The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. I. Somatostatin

Summary Using conventional immunocytochemical techniques, we have examined the morphology and distribution of somatostatin-like immunoreactive neurons in the visual cortex of albino rats between the first postnatal day and maturity. In the adult, somatostatin-immunoreactive neurons were observed in layers II to VI but were concentrated in layers II and III. These cells displayed morphological features characteristic of the multipolar and bitufted varieties of cortical non-pyramidal neurons as described in Golgi preparations of rat visual cortex.On the first postnatal day and in the subsequent few days, immunoreactivity was confined to immature bipolar and multipolar neurons concentrated in layers V and VI. Labelled cells first 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 at the end of this week. At this time they closely resembled their adult counterparts from which they appeared indistinguishable by the end of the third postnatal week. The late appearance of labelled cells in the superficial layers, where they are predominantly located in adult animals, suggests that the somatostatin immunoreactivity exhibited by most of these neurons develops several days after they have completed their migration and assumed their positions in the visual cortex.     

The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. II. Vasoactive intestinal polypeptide

Summary Using immunocytochemistry, we have examined the morphology and distribution of vasoactive intestinal polypeptide-like immunoreactive neurons in the visual cortex of albino rats whose ages were closely spaced in time between the first postnatal day and adulthood. In the adult, immunoreactive neurons were located in layers II to VI but were concentrated in layers II and III. All labelled neurons had the morphological characteristics of cortical non-pyramidal cells with the majority being of the bipolar variety as described in Golgi preparations. Some multipolar forms were also present.Vasoactive intestinal polypeptide-immunoreactivity appeared to develop in postnatal life. Labelled cells were first seen in layers V and VI at day 4. During the subsequent few days, some labelled cells were observed in the more superficial layers and by day 8 they were predominantly present in layers II and III. Although the distribution of immunoreactive cells at this time resembled that of adult animals, their morphology displayed immature features. The size and extent of their dendritic branching appeared to increase considerably during the second and third weeks and their morphological maturation was attained by the middle of the fourth postnatal week.     

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.     

The laminar distribution of glutamate decarboxylase and choline acetyltransferase in the adult and developing visual cortex of the rat

The activities of choline acetyltransferase and glutamate decarboxylase were measured in individual layers of the adult and developing rat visual cortex. In the adult, the level of choline acetyltransferase activity was highest in layer V followed by layers I, II & III, IV and VI. These measurements are in complete agreement with recent immunohistochemical observations in the same cortical area. Glutamate decarboxylase activity was highest in layer IV and declined significantly in the more superficial and deeper layers. The activities of both enzymes were low during the first postnatal week but increased dramatically between days 8 and 18. Choline acetyltransferase activity in all layers demonstrated a more gradual rise to adult levels from day 18 onward, while glutamate decarboxylase activity reached adult levels by day 24 in all layers, except layer IV, which showed a continuous increase to adulthood. The functional role of the differences in the laminar distribution of these enzymes remains unknown.     

Axosomatic synapses in the visual cortex of adult rat. A comparison between GABA-accumulating and other neurons

Summary Differences in axosomatic synapses between GABA-accumulating [G(+)N] or non-accumulating [G(–)N] neurons have been investigated in the visual cortex of adult rat. The neurons were classified and localized in light microscopic autoradiograms after [³H]GABA injections. The cells were then resectioned for electron microscopic identification of type 1 synapses (T1S) and type 2 synapses (T2S). A total of 167 neurons [45 G(+)N, 122 G(–)N] situated in laminae II-VI were evaluated. The two groups of neurons were not uniform populations. G(–)N included both pyramidal and non-pyramidal neurons, whereas no typical pyramidal neurons were found among G(+)N.A total of 691 synaptic contacts was evaluated for these groups of somata. The density of synapses was higher on G(+)N than on G(–)N. This was mainly due to a difference in the number of T1S. On G(–)N the frequency distributions of both types of synapses represented Poisson distributions, indicating that there were stochastic variations around mean values. In contrast, on G(+)N the distribution was exponential which suggests that G(+)N include several subpopulations with different densities of T1S.On all cortical neurons the average density of T2S was 50–60 T2S per 1000 m² of soma surface, which resembles the density in the neuropil. In contrast, T1S varied from zero to a mean of 12 per 1000 m² on G(–)N and to a mean of 51 per 1000 m² on G(+)N, i.e. the density of T1S, unlike T2S, is much smaller on neuronal somata than in the surrounding neuropil. It is suggested that the formation of axosomatic T1S, but not of T2S, is suppressed to a variable degree on almost all cortical neurons. Only on pyramidal neurons does the suppression of T1S seem to be complete.     

Cholinergic neurons and fibres in the rat visual cortex

Summary Choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was localized immunocytochemically in neurons and fibres in the rat visual cortex using a monoclonal antibody. ChAT-labelled cells were non-pyramidal neurons, primarily of the bipolar form, distributed in layers II through VI but concentrated in layers II & III. Their perikarya contained a large nucleus and a small amount of perinuclear cytoplasm. The somata and dendrites of all labelled cells received Gray's type I and type II synapses.ChAT-stained axons formed a dense and diffuse network throughout the visual cortex and particularly in layer V. Electron microscopy revealed that the great majority formed type II synaptic contacts with dendrites of various sizes, unlabelled non-pyramidal somata and, on a few occasions, with ChAT-labelled cells. However, a very small number of terminals appeared to form type I synaptic contacts. This study describes the morphological organization of the cholinergic system in the visual cortex, the function of which has been under extensive investigation.     

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.     

Projections of peptide-containing neurons in rat colon

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, substance P, neuropeptide Y, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or enkephalin were studied in the midcolon of the rat by immunocytochemistry and immunochemistry. Most of these nerve fibers had an intramural origin as was established by extrinsic denervation (serving of mesenterial nerves). Extrinsic denervation eliminated neuropeptide Y-containing fibers of presumably sympathetic origin together with sensory nerve fibers containing both substance P and calcitonin gene-related peptide. Co-existence of two peptides in the same neuron was studied by double immunostaining. This revealed co-existence of neuropeptide Y and vasoactive intestinal peptide in one population of intramural neurons; an additional population of intramural neurons was found to contain vasoactive intestinal peptide but not neuropeptide Y. All somatostatin-containing neurons in the submucous ganglia were found to harbor calcitonin gene-related peptide. A much larger population of submucous neurons containing calcitonin gene-related but not somatostatin was also detected. Some perivascular calcitonin gene-related peptide-containing nerve fibers (of intrinsic origin) harbored vasoactive intestinal peptide while others (of extrinsic origin) harbored substance P. The polarities and projections of the various peptide-containing intramural neurons in the transverse colon were studied by analysing the loss of nerve fibers upon local disruption of enteric nervous pathways (myectomy or intestinal clamping). Myenteric neurons containing vasoactive intestinal peptide, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or vasoactive intestinal peptide/neuropeptide Y gave off 5-10-mm-long descending projections while those containing substance P or enkephalin issued approx. 5-mm-long ascending projections. Submucous neurons containing calcitonin gene-related peptide, somatostatin/calcitonin gene-related peptide or gastrin-releasing peptide issued both ascending (2-6 mm) and descending (2-6 mm) projections, those containing vasoactive intestinal peptide issued ascending (approx. 2 mm) projections, while those containing galanin or vasoactive intestinal peptide/neuropeptide Y lacked demonstrable oro-anal projections. Enkephalin-containing fibers could not be detected in the mucosa and the mucosal substance P-containing nerve fibers were too few to enable us to delineate their projections.     

The distribution of clones of neurons in the rat somatosensory cortex

Summary We have labelled clones of neurons in the cerebral cortex of rats by introducing a retroviral vector, called BAG, into the cerebral vesicles of embryosin utero. BAG encodes the enzyme -galactosidase, which acts as a histochemical marker for the subsequent identification of clones derived from infected precursor cells. We have studied the distribution of neuronal clones in the rat somatosensory cortex, and have asked whether clonally-related neurons were dispersed randomly. We have discovered that they are not. Rather, clones disperse predominantly such that the earliest progeny of ventricular zone cell are found posterolateral to later generated cells. This distribution fits with what would be expected were neurons dispersed passively in accordance with the lateral to medial cortical neurogenic gradient.     

Vasoactive intestinal polypeptide-immunoreactive neurons in rat visual cortex

An antibody to vasoactive intestinal polypeptide (VIP) was used to examine the forms of VIP-positive neurons and the synapses made by VIP-positive axon terminals. Vasoactive intestinal polypeptide-positive cells are most common in layers II and III and the majority of them are typical bipolar neurons, with two primary dendrites which emanate from the upper and lower poles of the cell body. Their somata, which have only a few symmetric and asymmetric synapses, generally have a fusiform or "tear-drop" shape and contain nuclei with a vertically oriented cleft. The dendritic trees are arranged vertically and often extend through five cortical layers. The axons are thin and extend either from the soma or from one of the primary dendrites. The axons also follow a vertical trajectory. Other VIP-positive neurons are modified bipolar cells and a few of them are multipolar cells. The synapses formed by the VIP-positive axon terminals in the neuropil are symmetric in form, and although the synaptic clefts are narrow, the junctions are usually long and continuous, rather like those described for asymmetric synapses. Most of the VIP-positive axon terminals synpase with small dendritic shafts, but a few synapse with neuronal cell bodies. Since the majority of the VIP-positive neurons are bipolar cells it is concluded that these are the source of most of the VIP-positive axon terminals. If this is so, then the VIP-positive bipolar cells form symmetric synapses. This is in contrast to the observations of Peters and Kimerer (1981. J. Neurocytol. 10, 921-946) for the bipolar cells they examined in a Golgi-electron microscopic study had axon terminals forming asymmetric synapses. It is suggested that this disparity can be reconciled if it is assumed that the bipolar cell population consists of subgroups which have different biochemical characteristics and different synaptic relationships.     

Development of dendritic bundles of pyramidal neurons in the rat visual cortex.

The apical dendrites of pyramidal neurons in the cerebral cortex form vertical bundles whose distribution and density vary across species and areas. To understand their relationships with cortical columns, we labeled retrogradely neurons from the white matter underlying the visual cortex with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) at P3 and P10 and with biotinylated dextran amine at P30. We also mapped the distribution of apical dendrites in tangential sections, immunostained for microtubule-associated proteins (MAP2). Their composition and distribution were studied with Neurolucida and NeuroExplorer software. The apical dendrites of pyramidal neurons formed different bundle types: at P3 we found bundles formed (a) by neurons located in cortical plate; (b) by layer V neurons; and (c) by upper layer V neurons and cortical plate neurons. At P10, the amount of supragranular neurons participating in the bundles increased. The inter-dendritic and inter-bundle distances increased with age. These findings confirm that dendritic bundles are present in the rat visual cortex early in development and are formed by neurons belonging to different cortical layers. The existence of different types of bundles relative to the layer of location of their parent neurons suggests that they are heterogeneous from each other in nature and in the pattern of connectivity.     

Response properties of neurons in the visual cortex of the rat

Summary Response properties of neurons in the visual cortex, area 17, of Long Evans pigmented rats were investigated quantitatively with computer-controlled stimuli. Ninety percent of the cells recorded (296/327) were responsive to visual stimulation. The majority (95%, 281/296) responded to moving images and were classified as complex (44%), simple (27%), hypercomplex (13%) and non-oriented (16%) according to criteria previously established for cortical cells in the cat and monkey. The remaining 5% of the neurons responded only to stationary stimuli flashed on-off in their receptive field. Results of this study indicate that neurons of the rat visual cortex have properties similar to those of cells in the striate cortex of more visual mammals.Supported by grant EY02964, the Biological Humanics Foundation and the Bendix Corporation     

The development of excitatory transmitter amino acid-containing neurons in the rat visual cortex

The excitatory amino acids l-glutamate and l-aspartate are believed to be utilized as neurotransmitters by the pyramidal neurons in the mammalian cerebral cortex. These cells can be recognized early in development, while glutamate might play an important part in the maturation and plasticity of the cortex. Here, we used light and electron microscopic immunocytochemistry to study the time of appearance and maturation of glutamate and aspartate in neurons of the rat visual cortex. Glutamate- and aspartate-immunoreactive cells were first detected in deep cortical layers at postnatal day 3. During the next 3 weeks, labelled neurons were observed progressively in more superficial layers, but did not demonstrate their adult pattern of distribution until postnatal week 4. Electron microscopic analysis showed that glutamate- and aspartate-labelled neurons gradually develop their cytological and synaptic features during the first 4 postnatal weeks, with this process of differentiation originating in the deep cortical layers and progressively extending to the superficial layers. These findings suggest that cortical pyramidal neurons begin to express detectable levels of transmitter glutamate and/or aspartate after they have completed their migration. Their neurochemical differentiation follows an inside-out pattern similar to the pattern described for the genesis and morphological differentiation of this population of cortical neurons.     

Correlation of electrophysiology, morphology, and functions in corticotectal and corticopretectal projection neurons in rat visual cortex

In most mammals the superior colliculus (SC) and the pretectal nucleus of the optic tract (NOT) receive direct input from the ipsilateral visual cortex via projection neurons from infragranular layer V. We examined whether these projection neurons belong to different populations and, if so, whether it is possible to correlate the electrophysiological features with the suggested function of these neurons. Projection cells were retrogradely labeled in vivo by rhodamine-coupled latex beads or fast blue injections into the SC or the NOT 2–5 days prior to the electrophysiological experiment. Intracellular recordings of prelabeled neurons were made from standard slice preparations and cells were filled with biocytin in order to reveal their morphology. Both cell populations consist of layer V pyramids with long apical dendrites that form terminal tufts in layer I. In electrophysiological terms, 12 of the corticotectal cells could be classified as intrinsically bursting (IB), while two neurons showed a doublet firing characteristic and one neuron was classified as regular-spiking (RS). Intracortical microstimulation of cortical layer II/III revealed that SC-projecting neurons responded optimally to stimulation sites up to a distance of 1000 μm from the recorded cell. The morphological features of the SC-projecting cells reveal an apical dendritic tuft in layer I with a lateral extension of 300 μm, a mean spine density of 65 spines per 40 μm on the apical dendrites located in layer II/III, and a bouton density of 13 boutons per 100 μm on the intracortical axons. Sixteen NOT-projecting neurons exhibited an IB and five cells an RS characteristic. Intracortical microstimulation of cortical layer II/III showed that NOT-projecting neurons responded optimally to stimulation sites up to a distance of 1500 μm. Their morphological features consist of an apical dendritic tuft with a lateral extension of 500 μm, a mean spine density of 25 spines per 40 μm on the apical dendrites located in layer II/III, and a bouton density of 6 boutons per 100 μm on the intracortical axons. When the passive membrane parameters, responses to intracortical microstimulation in layer V, the extension of the basal dendritic field, and spine densities in layers I or V were compared between SC- and NOT-projecting cells, no differences were revealed. Differences were only consistently found in the supragranular layers, either for morphological parameters or for intracortical microstimulation. The results suggest that NOT-projecting and SC-projecting neurons, although biophysically similar, could integrate and transmit different spatial aspects of cortical visual information to their target structures. Received: 22 November 1996 / Accepted: 8 October 1997     

Receptive field properties of single neurons in rat primary visual cortex.

The rat is used widely to study various aspects of vision including developmental events and numerous pathologies, but surprisingly little is known about the functional properties of single neurons in the rat primary visual cortex (V1). These were investigated in the anesthetized (Hypnorm-Hypnovel), paralyzed animal by presenting gratings of different orientations, spatial and temporal frequencies, dimensions, and contrasts. Stimulus presentation and data collection were automated. Most neurons (190/205) showed sharply tuned (相似文献   

Projections of peptide-containing neurons in rat small intestine

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, neuropeptide Y, somatostatin, substance P, enkephalin and calcitonin gene-related peptide were studied in the rat jejunum by immunocytochemistry and immunochemistry. Their origin was determined by the use of various procedures for extrinsic denervation (chemical sympathectomy, bilateral vagotomy or clamping of mesenterial nerves). The terminations of the different types of intramural nerve fibers were identified by examination of the loss of nerve fibers that followed local disruption of enteric nervous pathways (intestinal myectomy, transection or clamping). The majority of the peptide-containing nerve fibers in the gut wall were intramural in origin, each nerve fiber population having its own characteristic distribution and projection pattern. Nerve fibers emanating from the myenteric ganglia terminated within the myenteric ganglia and in the smooth muscle layers: those storing vasoactive intestinal peptide/neuropeptide Y, somatostatin and substance P were descending, those storing enkephalin were ascending and those containing calcitonin gene-related peptide projected in both directions. Nerve fibers emanating from the submucous ganglia terminated mainly within the submucous ganglia and in the mucosa: those storing calcitonin gene-related peptide or vasoactive intestinal peptide/neuropeptide Y were ascending and those storing substance P or somatostatin were both ascending and descending. Enkephalin nerve fibers could not be detected in the mucosa.     

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.     

Distribution and morphology of Alz-50-immunoreactive cells in the developing visual cortex of kittens

Summary The population of interstitial cells of the white matter in the postlateral gyrus of the cat was studied at different postnatal ages using the antibody Alz-50. These neurons are among the first cells to develop in the cortex, and many of them are transitory, disappearing by cell death during the first postnatal days. In the present study, we found that immunoreactivity to Alz-50 is expressed during the first three postnatal weeks, and that positive neurons were not detected after postnatal day (P) 23. In addition to marking cells in the white matter, Alz-50 also recognizes many pyramidal cells in the cortical layers II-III and V of the visual cortex at postnatal day 4. The staining of cortical cells was not observed at other ages. We found that the number of positive cells in the white matter decreases by postnatal days 12 and 16, showing an apparent increase in number at postnatal day 23.In this study we also attempted to correlate the morphology of Alz-50-immunoreactive cells with the interstitial cells of the white matter, as seen in Golgi preparations. We conclude that Alz-50 immunoreactivity may be related to specific developmental changes and not particularly associated to the occurrence of cell death.