immunoreactive nerve fibers and terminals were identified in the celiac-superior mesenteric ganglion of the guinea pig at the ultrastructural level with the peroxidase-antiperoxidase technique. The immunostained material was localized in the large dense core vesicles of the terminals but was also present diffusely in the axoplasm. The terminals formed numerous axodendritic and a few axosomatic contacts, interpreted as synapses, with the principal ganglion cells. These findings suggest that dynorphin plays a role as a neurotransmitter or neuromodulator in the ganglion and, taken together with earlier findings, indicate an involvement of dynorphin neurons in the intestino-intestinal inhibitory reflex.     

Ultrastructure of cholinergic neurons in the laterodorsal tegmental nucleus of the rat: Interaction with catecholamine fibers

The ultrastructure of choline acetyltransferase (ChAT)-immunoreactive neurons in the laterodorsal tegmental nucleus (TLD) of the rat was investigated by immunohistochemical techniques. The immunoreactive neurons were medium to large in size, with a few elongated dendrites, contained well-developed cytoplasm, and a nucleus with deep infoldings. They received many nonimmunoreactive, mostly asymmetric synaptic inputs on their soma and dendrites. ChAT-immunoreactive, usually myelinated, axons were occasionally seen in TLD. Only one immunoreactive axon terminal was observed within TLD, and it made synaptic contact with a nonimmunoreactive neuronal perikaryon. The synaptic interactions between ChAT-immunoreactive neurons and tyrosine hydroxylase (TH)-immunoreactive fibers in the TLD were investigated with a double immunohistochemical staining method. ChAT-immunoreactivity detected with a beta-galactosidase method was light blue-green in the light microscope and formed dot-like electron dense particles at the electron microscopic level. TH-immunoreactivity, visualized with a nickel-enhanced immunoperoxidase method, was dark blue-black in the light microscope and diffusely opaque in the electron microscope. Therefore, the difference between these two kinds of immunoreactivity could be quite easily distinguished at both light and electron microscopic levels. In the light microscope, TH-positive fibers were often closely apposed to ChAT-immunoreactive cell bodies and dendrites in TLD. In the electron microscope, the cell soma and proximal dendrites of ChAT-immunoreactive neurons received synaptic contacts from TH-immunoreactive axon terminals. These results provide a morphological basis for catecholaminergic regulation of the cholinergic reticular system.     

Met-enkephalin-like immunoreactivity in rat forebrain and spinal cord using hydrogen peroxide and Triton X-100. Ultrastructural study

Using two immunocytochemical methods, we have shown in light microscopy that the met-enkephalin-like immunoreactivity within striatum and spinal cord of the rat is differentially distributed in either perikarya or nerve terminals according to the technical conditions used [1]. The present electron microscopic study has been undertaken in order to elucidate the subcellular localization of immunoprecipitates according to the same technical conditions. In the neostriatum, numerous met-enkephalin-containing perikarya were stained (principally at the level of rough endoplasmic reticulum) when tissue sections were treated with hydrogen peroxide (H2O2) only, prior to the immunocytochemical procedure. However, injections of colchicine were required to demonstrate perikarya in the dorsal horn of the spinal cord. At variance with previous results, numerous dendritic profiles and nerve terminals were also reactive in this condition. Neurotubules, mitochondria, large granular vesicles (LGVs) and small synaptic vesicles were stained within these structures. The addition of a low concentration of Triton-X-100 (0.02%) in the first incubation medium often resulted in the disappearance of most perikarya and in the staining of only LGVs in nerve terminals. The addition of a higher concentration of Triton-X-100 (0.1%) produced diffusion of immunoprecipitates at the level of nerve terminals, which was probably responsible for the increased intensity of staining and, subsequently, for the better demonstration of fibre varicosities in light microscopy. On the contrary, the disappearance of reactive perikarya seemed to result from the diffusion of the non-protected peptide out of the cytoplasm. The diverse ultrastructural localizations of met-enkephalin-like immunoreactivity in striatum and spinal cord are finally discussed in light of intrinsic connections or afferents described in the literature.     

An electron microscopic observation of the vesicular acetylcholine transporter-immunoreactive fibers in the rat dorsal raphe nucleus

By using immunocytochemistry with an antibody directed against the vesicular acetylcholine transporter, many cholinergic neuronal processes were found to be immunopositive in the dorsal raphe nucleus. At the electron microscopic level, most of these processes were found to be axons. The immunopositive axon terminals made synapses on immunonegative dendrites and their spines whereas rare synapses were found between the immunopositive axon terminals and the immunonegative neuronal perikarya. Occasionally, the dendrites postsynaptic to an immunopositive axon terminal also received a synapse from an immunonegative axon terminal. The synapses made by the immunopositive axon terminals were usually symmetric and had a short active zone. Fewer immunostained dendrites were found, and they usually received asymmetric synapses from nonimmunostained axon terminals. The existence of cholinergic axon terminals and the synapses made by these terminals support the physiological data indicating that acetylcholine plays a role in the pain inhibition system in the dorsal raphe nucleus.     

Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons

The serotoninergic (5-hydroxytryptamine, 5-HT) innervation of the rat ventral tegmental area (VTA) was examined by light and electron microscopic radioautography following intraventricular infusion of [³H]5-HT. The [³H]5-HT labeled processes were characterized with respect to their regional distribution, ultrastructure and relationships with all neurons, including dopaminergic neurons, identified in the same sections using immunocytochemistry for the localization of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). By light microscopy, [³H]5-HT labeled axons and axonal varicosities were detected throughout the interfascicular nucleus and ventral portion of the VTA. By electron microscopy, [³H]5-HT-labeled axons were found to be mainly small and unmyelinated, although a few showed several lamellae of myelin. The labeled varicosities measured 0.6 μm in mean diameter and contained many small, round or flattened agranular vesicles and a few large granular vesicles. More than 18% showed synaptic specializations in single thin sections. Most of these synapses were asymmetric and established on dendritic shafts. Based on the probability of seeing such synaptic specializations in single thin sections, it was estimated that as many as 50% of the labeled 5-HT terminals formed synaptic contacts in the VTA. In dually labeled light microscopic sections, [³H]5-HT-accumulating processes often appeared adjacent to TH-immunoreactive perikarya and proximal dendrites. Electron microscopy demonstrated that terminals with radioautographic labeling for 5-HT formed conventional synapses both with TH-labeled and unlabeled dendrites in the VTA. Many additional 5-HT terminals lacking recognizable synaptic densities were directly apposed to TH-labeled dendrites and were isolated from the rest of the neuropil by thin glial leaflets. These results suggest that 5-HT neurons innervate both dopaminergic and non-dopaminergic neurons in the VTA and may influence mesocortical and mesolimbic efferent systems through synaptic as well as non-synaptic mechanisms.     

Lafora-like inclusion bodies in the CNS of aged dogs

Summary Forty randomly selected dogs, arranged into three groups according to age, were examined light and electron microscopically. Lafora-like inclusion bodies were found in all cases of aged dogs without neurological signs. They appeared as PAS positive, round bodies measuring up to 15m in diameter. Typically they had central cores and radiating peripheral lines. Electronmicroscopically, the inclusions consisted of irregularly clustered, short branching filaments measuring about 80–120 Å in diameter or of electrondense homogeneous or granular central cores with filamentous peripheral areas and located both in the perikarya and neuronal processes. The inclusions were disseminated all over the brain and spinal cord. The thalamic nuclei, a circumscribed area dorsal to the aqueduct, and the molecular layer of the superior colliculus were the most severely affected. The frequency of the inclusions showed age-dependency; the inclusions were not found in dogs younger than 2 years, but were extensive in all dogs of more than 8 years of age. The occurrence of the inclusions may therefore represent an age-dependent phenomenon. The relationship between the present findings and Lafora's disease is discussed.     

Morphometric study of synaptic patterns in the rat caudate nucleus and hippocampus under haloperidol treatment

A morphometric study of the synapses on dendritic shafts and spines was performed in the rat caudate nucleus and the CAI area of the hippocampus under chronic haloperidol treatment. In the nucleus caudatus, the synaptic density on dendritic shafts increased by 83% and those on spines by 53%. Most of the parameters measured in axospinous synapses were significantly increased: the area of presynaptic axon terminals (20%), the number of mitochondria per axon terminal (51%), the length of active zone (11%), the area of postsynaptic density (23%), and the perimeter of postsynaptic density (12.5%). The area of postsynaptic spines showed no changes. In the synapses on dendritic shafts, the area of presynaptic terminals decreased (31%), the area of mitochondria per terminal decreased (40%), the length of active zone increased (14%), and other parameters were unchanged. There were no significant differences in the same parameters measured in the hippocampus. The data are discussed as morphological correlates of behavioral supersensitivity and dopamine D2 receptor up-regulation.     

The use of semiautomatic morphometry in the study of normal rat gastrocnemius muscle fibers

Summary Based on a fixed number of EM photomicrographs a semiautomatic image analysis is made of normal gastrocnemius muscle fibers to obtain reference values for comparison with pathologic data. Z disc, mitochondria, tubular system, and sarcoplasmic reticulum are measured. It is stated that no definite fiber type differentiation is possible based on these quantitative data. However, measurements of Z disc and mitochondria are suggestive of such a differentiation. On the other hand, all data reach a Gaussian distribution on a logarithmic scale after a 5% correction factor is introduced.Supported by Fonds voor Geneeskundig Wetenschappelijk Onderzoek, FGWO no. 3.0004.82     

Glutamate-like immunoreactivity in ascending spinofugal afferents to the rat periaqueductal grey

The midbrain periaqueductal gray is a key structure for the mediation of an integrated defence behaviour. Although a prominent role for glutamate in PAG mechanisms is supported by both behavioural and morphological studies, whether PAG afferents conveying somatosensory information constitute a source of glutamatergic input to the PAG remains unknown. Here, we have compared the projection pattern of orthogradely-labelled spinoannular fibres with the distribution of glutamate-like immunoreactivity in the PAG at the light microscopic level. Transaxonal labelling was observed throughout the whole rostrocaudal axis of the PAG except for the dorsolateral regions. Cell-processes and terminal-reminiscent puncta were strongly immunoreactive in all PAG regions, including the dorsolateral areas. To ascertain whether glutamate-immunoreactive puncta observed at light microscopy indeed constituted axon terminals of the spinoannular system, glutamate-like immunoreactivity was assessed in orthogradely-labelled synaptic terminals using a post-embedding immunogold procedure for electron microscopy. Quantitative analysis of gold particle densities revealed over twice as strong an immunoreactivity in anatomically-identified spinoannular axon terminals as in dendrites postsynaptic to them, perikarya and inhibitory Gray II synapses, as well as an over 5-fold heavier immunolabelling than in glial profiles. These findings reveal that glutamate is accumulated in synaptic terminals of the spinoannular system, supporting a neurotransmitter role for this acidic amino acid in spinofugal afferents to the PAG.     

Regeneration of central catecholamine fibers in young and aged rat brain

Catecholamine histofluorescence patterns were examined in brains of young and aged rats, 1 to 14 days following neurosurgical transection of the medial forebrain bundle. At all ages examined, two phenomena were observed: degeneration of nerve fibers and vigorous regrowth of catecholamine-containing fibers in the lesion site. Regenerated catecholamine fibers invaded the area of scarred tissue. This invasion of the scarred area implies that the robust plasticity of catecholaminergic pathways, known to exist in young animals, persists in aged brain.     

Immunocytochemical study on the distribution of p53 in the hippocampus and cerebellum of the aged rat

A role for p53-mediated modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. P53 gene upregulation precedes apoptosis in many cell types, and a potential role for this molecule in apoptosis of neurons has already been demonstrated in Alzheimer's disease. Recent studies suggest that p53-associated apoptosis may be a common mechanism of cell loss in several important neurodegenerative diseases. In the present study, we examined changes in p53-immunoreactive (IR) neurons in the brains of aged rats for the first time employing immunocytochemical and in situ hybridization methods. P53-IR neurons were found in the CA1 region of hippocampus, septal region and cerebellum in the aged rats, but there was no p53-IR cell in the brains of adult rats. In the hippocampus of the aged rat, p53-IR cells predominated in the stratum oriens and pyramidal layers, while the molecular layer contained relatively few p53-IR cells. The most prominent population of immunoreactive labeling in cerebellar cortex was localised within the cell bodies of Purkinje cells and dendrites in molecular layers. Upregulation of p53 in the Purkinje cells observed in this study suggests that significant loss of Purkinje cells with aging may be regulated with several apoptosis-controlling factors including p53 and oxidative stress mechanism. Further investigations are required to establish whether direct functional relations exist between p53 and the apoptotic neuronal death in normal aging or Alzheimer brains.     

Ultrastructural alterations in caudate nucleus in aged cats

These studies provide information on the changes in the ultrastructure in the caudate nucleus of aged cats. The major findings was that there was a decrease in the density of synapses in caudate neuropil. This decrease occurred in animals after 3 years of age and remained relatively constant in older animals. In conjunction with this change a population of unusually long synapses also occurred. These larger synaptic appositions were associated with enlarged spine heads. The caudate also showed a number of qualitative ultrastructural alterations. Many neurons contained accumulations of lipofuscin or lipopigment granules in aged animals. These inclusions occurred in both soma and dendrites of neurons and all types of glial cells. A unique configuration of collapsed agranular cisterns also was observed in aged animals. The present results indicate that decreases in synaptic density may by one morphological event underlying functional alterations observed in caudate neurons in aged cats.     

Ultrastructural organization of the noradrenergic innervation of the superficial gray layer of the hamster's superior colliculus

Immunocytochemistry with an antibody-directed tyrosine hydroxylase (TH) was combined with electron microscopy and serial-section analysis to examine the synaptic organization of the catecholaminergic projection to the stratum griseum superfciale (SGS) of the hamster's superior colliculus (SC). A total of 250 TH-immunoreactive profiles within SGS were examined. Of these, 114 (45.6%) made synaptic contacts; 81 (71.1%) were axodendritic, and the remainder (33, 28.9%) were axo-axonic. Serial-section analysis was employed to evaluate the presence or absence of synaptic contacts for 26 profiles. Overall, 19 (73.1%) of the profiles followed through serial sections exhibited synaptic contacts. Double staining of single sections with antibodies directed against TH and dopamine-ß-hydroxylase (DßH) and examination in the light microscope indicated that virtually all TH-positive fibers also contained DßH. This indicated that the fibers examined at the electron microscopic level were noradrenergic rather than dopaminergic. These results suggest that norepinephrine may have both pre- and postsynaptic actions in the hamster's SC and that at least some of these effects are mediated by conventional synapses.     

Ultrastructure of the Gunn rat substantia nigra I. Cytoplasmic changes

Summary The substantia nigra of various aged hyperbilirubinemic (Gunn) rats was studied by means of electron microscopy. The cytological features observed in the neuronal somata were the presence of (1) complex membranous bodies (CMBs), (2) dilated cisternae of granular endoplasmic reticulum, (3) single membrane bound vacuoles and (4) enlarged mitochondria. Nearly every neuronal soma studied in two week old Gunn rats contained CMBs, which consisted of several layers of membrane that usually, but not always, surrounded small islands of cytoplasm. On occasion CMBs were seen to be directly connected with granular endoplasmic reticulum and, in a few instances, they were located within a cistern of endoplasmic reticulum. There were significantly fewer CMBs in the neuronal somata of adult Gunn rats. They were located peripherally in the somata or in the proximal portions of dendrites. Furthermore, in these animals the cytoplasm appeared normal and did not exhibit the features commonly seen in younger animals. Only a few hyperchromatic neurons were observed and no pronounced gliosis was evident. Therefore it is assumed that the majority of substantia nigra neurons recovered from the toxic effect of bilirubin or that the concentration of bilirubin deposited in the substantia nigra is not sufficient to be lethal. The hypothesis that is considered is that CMBs represent autophagic activity which results from exposure of neurons to bilirubin. The adjacent neurites and glia did not demonstrate the cytoplasmic changes that were characteristic of the neuronal somata.     

Increase in somatostatin immunoreactivity in the suprachiasmatic nucleus of aged Wistar rats

Decreased immunoreactivity has been reported for several neuropeptides in the aged suprachiasmatic nucleus (SCN). We compared somatostatin (SS) and substance P (SP) immunoreactivity (ir) in aged (20-26 months) and young (6 months) Wistar rats. The old rat SCN revealed a significant increase in SSir (2.6-fold) and SPir. The results show that not all SCN-neuropeptidergic systems decline with age, and suggest a specific age-related role for SS in the SCN.     

谷氨酸钠及r-氨基丁酸与大鼠尾壳核的铁代谢

BACKGROUND: Previous studies suggest that neurotransmitters can affect iron content, but the molecular mechanism is unclear. OBJECTIVE: To observe the regulation of Glutamate and GABA on iron metabolism in nigrostriatum of rats. DESIGN, TIME AND SETTING: Randomized grouping and immunohistochemistry study was performed at the institute of molecular neurobiology and neuropharmacology in Hebei normal university from July 2008 to October 2009. MATERALS: A total of 36 Male Sprague–Dawley rats were used to inject GMS or GABA into substantial nigra. METHODS: All the rats were divided into control group, GMS group and GABA group. Rats were anesthetized and positioned in a small-animal stereotaxic apparatus with a following injection of GMS and GABA. MAIN OUTCOME MEASURES: DMT1(-IRE) and HP content in substantial nigra and caudate putmanarea were studied by immunohistochemistry. RESULTS: The iron content in the caudate putamen nucleus of GMS group significantly increased, while iron content in GABA group decreased with no obvious change. At the meantime, the study showes that the expression of tyrosine hydroxylase (TH) in GMS group and GABA group shows no change. GMS significantly increase the level of DMT1(-IRE) while GABA decrease it. However, HP expression decreased in GMS group and increased in GABA group. CONCLUSION: Glutamate and GABA can affect iron metabolism through DMT1(-IRE) and HP in the nigrostriatum.