Cyto- and chemoarchitecture of the cerebral cortex of an echidna (Tachyglossus aculeatus). II. Laminar organization and synaptic density

We have examined the distribution and morphology of neurons immunoreactive for nonphosphorylated neurofilament protein (SMI-32 antibody), calcium-binding proteins (parvalbumin, calbindin, calretinin), and neuropeptide Y as well as neurons reactive for NADPH diaphorase in the cerebral cortex of the Australian short-beaked echidna (Tachyglossus aculeatus). We have also studied synaptic morphology and density in S1 somatosensory cortex and assessed parameters associated with metabolic activity of the cerebral cortex (vessel volume density, mitochondrial volume density, and mitochondrial numerical density) in semi- and ultrathin sections. SMI-32 immunoreactivity was found mostly in layer V pyramidal neurons in selected cortical regions (S1, PV, V1, A). These neurons often showed atypical morphology compared with therian cortex. Neurons immunoreactive for calcium-binding proteins were broadly similar in both morphology and distribution to those seen in therian cortex, although calretinin-immunoreactive neurons were rare. Both Gray type I and Gray type II synapses could be identified in echidna S1 cortex and were similar to those seen in therian cortex. Peak synaptic density was in upper layer IV, followed by layer I, lower layer II, and upper layer III. Most synapses were of type I (72%), although types I and II were encountered with similar frequency in lower layer II and upper layer III. The capillary volume fraction values obtained for the echidna (from 1.18% in V1 to 1.34% in S1 cortex) fall within the values for rodent cortex. Similarly, values for mitochondrial volume fraction in echidna somatosensory cortex (4.68% +/- 1.76%) were comparable to those in eutherian cortex.     

Deafferentation-induced abnormal neurofilament phosphorylation in red nucleus neurones

Hippocampal deafferentation has been proposed as a pathogenetic mechanism for neurofibrillary tangle (NFT) formation in human mesolimbocortical dementia. We previously developed a rodent model of hippocampal deafferentation involving bilateral destructive lesions of the ventrotegmental area (VTA), septum of the medial forebrain and entorhinal cortex combined with pharmacological inhibition of serotonin 5-HT2 and dopamine D1 receptors. Unexpectedly, we observed an alteration in phosphorylated neurofilament protein immunoreactivity and argyrophilia in magnocellular neurones of the red nucleus. Here, we determined the neuroanatomical, pharmacological and temporal requirements for this effect on red nucleus neurones. We found that abnormal phosphorylation and argyrophilia were critically dependent on bilateral destruction of the VTA and antagonism of 5-HT2 receptors. Although extensive neurofilament hyperphosphorylation and argyrophilia were observed in red nucleus magnocellular neurones within nine days of treatment, no NFTs were formed and these effects were transitory. Resolution of these cytoskeletal abnormalities was accompanied by increased expression of the calcium binding protein, parvalbumin, suggesting that alterations in intraneuronal calcium levels may modify the deafferentation response.     

大鼠三叉神经脊束核尾侧亚核Ⅱ层内CB样,PV样阳性结构的分布及？…

本研究用免疫组织化学方法观察了Ｃａｌｂｉｎｄｉｎ Ｄ－２８ｋ（ＣＢ）样和Ｐａｒｖａｌｂｕｍｉｎ（ＰＶ）样胞体、纤维和终末在三叉神经脊束核尾侧亚核（Ｖｃ）Ⅱ层内的公布及它们的突触联系。在光镜下观察到ＣＢ样和ＰＶ样阳性胞体、纤维和终末在ＩＩ层内侧带（ＩＩｉ）最为密集，ＰＶ样阳性神经元的胞体稍大，但数量少于ＣＢ样阳性神经元。在电镜下观察到ＣＢ样或ＰＶ样阳性结构主要形成下列４种突触联系：⑴阳性轴突终末与阳     

Glutamic-acid-decarboxylase-and parvalbumin-like-immunoreactive structures in the olfactory bulb of the human adult

This study examines the distribution and morphological characteristics of glutamic-acid-decarboxylase-like (GAD)- and parvalbumin-like (PA)-immunoreactive structures in the olfactory bulb of the human adult. GAD-immunoreactive somata occurred in the glomerular layer, the external granule cell layer, the more superficial portion of the external plexiform layer, and the internal granule cell layer. The cells were small- to medium-sized. Demonstration of lipofuscin pigment revealed the presence of unpigmented as well as pigmented neurons, thus suggesting the existence of two subpopulations of GAD-positive neurons. GAD-immunoreactive puncta and/or fibers were mainly seen in the periglomerular region and the internal granule cell layer. All other layers of the bulb, as well as the intrabulbar portion of the anterior olfactory nucleus, displayed considerably less of these puncta and/or fibers. The olfactory nerve layer remained practically clear of immunoreactive material. PA-immunoreactive somata occurred in the glomerular layer and both the external and internal granule cell layer. Only a small number of immunoreactive nerve cells were encountered within the white matter or the olfactory tract. Most PA-positive neurons displayed characteristics of short axon cells whereas a few others resembled van Gehuchten cells. All of the PA-immunoreactive neurons were devoid of lipofuscin pigment. Immunoreactive puncta and fibers were present in all layers though predominating in the periglomerular region, the olfactory nerve layer, and the internal granule cell layer. The intrabulbar portions of the anterior olfactory nucleus did not show any immunoreactive structures.     

大鼠脑干内三叉神经本体觉中枢通路中小白蛋白样阳性神经元的分布与发育

应用免疫组织化学技术对脑干内三叉神经本体觉中枢通路中PV样阳性神经元的分布与发育进行了观察.结果发现:①早在胚胎13 d时,首先在三叉神经中脑核(Vme)内观察到许多含小白蛋白(Parvalbumin,PV)样阳性神经元,主要为大的假单极神经元,呈中等阳性反应.②生后3 d时,Vme内PV样阳性神经元的数量明显增多,免疫反应呈强阳性,并可观察到Probst束呈强阳性反应.③生后10 d时,在三叉神经脊束核吻侧亚核背内侧部(Vodm)及三叉神经感觉主核背内侧部(Vpdm)均出现少量中等强度的PV样阳性神经元.④生后14 d时,三叉神经脊束核吻侧亚核邻接的外侧网状结构(LRF),三叉上核尾外侧部(Vsup-CL),三叉神经运动核腹侧区(AVM)及上橄榄核背侧区(ADO)均出现PV样阳性神经元.⑤生后21 d时,Vodm和LRF(Vodm-LRF)区、"带状区"包括Vpdm,Vsup-CL,ADO,AVM及Probst束内的PV样阳性神经元及纤维的分布均达成年大鼠水平.上述结果表明,大鼠脑干内三叉神经本体觉中枢通路中PV样阳性神经元的分布、发育以及PV样阳性纤维的形成可能与胚胎及生后发育期该通路中神经元的功能成熟有关.     

Neuropeptide Y (NPY)-immunoreactive neurons in the primate fascia dentata; occasional coexistence with calcium-binding proteins: a light and electron microscopic study.

Neuropeptide Y (NPY)-containing neurons are known to be highly vulnerable following sustained electrical stimulation in rats and in humans suffering from temporal lobe epilepsy. This has been related to a strong excitatory input. In contrast, there is evidence that neurons containing calcium-binding proteins exhibit a high resistance under experimental seizure and hypoxia conditions. The aim of this study was to determine the coexistence of NPY and calcium-binding proteins in inhibitory neurons of the primate fascia dentata and their synaptic connections. Vibratome sections of hippocampi of African green monkeys (Cercopithecus aethiops) were immunostained with antibodies against NPY, PARV, and CB. A quantitative coexistence study was performed for NPY and PARV on consecutive semithin sections. In contrast to the rodent hippocampus, NPY-immunoreactive neurons were found exclusively in the hilus of fascia dentata with horizontally oriented dendrites which did not extend into the granular and molecular layer. Conversely, PARV-immunoreactive neurons were also present in the granular and inner molecular layer and extended their dendrites far out in the molecular layer and the hilus. Axon terminals immunoreactive for NPY were mostly concentrated in the middle and outer molecular layer and the hilar region and were rare in the granular layer. PARV-immunoreactive boutons were basically restricted to the granular layer where they formed typical baskets. The antibody against calbindin stained almost exclusively granule cells. Coexistence of NPY- and PARV-immunoreactivity was found only in hilar neurons and was rare (9 out of 152 cells analyzed). These results suggest that most NPY-immunoreactive neurons do not contain calcium-binding proteins. NPY-containing neurons exhibited ultrastructural characteristics as described for inhibitory neurons. Their dendrites were only sparsely contacted by mostly asymmetric synaptic terminals, including a very small number of mossy fiber axon terminals. In turn, numerous NPY-immunoreactive axon terminals formed symmetric synapses with spines and dendritic shafts of unlabeled neurons in the middle and outer molecular layer, whereas no contact with granule cell bodies was evident. Thus, we conclude that the vulnerability of NPY-containing inhibitory neurons may be due more to the lack of calcium-binding proteins than to a strong excitatory innervation. As their axons may contribute to the inhibitory control of the major excitatory input from the entorhinal cortex, their loss following overstimulation may play a role in perpetuating hippocampal seizure activity.     

Postnatal subventricular zone progenitors give rise not only to granular and periglomerular interneurons but also to interneurons in the external plexiform layer of the rat olfactory bulb

Interneurons in the granule cell layer (GCL) and glomerular layer (GL) of the olfactory bulb (OB) are generated from progenitors in the subventricular zone (SVZ) of the lateral ventricle. However, little is known about the origin of interneurons in the external plexiform layer (EPL) of the OB. On the basis of the concept of corticogenesis, I hypothesized that interneurons in the EPL of the rodent OB also originate in the SVZ. In the present study, replication-incompetent retroviruses encoding a marker gene, human placental alkaline phosphatase (AP), were injected into the lateral ventricles of postnatal day 4 Wistar rats to label dividing cells in the SVZ. Two days after injection, some of the AP-labeled cells had migrated into the OB. Five weeks after injection, AP/NeuN double-labeled cells were found not only in the GCL and GL but also in the EPL of the OB. In the EPL, most AP-labeled cells were calcium-binding protein parvalbumin (PV)-immunoreactive (+) interneurons. A subset of these cells was made up of calcium-binding protein calretinin (CR)(+) interneurons. According to their structural features, AP-labeled cells in the EPL were Van Gehuchten cells, multipolar cells, and superficial short-axon cells. Thus, postnatal SVZ progenitors give rise not only to granular and periglomerular interneurons but also to interneurons in the EPL of the OB. Furthermore, these results suggest that SVZ progenitors give rise to virtually all subpopulations of interneurons in the OB.     

Time dependent changes of striatal interneurons after focal cerebral ischemia in rats

Summary. The cellular damage over time and the alterations of neuronal subtypes was characterized in the striatum after 90-min middle cerebral artery occlusion and reperfusion in rats. We investigated the immunohistochemical alterations of choline acetyltransferase (ChAT)-positive (cholinergic-positive), γ-aminobutyric acid (GABA)ergic parvalbumin (PV)-positive, GABAergic nNOS (neuronal nitric oxide synthase)-positive interneurons, neuronal nuclei (NeuN)-positive spiny projection neurons, glial fibrillary acidic protein (GFAP)-positive strocytes and microglial response factor-1 (MRF-1)-positive microglia in the striatum after focal cerebral ischemia in rats. In the present study, transient focal cerebral ischemia in rats caused severe damage against interneurons as well as spiny projection neurons in the striatum. In contrast, a significant increase in the number of GFAP-immunopositive astrocytes was observed in the ipsilateral striatum 15 days after focal cerebral ischemia. Furthermore, a significant increase of MRF-1 immunoreactivity was observed in microglia of the ipsilateral striatum 7 days and 15 days after focal cerebral ischemia. Among three types of cholinergic interneurons, GABAergic PV-positive interneurons and GABAergic nNOS-positive interneurons, the severe damage of cholinergic and GABAergic PV-positive interneurons was more pronounced than that of GABAergic nNOS-positive interneurons after transient focal cerebral ischemia in rats. Furthermore, the present results suggest that GABAergic nNOS-positive interneurons in the striatum after focal cerebral ischemia undergo cellular death in a delayed manner. Correspondence: Tsutomu Araki, Department of Neurobiology and Therapeutics, Graduate School and Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan