The development of non-pyramidal neurons in the visual cortex of the rat

Summary We have studied the maturation of non-pyramidal cells in layers II–VI of the visual cortex of albino rats from birth to maturity, using Golgi-Cox and rapid Golgi preparations. At birth, non-pyramidall cells are sparse, immature and concentrated in the deep part of the cortical plate: their number increases towards the end of the first week but they remain sparse and immature in the upper part of the cortical plate. During the second postnatal week, the number, size and extent of dendritic and axonal branching of these cells undergo considerable increases and the cells become conspicuous in layer IV and apparent in the supragranular layers: this growth spurt occurs just after (and may be related to) the arrival and establishment in the cortex during the second half of the first postnatal week, of extrinsic afferents.During the third postnatal week, most of the cells complete their maturation. At the end of this week, the number of spinous cells is greater and the spine density of some cells is higher than in the adult, falling to adult values during the fourth postnatal week. It is noteworthy that the non-pyramidal cells appear to reach maturity at about the same time in all the layers studied, and at the same time as the pyramidal cells with which they are associated. These observations are not in accord with the prevalent view that non-pyramidal cells complete their differentiation much later than pyramidal cells.     

Silent synapses in the immature visual cortex: layer-specific developmental regulation

Central glutamatergic synapses are thought to initially form as immature, so-called silent synapses showing exclusively N-methyl-d-aspartate receptor-mediated synaptic transmission. Postsynaptic insertion of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors during further development leads to a conversion into functional, mature synapses. Here, we tested the hypothesis that, according to the "inside first-outside last" pattern of neocortical layer formation and synaptogenesis, pyramidal cells in the superficial layers might show a higher fraction of silent synapses compared with pyramidal cells in the deep layers. We performed an electrophysiological analysis of glutamatergic synapses in acute rat visual cortex slices during postnatal development. In layer VI pyramidal neurons the incidence of silent synapses was high during the first postnatal week and strongly declined during further development. Surprisingly, in superficial cortical plate pyramidal neurons (immature layers II/III), the fraction of silent synapses was initially very low and increased up to the second postnatal week. Thereafter, a similar decline as found in layer VI pyramidal neurons was observed. Thus the developmental regulation of silent synapses was clearly different in pyramidal neurons from different neocortical layers. The almost complete absence of silent synapses at early stages in layer II/III pyramidal neurons indicates that an initially formed subset of synapses is constitutively functional. This might be important to enable spontaneous activity and latter activity-dependent maturation of synapses.     

Morphological and electrical description of medullary respiratory neurons of the cat

Summary Synaptic currents were measured in voltage clamped crayfish muscle fibers which were triggered either by stimulation of the motor axon (EPSC), or byl-gutamate (gEPSC) applied by microiontophoresis or superfusion. Among a number of analogues of glutamate,l-glutamic-acid--methyl ester,l-glutamic-acid-dimethyl ester andl-aspartate, were reasonably specific antagonists at the motor synapses, although at relatively high concentrations. Also, 2-amino-4-phosphono-butyric acid and morphine were effective antagonists; the action of morphine, however, seemed to be unspecific.Aspartate was further shown to decrease the size of the quantum EPSC, without affecting the probability of release of transmitter or the potential change recorded from the presynaptic nerve terminal. The results also indicate that aspartate, after longer incubations, is released as a false transmitter. The dose-response curve to short glutamate pulses is shifted by aspartate to higher glutamate concentrations, without affecting the steep slope of the dose-response curve or the saturation level. This effect can be interpreted as competitive inhibition by aspartate, with an equilibrium concentration of aspartate at the receptor of 0.3–1.5 mmol/l. In longer glutamate applications the receptor desensitizes rapidly. Aspartate reduces this desensitization in addition to its competitive inhibitory effect. Suppression of desensitization can be more effective than inhibition in long glutamate applications; in this case aspartate apparently potentiates the effects of glutamate.This work was supported by the Deutsche Forschungsgemeinschaft     

Spatial distribution of inhibitory synaptic connections during development of ferret primary visual cortex

Intracortical inhibition in the primary visual cortex plays an important role in creating properties like orientation and direction selectivity. However, the development of the spatial pattern of inhibitory connections is largely unexplored. This was investigated in the present study. Tangential slices of layers 2/3 of ferret striate cortex were prepared for whole-cell patch clamp recordings, and presynaptic inhibitory inputs to pyramidal neurons were scanned by local photolysis of Nmoc-caged glutamate. Inhibitory synaptic currents (IPSCs) were first detected around postnatal day (P) 17. They originated locally around the recorded cells. Both the number and the total areas supplying the inhibitory inputs increased thereafter and peaked at the time around and shortly after eye opening (P29–37). A refinement period then followed in which the areas providing the majority of inhibitory inputs shrank from 600 µm around the recorded neurons to 200–300 µm in more mature animals (P38). The amplitude of IPSCs increased progressively with increasing age. Long-range inhibitory inputs (>600 µm) were present around eye opening and they often developed into a clustered patchy pattern in more mature animals (P38). In summary, our results show a refinement and clustering in the spatial pattern of inhibitory connections during postnatal development of ferret visual cortex.     

Postnatal development of the telencephalon of the tammar wallaby (Macropus eugenii). An accessible model of neocortical differentiation

Summary The sequence of development of cell layers in the neocortex of the tammar has been followed from 24 days gestation to 213 days postnatal. The tammar is born at 27 days gestation and the major period of its development occurs during the subsequent 250 days, most of this time being spent within the pouch. Although the pattern of differentiation of the cell layers appears to resemble that described for many Eutherian mammals, the neocortex is at an embryonic 2 layered stage at birth and a cortical plate is not present throughout the telencephalon until 10–15 days postnatal. A transient subplate zone, presenting a characteristic appearance with widely spaced rows of cells aligned parallel to the cortical surface, develops between 20 and 70 days postnatal, but no secondary proliferative region is seen in the subventricular zone of the dorso-lateral wall.Preliminary experiments with (³H)-thymidine injections indicate that the cortical plate follows the inside-out pattern of development described in many Eutherian mammals and that the oldest neurons are found in the parallel cell rows of the subplate zone. The importance of the late differentiation of the neocortex in relation to the time of birth and the resulting usefulness of the tammar as an experimental model of cortical development is discussed.     

Induction of mature neuronal properties in immortalized neuronal precursor cells following grafting into the neonatal CNS

Summary RN33B, a conditionally-immortalized neuronal cell line, survives and differentiates following grafting into the neocortex and hippocampus of adult and neonatal rat hosts. We have previously shown that these cells assume shapes characteristic of endogenous neurons at the integration site and persist up to 24 weeks post-grafting. In the present study we use electron microscopy and immunohistochemistry to characterize such cells. Differentiated RN33B cells were identical in size to endogenous neurons and their sizes depended on the specific location of integration. RN33B cells in the granule cell layer of the dentate gyrus and CA3 and CA1 pyramidal layers were 9.0, 15.3, and 12.6 m in diameter, respectively. Grafted RN33B cells received synapses from fibres of host origin. Differentiated cells expressed neuronal markers, but not glial markers. Some differentiated cells expressed glutamate bothin vitro andin vivo whereas undifferentiated cells did not. Grafted RN33B cells that differentiated with morphologies similar to CA3 pyramidal neurons and pyramidal cortical neurons expressed Py antigen, a neuronal marker that is differentially expressed in endogenous large pyramidal neurons of the cerebral cortex and large pyramids of hippocampal field CA3. This Py immunoreactivity was region-specific and corresponded to the endogenous pattern of Py immunostaining. Collectively, these data indicate that RN33B cells are capable of region-specific differentiation and have the potential to integrate functionally into the host CNS.     

The neocortex

By way of introduction, an outline is presented of the origin and evolutionary development of the neocortex. A cortical formation is lacking in amphibians, but a simple three-layered cortex is present throughout the pallium of reptiles. In mammals, two three-layered cortical structures, i.e. the prepiriform cortex and the hippocampus, are separated from each other by a six-layered neocortex. Still small in marsupials and insectivores, this new structure attains amazing dimensions in anthropoids and cetaceans. Neocortical neurons can be allocated to one of two basic categories: pyramidal and nonpyramidal cells. The pyramidal neurons form the principal elements in neocortical circuitry, accounting for at least 70% of the total neorcortical population. The evolutionary development of the pyramidal neurons can be traced from simple, extraverted neurons in the amphibian pallium, via pyramid-like neurons in the reptilian cortex to the fully developed neocortical elements designated by Cajal as psychic cells. Typical mammalian pyramidal neurons have the following eight features in common: (1) spiny dendrites, (2) a stout radially oriented apical dendrite, forming (3) a terminal bouquet in the most superficial cortical layer, (4) a set of basal dendrites, (5) an axon descending to the subcortical white matter, (6) a number of intracortical axon collaterals, (7) terminals establishing synaptic contacts of the round vesicle/asymmetric variety, and (8) the use of the excitatory aminoacids glutamate and/or aspartate as their neurotransmitter. The pyramidal neurons constitute the sole output and the largest input system of the neocortex. They form the principal targets of the axon collaterals of other pyramidal neurons, as well as of the endings of the main axons of cortico-cortical neurons. Indeed, the pyramidal neurons constitute together a continuous network extending over the entire neocortex, justifying the generalization: the neocortex communicates first and foremost within itself. The typical pyramidal neurons represent the end stage of a progressive evolutionary process. During further development many of these elements have become transformed by reduction into various kinds of atypical or aberrant pyramidal neurons. Interestingly, none of the six morphological characteristics, mentioned above under 1–6, has appeared to be unassailable; pyramidal neurons lacking spines, apical dendrites, long axons and intracortical axon collaterals etc. have all been described. From an evolutionary point of view the typical pyramidal neurons represent not only the principal neocortical elements, but also the source of various excitatory local circuit neurons. The spiny stellate cells, which are abundant in highly specialized primary sensory areas, form a remarkable case in point. In these elements only two of the six original pyramidal attributes, i.e. spiny dendrites and an intracortical axonal arbor, are retained. The nonpyramidal neurons display a diverse morphology, but share a number of important morphological and functional features: (1) their dendrites bear only a few spines or none, (2) their axons do not leave the cortex, (3) their terminals make synapses of the flat vesicle/symmetric variety, (4) they use the inhibitory neurotransmitter GABA, and (5) almost all types make synaptic contacts with pyramidal neurons. Several subclasses of nonpyramidal neurons are selectively immunoreactive for particular calcium-binding proteins. The widely held notion that the pyramidal neurons constitute the relatively constant basic framework of the cortex, whereas the local circuit neurons are variable and increase during phylogenetic development in number as well as in diversity is untenable. A survey is presented of the structure, synaptology and chemodifferentiation of the various neocortical cell types, allocating them to three groups: pyramidal neurons, excitatory interneurons and inhibitory interneurons. The synaptic relations of the various neocortical neurons are pictorially summarized in two microcircuitry diagrams, which together form the pièce de résistance of the present treatise. The various approaches to the structure of the neocortex are discussed. It is emphasized that correlative structural, ultrastructural and electrophysiological studies of pyramidal neurons known to project to a given cortical or subcortical target form a promising field of interdisciplinary research.Dedicated to the memory of Hendrik van der Loos.     

Maturation of rat visual cortex. I. A quantitative study of Golgi-impregnated pyramidal neurons

Summary The early postnatal maturation of pyramidal neurons in layers II/III and V of the rat visual cortex has been examined in an attempt to elucidate some determinants of their mature morphology. Three indices have been quantified using Golgi-impregnated pyramidal cells: densities of spines along apical dendrites, numbers of primary basal dendrites and volumes of cell bodies. The mean density of spines on the apical dendrites of all pyramidal neurons increases in a stepwise fashion. The first significant increase occurs between days 6 and 9 and the second, between days 12 and 15; these increases may correlate with the arrival of geniculate afferents and with the opening of the eyes, respectively. In younger animals, the distribution of spines along the apical shafts is relatively even, whereas in older animals, spine density increases significantly over the proximal 125 m portion and is relatively constant over the remaining distal portion. By day 21, layer V pyramidal cells have acquired more primary basal dendrites and larger somatic volumes than layer II/III cells. Furthermore, as the cells mature the rates of change in these characteristics are significantly different for neurons in layer II/III and in layer V. For both cell populations, the mean number of primary basal dendrites increases to a maximum before falling to a steady level, but for neurons in layer V, the maximum is higher and attained three days earlier than for layer II/III cells. Moreover, the increase in volume of cell bodies of layer V neurons begins three days before that of layer II/III cells. This three day phase difference in maturation may reflect the cell birth dates, since autoradiographic evidence indicates that layer V pyramidal neurons reach the cortical plate about three days prior to those which occupy layer II/III in the adult visual cortex.     

Immunohistochemical studies of localization and co-localization of glutamate, aspartate and GABA in the anterior thalamic nuclei, retrosplenial granular cortex, thalamic reticular nucleus and mammillary nuclei of the rat

Localization and possible co-localization of glutamate, aspartate and GABA immunoreactivities was examined in the anterior thalamic nuclei, retrosplenial granular cortex, thalamic reticular nucleus and mammillary nuclei of the rat by double antigen immunohistochemistry using diaminobenzidine and benzidine dihydrochloride in one series and double immunofluorescence labelling with rhodamine and fluorescein in a second series of animals. In three of these regions, retrosplenial granular cortex, anterior thalamic nuclei, and mammillary nuclei, glutamate immunoreactivity was co-localized with aspartate immunoreactivity in a majority of the projection neurons (pyramidal neurons, predominantly in layers V and VI in retrosplenial granular cortex; rounded polygonal multipolar neurons throughout the rostrocaudal extent of the anterior thalamic and mammillary nuclei). None of the cells showing glutamate and/or aspartate immunoreactivity in these regions also displayed GABA immunoreactivity, which was present in non-pyramidal cells in the retrosplenial granular cortex (chiefly in layers I–III) and in small numbers of cells within the anterior thalamic nuclei. In the thalamic reticular nucleus, in contrast, most neurons were immunoreactive for GABA and in the majority of these neurons glutamate (and/or aspartate) immunoreactivity was co-localized with GABA.     

小鼠大脑新皮质片层化形成过程和细胞周期变化

目的 探讨小鼠大脑新皮质片层化的组织发生过程和细胞周期的关系,对有丝分裂后神经元在迁移中的细胞周期变化、神经细胞的增殖、神经元的迁移进行观察.方法 各日龄共计200只小鼠,应用免疫荧光法、5′-溴脱氧尿嘧啶核苷(BrdU)检测和DiI标记技术对胚胎期和出生后小鼠的大脑皮质进行形态学观察,对皮质BrdU和Cyclin D1阳性细胞密度进行测量.结果 皮质板最早在胚龄15d(E15)时形成,小鼠大脑新皮质深层(第Ⅵ～Ⅴ层)片层化进程开始于生后0 d(P0),皮质浅层(第Ⅳ～Ⅱ层)的片层化趋势开始于P5,P7时6层结构完全形成,但未呈现片层化特点,P14时小鼠大脑新皮质片层化完全形成,P30时片层化结构趋于稳定.在大脑新皮质片层化过程中,锥体细胞在E17时呈椭圆形,树突有小分支,在P15时发育成熟,呈锥形并有复杂的顶树突和基树突.BrdU检测发现,室管层和室管层下区有大量增殖的干细胞,在此期间由BrdU阳性细胞增殖生成的有丝分裂后神经元可以迁移到大脑新皮质;P0至P30,迁移到皮质板的有丝分裂后神经元逐渐减少.利用G1期特异性标记物Cyclin D1对有丝分裂后神经元的细胞周期进行分析发现,有丝分裂后神经元处于G1期,它们一旦定居到皮质板将退出细胞周期.新皮质中Cyclin D1阳性细胞数量呈抛物线变化,在P12达到峰值,P30后在皮质板只能发现少量的Cyclin D1阳性细胞.结论 小鼠大脑新皮质片层化过程经历了细胞增殖、分化与迁移,同时伴随着皮质板锥体细胞的成熟.神经细胞的增殖和迁移主要发生在胚胎期和生后早期,迁移的细胞主要处于G1期.有丝分裂后神经元的分化过程实际上是G1期到G0期的过渡,一旦在皮质板定居下来,有丝分裂后神经元将退出细胞周期,进入G0期.     

An ultrastructural study of the maturation of neuronal somata in the visual cortex of the rat

Summary The postnatal development of neuronal perikarya in layers II–VI of the visual cortex of perfusion-fixed albino rats, 12 h to 180 days old, has been studied by electron microscopy. Particular attention was paid to cells in photographic montages of 75m wide strips extending through the full depth of the occipital cortex, cut from 100 m vibratome sections of the brain.At birth, and during the first few postnatal days, most of the neurons present in the cortex are small, tightly packed indifferent cells with scanty cytoplasm containing mitochondria and chiefly free ribosomes; a few presumptive pyramidal cells with a developing apical dendrite and more voluminous cytoplasm can be recognized in deep cortex. Non-pyramidal cells can be identified on postnatal day 6, when although scarce and with immature cytoplasmic features, they already display a more electron opaque chromatin pattern than developing pyramidal cells and receive axo-somatic contacts of Gray's type I.During the second postnatal week there are conspicuous increases in the maturity of the cells, which acquire a rich complement of cytoplasmic organelles: in general cells situated in the deep cortical plate are larger and better differentiated than those in the superficial plate, and non-pyramidal cells are less well differentiated than the associated pyramidal cells. By the end of the second week, differences in cytoplasmic maturity between superficial and deep, and between pyramidal and non-pyramidal cells are less evident.Maturation proceeds during the third postnatal week; both types of cells acquire an adult complement of axo-somatic synapses and their mature nuclear and cytoplasmic features, and by day 24 are indistinguishable from their adult counterparts. In keeping with previous Golgi studies of this same cortex, the non-pyramidal cells did not acquire mature ultrastructural features significantly later than the pyramidal cells. A possible correlate of particularly active synaptogenesis and plasticity in the population of nonpyramidal, cells during the third postnatal week (immediately after eyeopening), was that at this time these cells contained very prominent accumulations of granular reticulum, ribosomes and Golgi apparatus, and appeared hypertrophic.     

Immunocytochemical localization of calcineurin in the adult and developing primary visual cortex of cats

An immunocytochemical method was used to localize calcineurin, a calcium-dependent calmodulinstimulated protein phosphatase, in the primary visual cortex of developing and adult cats. In the adult calcineurin immunoreactivity exhibits a laminar distribution with dense labeling in the upper half of layers II/III and two lightly labeled bands in lower layer IV and in layer VI. Most of the immunoreactive neurons are pyramidal in shape and appear to form a subpopulation of cortical neurons, but non-pyramidal neurons were also labeled, especially during early stages of postnatal development. The distribution pattern of calcineurin immunoreactivity showed developmental changes until at least 3 months of age. The number of calcineurin-positive cells abruptly increased at 3 weeks, and heavily labeled neurons appeared in a well-delineated band in layer IV between 3 and 5 weeks of age. At 6 to 10 weeks, neurons in layers II/III also became strongly immunoreactive. At this developmental stage intensely stained cells were thus distributed throughout layers II to IV. Thereafter, there was a marked decrease in the number of immunoreactive cells in layer IV and beyond 12 weeks the distribution pattern of calcineurin immunoreactivity became similar to that of adult animals. These changes of calcineurin expression show some relation with the inside-out pattern of cortical maturation and with the time course and the laminar selectivity of use-dependent malleability. Therefore, we suggest that calcineurin may be involved in processes of neuronal differentiation and experience-dependent plasticity.     

Aspinous and sparsely-spinous stellate neurons in the visual cortex of rats contain glutamic acid decarboxylase

Summary Glutamic acid decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter -aminobutyric acid (GABA), has been localized in the rat visual cortex by immunocytochemical methods with both light and electron microscopy. In both colchicine-injected and non-injected preparations of the visual cortex, GAD-positive reaction product was observed in somata, proximal dendrites and axon terminals of non-pyramidal neurons. The GAD-positive terminals were observed to form symmetric synaptic junctions most commonly with dendritic shafts and somata of pyramidal and stellate neurons and less frequently with initial axon segments of pyramidal neurons and dendritic spines. In colchicine-injected preparations, GAD-positive somata were located in all cortical layers including the immediately subjacent white matter. In contrast, sections from non-injected rats displayed GAD-positive somata within a superficial and a deep cortical band. The GAD-positive somata observed in both types of preparations received both symmetric and asymmetric synaptic junctions, lacked apical dendrites, and had radially oriented dendrites of small diameter. These characteristics of GAD-positive neurons indicate that they are aspinous and sparsely-spinous stellate neurons. The localization of GAD within these neurons in combination with physiological and pharmacological data indicate that these local circuit neurons mediate GABA-ergic inhibition in the neocortex.     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

 We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND 10, decreased until PND 30, and thereafter remained at the same level as on PND 0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND 10 and PND 15, after which the immunoreactivity gradually decreased and reached a plateau at PND 30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED 20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND 0. Immunoreactivity in the neurons of the subiculum was found after PND 5 and that of the polymorphic cell layers was found after PND 15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions. Accepted: 13 June 1996     

Developmental changes of glutamate receptors in the rat cerebral cortex and hippocampus

We studied the immunohistochemial localization of the glutamate receptors (GluR-1, -2, and -3,) in the developing rat cerebral cortex and hippocampus using antibodies to GluR1 and to an epitope common to GluR2 and GluR3 (GluR2/3) subunits. In the cerebral cortex, GluR1 immunoreactivity appeared in the neurons from postnatal day (PND) 0, increased with maturation, was highest at PND?10, decreased until PND 30, and thereafter remained at the same level as on PND?0. GluR2/3 immunoreactivity appeared earlier in scattered neurons on embryonal day (ED) 18, increased with maturation and reached a peak between PND?10 and PND?15, after which the immunoreactivity gradually decreased and reached a plateau at PND?30. For both GluR1 and GluR2/3, some of the pyramidal neurons showed intense staining. In the pyramidal layers of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in all the pyramidal neurons of the CA1–4 area from ED?20. In the dentate gyrus of the hippocampus, GluR1 and GluR2/3 immunoreactivity was found in the neurons of the granule cells after PND?0. Immunoreactivity in the neurons of the subiculum was found after PND?5 and that of the polymorphic cell layers was found after PND?15–20. Our results indicate that the development of glutamate receptor subunits in the rat cerebral cortex and hippocampus is expressed in different spatial patterns and distinct temporal patterns throughout development and is scheduled during the early postnatal period, when synaptic plasticity or synaptic connection occurs in these regions.     

The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. IV. Avian pancreatic polypeptide

Summary Immunocytochemical techniques were used to investigate the morphology and distribution of avian pancreatic polypeptide-like immunoreactive neurons in the visual cortex of albino rats at various ages from the first postnatal day to adulthood. In the adult, immunoreactive neurons were located in layers II to VI but were somewhat concentrated in the deeper cortical layers. The overwhelming majority of labelled cells exhibited morphologies characteristic of multipolar, bitufted and bipolar varieties of non-pyramidal neurons as described in Golgi preparations of rat visual cortex. However, a few immunoreactive pyramidal neurons were also observed.On the first postnatal day, a small number of immature non-pyramidal neurons were observed in the subplate region. Labelled cells appeared in the more superficial layers at the beginning of the second postnatal week and attained a distribution similar to that observed in adult animals during the third week. The morphological maturation of immunoreactive neurons occurred gradually during the first two postnatal weeks and at day 21, they appeared qualitatively indistinguishable from their adult counterparts.     

Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development

The rat retrosplenial cortex is part of a heavily interconnected limbic circuit, considered to have an important role in spatial memory. Interestingly, the granular retrosplenial cortex has an exceptionally distinct system of dendritic bundles, originating from callosally projecting pyramidal neurons in layer II. These can be detected as early as postnatal day 5; and, although their functional significance remains to be elucidated, the existence of these bundles makes the granular retrosplenial cortex an attractive model system for a wide range of development and functional investigations. Here, we report four results concerning the development of modularity in the granular retrosplenial cortex in rats as investigated by neurochemical markers associated to cortico-cortical and thalamo-cortical connections. Emphasis is placed on zinc, an activity-related substance associated with glutamatergic, non-thalamic terminations. 1) Zinc shows a transient strong expression during early postnatal development, but later than the appearance of the upper layer bundles (at postnatal day 5). By postnatal day 11 to postnatal day 15 staining for zinc achieved its most complex pattern; such that layer I had an elaborate organization both in the tangential and radial dimensions. Three sublaminae were distinguished (layers Ia-c): a superficial, thin tier (Ia) with patchy, moderate staining which periodically intruded into the underlying layer Ib ("funnel" modules), a middle band of variable width and light staining (Ib), and a deep, thin band with heavy and patchy staining (Ic) which, at rostral levels, spread upward into layer Ib (as "dome-like" modules). 2) At postnatal day 15, immunohistochemical methods showed that layers Ia, b zinc-funnels were co-localized with glutamate receptor subunits 2/3, GABA receptor type A alpha1 subunit and the thalamo-cortical marker, vesicular glutamate transporter 2. Layer Ic and the zinc dome-like modules were co-labeled for the cortico-cortical marker, vesicular glutamate transporter 1 and calretinin. 3) The spatial coincidence between zinc funnels in layers Ia, b and vesicular glutamate transporter 2 was further investigated by electron microscopy, which demonstrated co-localization of zinc and vesicular glutamate transporter 2 in synaptic boutons. The unusual co-localization of zinc and thalamo-cortical terminations was confirmed by retrograde transport of zinc to neurones in the anterodorsal thalamic nucleus at postnatal day 9 and postnatal day 13, and can thus be considered a transient zinc expression in thalamo-cortical boutons. This was not observed at postnatal day 28 or later. 4) After postnatal day 18, zinc staining started to fade in all layers. Before postnatal day 21, the heavy staining for zinc in the domes had completely disappeared. Zinc staining in layer Ia and the funnels virtually disappeared after postnatal day 28. A transient expression of zinc is reported in at least one other cortical area (layer IV of barrel cortex from postnatal day 5 to postnatal day 14, maximal at postnatal days 9-11). We conclude that the transient expression of zinc can occur in both limbic and sensory areas, and that down-regulation of zinc in cortical modules might be related to synaptic plasticity and remodeling during development.     

The appearance of long-latency responses to a conditioned signal in the cortex is explained by strengthening of collateral connections between pyramidal neurons

Experimental analysis and computer simulation of the neurophysiological processes underlying the stable and local electrophysiological expression of conditioned reflexes in the cerebral cortex, a phenomenon discovered in Asratyans laboratory in the 1960s, showed that the long-latency components of cortical evoked potentials to a conditioned signal correspond to the late phases of the responses of motor cortex neurons, which are analogous to and probably generated by the same mechanism as long-latency epileptiform reactions of neurons in the epileptogenic cortex. Late long-latency components are generated via activation of NMDA receptors in the collateral connections between pyramidal neurons. The delay in the generation of responses depends on the initial activation of GABA_A receptors and the slow kinetics of the current through NMDA channels. The appearance of late components as a result of training is explained by increases in the efficiency of collateral excitatory connections between pyramidal neurons.Translated from Zhurnal Vysshei Nervnoi Deyatelnosti, Vol. 54, No. 3, pp. 403–408, May–June, 2004.     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter     

Postnatal development of parvalbumin and calbindin D28K immunoreactivities in the cerebral cortex of the rat

Parvalbumin and calbindin D28k immunoreactivities were examined in the neocortex of the rat during postnatal development. Parvalbumin-immunoreactive nonpyramidal neurons first appear in layer V and later in layers VI and IV, and then in II and III. Immunoreactive terminals forming baskets surrounding unlabelled somata appear about 2 days later. The first parvalbumin-immunoreactive neurons appear in the retrosplenial and cingulate cortices, and the rostral region of the primary somatosensory cortex at postnatal days 8 or 9 (P8–P9). These regions are followed by the primary visual, primary auditory and motor cortices at P11. Parvalbumin immunoreactivity appears last in the secondary areas of the sensory regions and association cortices. Adult patterns are reached at the end of the 3rd week. Calbindin D28K-immunoreactive nonpyramidal neurons are found at birth in all cortical layers excepting the molecular layer. The intensity of the immunoreaction increases during the first 8 or 11 days of postnatal life, first in the inner and later in the upper cortical layers, following, therefore, an inside-out gradient. Heavily-labelled calbindin D28K-immunoreactive nonpyramidal cells dramatically decrease in number from P11 to P15 due mainly to a decrease of the multipolar subtypes. This suggests that two populations of calbindin D28k-immunoreactive nonpyramidal neurons are produced in the neocortex during postnatal development: one population of neurons transitorily expresses calbindin D28k immunoreactivity; the other population is composed of neurons that are permanently calbindin D28k immunoreactive. In addition to heavily labelled nonpyramidal cells, a band of weakly labelled pyramid-like neurons progressively appears in layers II and III throughout the cerebral cortex, beginning in layer IV in the somatosensory cortex by the end of the 2st week. Adult patterns are reached at the end of the 3rd week. These results indicate that parvalbumin and calbindin D28k immunoreactivities in the cerebral neocortx follow different characteristic patterns during postnatal development. The appearance of parvalbumin immunoreactivity correlates with the appearance of the related functional activity in the different cortical regions, and, probably, with the appearance of inhibitory activity in the neocortex. On the other hand, the early appearance of calbindin D28k immunoreactivity in the neocortex may be related to the early appearance of calbindin immunoreactivity in many other brain regions, and suggests another, as yet unknown, role for this calcium-binding protein during development of the cerebral cortex.