Selective inhibitory effects of pregabalin on peripheral C but not A-delta fibers mediated nociception in intact and spinalized rats

Effects of pregabalin (PGB, 20–80 mg/kg i.v. injection) on spinally-organized nociception were investigated in isoflurane-anesthetized intact and spinalized rats. Responses of single deep spinal dorsal horn (DH) (laminae IV–V) nociceptive-specific (NS) neurons receiving peripheral inputs from A-δ and C fibers to repetitive electrical stimulation (intensity: 3–5 mA; frequency: 1 Hz; pulse duration: 1 ms), mechanical/heat stimulation were recorded extracellularly during physiological condition and s.c. bee venom (BV) induced inflammation. PGB significantly inhibited C-fiber mediated spinal NS neurons' late responses including phenomena of wind-up (temporal summation) and after-discharge. However, the antinociceptive effects of PGB on nociception were not observed until 30 min after its administration. In contrast, no significant inhibitory effect of PGB on A-δ fiber mediated early responses was observed during the experiments. Compared with intact rats, the inhibitory effects of PGB upon nociception vanished in the spinalized animals. This suggests that PGB-induced selective antinociceptive effect on C-fiber mediated nociception is mainly central effects involving supraspinal centers via descending inhibitory controls. Furthermore, pre-treatment, but not post-treatment, with PGB (80 mg/kg) markedly inhibited s.c. BV elicited spontaneous neuronal responses, and noxious mechanical/heat stimuli evoked hyperactivities of spinal NS neurons, indicating that PGB has efficacy of pre-emptive analgesia on pathological pain associated with central sensitization.     

Zinc-positive afferents to the rat septum originate from distinct subpopulations of zinc-containing neurons in the hippocampal areas and layers

The purpose of the present study was to examine whether zinc-positive and zinc-negative hippocampal neurons in rats differed with respect to their projections to the septum. By combining retrograde axonal transport of the fluorescent tracer Fluoro-Gold with histochemical demonstration of zinc selenide complexes in zinc-containing neurons after intraperitoneal injection of sodium selenite, we were able to visualize the distribution of retrogradely Fluoro-Gold labeled neurons and zinc-containing neurons in the same sections. After unilateral injection of Fluoro-Gold into the rat septum a few retrogradely labeled cells were observed in layer IV of the ipsilateral medial entorhinal area, and numerous labeled cells were observed mainly in the superficial layers of the ipsilateral subicular areas and throughout the CA1 and CA3 pyramidal cell layers, as well as in the contralateral CA3 pyramidal cell layer. Zinc-containing neurons were observed in layers IV–VI of the medial entorhinal area, layers II and III of the parasubiculum, layers II, III and V of presubiculum, and in the superficial CA1 and deep CA3 pyramidal cell layers. Cells double-labeled with Fluoro-Gold and zinc selenide complexes were primarily located in distal (relative to the area dentata) parts of the superficial CA1 pyramidal cell layer and distal parts of the deep CA3 pyramidal cell layer and in layers II and III of presubiculum. Only a very few double-labeled cells were seen in the contralateral CA3. The result demonstrates that the hippocampo-septal projection of rats is a mixture of zinc-positive and zinc-negative fibers. Where-as zinc-negative fibers originate from neurons throughout the hippocampal and retrohippocampal areas, zinc-positive fibers originate from distinct subgroups of zinc-containing cells in different areas and layers.     

Distribution of neuronal populations containing neurofilament protein and calcium-binding proteins in the canine neocortex: regional analysis and cell typology

Neurophysiological experiments in carnivores have revealed the existence of a large number of cortical regions and an organization of sensory systems quite similar to that found in primates. However, the cyto- and chemoarchitecture of the cerebral cortex is relatively poorly known in carnivores. We analyzed the distribution and typology of classes of neurons containing neurofilament protein or the calcium-binding proteins parvalbumin, calbindin, and calretinin in six neocortical regions of the dog. In all these areas, neurofilament protein was present in a subpopulation of medium-to-large size pyramidal neurons predominantly distributed in layers III and V. Parvalbumin was present in a large population of morphologically diverse interneurons. Small ovoid and multipolar neurons were observed throughout the cortical layers, but predominated in layers II and IV. Layers III and V–VI were characterized by the presence of larger and intensely immunoreactivity neurons with bitufted or multipolar morphology, and layers V–VI also contained large multipolar neurons. Calbindin was observed in small round and multipolar interneurons in layer II, and typical double bouquet cells in layer III. Layers IV–VI contained isolated double bouquet cells and large multipolar neurons. A few calbindin-immunoreactive pyramidal neurons were also observed in layer V. Calretinin was localized in bipolar and double bouquet cells in layers II and upper III. The lower part of layer III and layers IV–VI contained rare calretinin-immunoreactive neurons. In some areas, layer III displayed a few large isolated multipolar neurons and pyramidal neurons containing calretinin. In addition, the results show that there is a substantial degree of variability in the distribution of these proteins among cortical regions, and that although they are found in morphologically comparable neuronal types in dog, monkeys, and humans, many difference exist in their regional distribution patterns between carnivores and primates.     

Postnatal development of the visual cortex of the mouse after enucleation at birth

Summary Quantitative data in the neocortex up to the age of 180 days (neuronal densities, number of neurones, glial cells, dendritic intersections and spines) were compared in normal mice and mice enucleated at birth.Bilateral enucleation induced an increase of neuronal density in all cortical layers of areas 17, 18a, and 41, the supragranular layers II–III being more affected than layers IV–VI. This was noticed in layer II 10 days after the operation and was maximal in all layers between 30 and 60 days; at 180 days there was some return to normal of the neuronal density in all layers. The total number of neurones and glial cells were the same in the bilaterally enucleated mice as in the controls. No reaction in dendritic branching was evident for pyramids of layers III and V in areas 17 and 41 after bilateral enucleation. In contrast the number of spines was reduced on the apical dendrites of pyramids from layers III and V in area 17, but not in area 41.After unilateral enucleation the reaction was less severe and delayed compared with bilateral enucleation, the first signs of increase of neuronal density appearing 30 days after the lesion in the contralateral hemisphere. The contralateral areas 17 and 18a were more affected than the ipsilateral ones and area 41 showed no change compared to the control. As after bilateral enucleation, layers IV and V were least affected by unilateral enucleation in both ipsi- and contralateral cortices.These results suggest that deafferentation in an immature system affects the development of all cortical layers but with a greatest intensity in supragranular layers, which are not the main direct targets of thalamo-cortical input.Supported by the Swiss National Research Foundation, Grants no. 3-641-71 and 3-434-74     

Development of the ultrastructural features of neuropeptide Y-immunoreactive neurons in the rat visual cortex

Summary Immunohistochemical studies have localized neuropeptide Y into a small population of non-pyramidal neurons in the mammalian cerebral cortex. In the rat, these cells are distributed in layers II–VI and are characterized at the ultrastructural level by an abundance of cytoplasm containing a plethora of organelles, most conspicuous of which are cisternae of granular endoplasmic reticulum stacked in parallel arrays. In the present study, we used electron microscopic immunocytochemistry to examine the ultrastructural development of neuropeptide Y-labelled neurons in the rat visual cortex from birth, when they first appear in this cortical area, until postnatal day 32. At birth and in the subsequent few days, neuropeptide Y neurons, found exclusively in layers V and VI, often show a deeply infolded nucleus and little cytoplasm containing few organelles. At the end of the first postnatal week, labelled cells are still restricted to layers V and VI and display immature features. However, at this stage, cells often show irregularly enlarged proximal dendrites filled with organelles. During the second postnatal week, neuropeptide Y-immunoreactive cell bodies appear for the first time in layers II and III, and at the end of this week they have a distribution similar to that observed in the adult. Labelled cells are overall more differentiated than at earlier ages showing some of the ultrastructural features which distinguish them in the adult. No differences in maturation are evident between immunoreactive neurons located in the superficial layers and those in the deep layers, suggesting that the neuropeptide Y neurons in the more superficial layers express the peptide after having completed their migration and have acquired their characteristic ultrastructural features. Maturation proceeds during the third postnatal week. At the end of this stage, neuropeptide Y-containing cells acquire their mature nuclear and cytoplasmic features and an adult complement of synapses.     

Morphology of spiny neurons in the human entorhinal cortex: intracellular filling with lucifer yellow

The present study was designed to investigate the morphology of spiny neurons in the human entorhinal cortex. Coronal entorhinal slices (n = 67; 200 microm thick) were obtained from autopsies of three subjects. Spiny neurons (n = 132) filled with Lucifer Yellow were analysed in different subfields and layers of the entorhinal cortex. Based on the shape of the somata and primary dendritic trees, spiny neurons were divided into four morphological categories; (i) classical pyramidal, (ii) stellate, (iii) modified stellate, and (iv) horizontal tripolar cells. The morphology of filled neurons varied more in different layers than in the different subfields of the entorhinal cortex. In layer II, the majority (81%) of spiny neurons had stellate or modified stellate morphology, but in the rostromedial subfields (olfactory subfield and rostral subfield) there were also horizontal tripolar neurons. Dendritic branches of layer II neurons extended to layer I (94%) and to layer III (83%). Unlike in layer II, most (74%) of the filled neurons in layers III, V and VI were classical pyramidal cells. The majority of pyramidal cells in the superficial portion of layer III had dendrites that extended up to layer II, occupying the space between the neuronal clusters. Some dendrites reached down to the deep portion of layer III. Apical dendrites of layer V and VI pyramidal cells traveled up to the deep portion of layer III.Our data indicate that the morphology of spiny neurons in different layers of the human entorhinal cortex is variable. Vertical extension of dendritic branches to adjacent layers supports the idea that inputs terminating in a specific lamina influence target cells located in various entorhinal layers. There appears to be more overlap in the dendritic fields between superficial layers II and III than between the superficial (II/III) and deep (V/VI) layers, thus supporting the idea of segregation of information flow targeted to the superficial or deep layers in the human entorhinal cortex.     

Comparison of neuronal and glial numerical density in primary and secondary visual cortex of man

Summary The numerical density of neurons and glial cells was estimated in visual area 18 of the adult human cerebral cortex and compared with that of area 17. Blocks of areas 17 and 18 came from the same brains and this allowed the comparison of 1) neuronal and glial numerical densities through the whole cortical depth with calculation of the neuron/glia ratio, 2) neuronal and glial numbers under one square millimeter of cortical surface, and 3) neuronal numerical densities in three groups of identified layers. The mean neuronal density is approximately 40000 neurons/mm³ in area 17 and 31500/m³ in area 18. The mean glial density is around 27000/mm³ in area 17 and 32000/mm³ in area 18. This gives a neuron/glia ratio of approximately 1.5 in area 17 and of 1.0 in area 18, but the total cellular density is similar in both areas. There are about 90000 neurons and 64000 glial cells under one square millimeter of cortical surface in area 17, and some 73000 neurons and 74000 glial cells in area 18. The higher neuronal density in area 17 is found through the whole depth of cortex and does not seem to be more pronounced in layer IVc of area 17 compared to layer IV in area 18 than in the groups of layers II–III and V–VI.     

Inhibitory effects of excitatory amino acids on pyramidal cells of the in vitro turtle medial cortex

Summary Peripheral information reaches the motor cortex partly through corticocortical pathways that arise from two functional subdivisions, area 2 and area 3a, of the sensory cortex. These sensory areas are synaptically linked with one another. The patterns of connectivity and the different submodality input that each area receives suggest that they send different efferent signals to the motor cortex. The projections from area 2 to area 3a and to the motor cortex were studied with retrogradely transported fluorescent tracers. The pattern and distribution of neuronal labeling in area 2 was determined following injections of different tracers into the forelimb regions of area 3a and the motor cortex. The results showed that the projections from area 2 to the two target regions were topographically and somatotopically related. Multiple clusters of motor cortex projection neurons were found in area 2, and these clusters overlapped extensively with clusters of area 3a projection neurons. Although cells labeled with one of the dyes were often in close proximity to cells labeled with the other dye, no double-labeled cells were found. Two different laminar patterns were seen for the two populations of neurons. The projection to area 3a originated from cells located in layers II–III and layers V–VI. The projection to the motor cortex originated from cells spread throughout layers II–IV, but predominately in layer III. Differences in laminar arrangement of the two populations of cells suggest a directional flow of information processing in the sensorimotor cortex. While sensory feedback is essential for the execution of skilled motor tasks, the flow of information processing and connectivity in the cortex is not well understood. The data presented here offer insight into some aspects of the mechanisms of sensorimotor integration.     

Acute bladder inflammation differentially affects rat spinal visceral nociceptive neurons

The present investigation examined the effect of inflammation produced by intravesical zymosan on spinal dorsal horn neuronal responses to urinary bladder distension (UBD). Extracellular single-unit recordings of neurons excited by UBD were obtained in spinalized female Sprague–Dawley rats. Neurons were classified as Type I—inhibited by heterotopic noxious conditioning stimuli (HNCS) or as Type II—not inhibited by a HNCS. In Experiment 1—following neuronal characterization, 1% zymosan was infused into the bladder and after 2 h spinal units were recharacterized. Control rats received intravesical saline or subcutaneous zymosan. In Experiment 2—rats were pretreated with intravesical zymosan 24 h prior to surgical preparation. Control rats received anesthesia only. 137 spinal dorsal horn neurons excited by UBD were characterized. In comparison with controls, Type II neurons demonstrated increased spontaneous and UBD-evoked activity following intravesical zymosan treatment (both Experiments 1 and 2) whereas Type I neurons demonstrated either no change (Experiment 1) or decreased activity (Experiment 2) following bladder inflammation. No significant changes were noted in neuronal activity in control experiments. Inflammation differentially affects subpopulations of spinal dorsal horn neurons excited by UBD that can be differentiated according to the effect of HNCS. This results in an altered pattern of spinal sensory transmission that may serve as the mechanism for the generation of visceral nociception.     

Morphological changes of c-Fos-like immunoreactivity in rat cerebral cortex after cerebral ischemia and reperfusion with special reference to vasoactive intestinal polypeptide

We investigated morphological changes in neurons with c-Fos-like immunoreactivity (c-Fos-LI) after cerebral ischemia by light and electron microscopic immunocytochemistry. Strong c-Fos-LI was observed in layers II–VI of the cerebral cortex with an especially abundant distribution in the nuclei of layers II, IV, and VI ipsilateral to the lesioned side. Reperfusion after ischemia had a greater effect on the expression of c-Fos-LI than did permanent ischemia. Vasoactive intestinal peptide (VIP)-positive neurons were seen scattered in layers II–V of the cerebral cortex. Some VIP-positive neurons showed c-Fos-LI after ischemia. Electron microscopy revealed c-Fos-LI in euchromatin in the nuclei of c-Fos-positive cells. Dilatation of the cisternae of the rough endoplasmic reticulum and the presence of numerous secondary lysosomes were found in neurons on the lesioned side after 12h of reperfusion. Some VIP-containing neurons revealed c-Fos-LI with reperfusion after ischemia by a double immunostaining method on the same tissue section. These findings suggest that ischemia potentiates c-fos expression in VIP- or other transmitter- or modulator-containing neurons, thereby protecting from neuronal cell death.     

Neuronal composition and interneuronal connection of area 5 in the cat parietal association cortex

Area 5 of the cat cortex was studied by Nissl's method and by Golgi's chromate-silver impregnation method. Its typical six-layered structure with well-developed layers of pyramidal cells was revealed. The characteristic features of area 5 are: predominance of pyramidal cells in layers II–III and the presence of large forms (40×26) among them (in layer III); giant pyramidal neurons (70×23) arranged singly or nidally in layer V; large (diameter 25–30) and giant (diameter 40–45) stellate cells with radial dendrites, arranged singly or in groups in layers V–VI; infrequent efferent fusiform neurons (40×20) in layers V–VI. Stellate cells connecting pyramidal neurons in the same or in different layers were found in layers II–VI. Some stellate cells in layers II–III form long horizontal connections within area 5. Interneuronal connections are effected by axosomatic and axodendritic terminals, the latter being more numerous; Dendrodendritic and axoaxonal synapses are less common.Translated from Neirofiziologiya, Vol. 11, No. 1, pp. 35–42, January–February, 1979.     

A comparison of frailty of primary neurons,embryonic, and aging mouse cortical layers

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ_1–42, okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.     

Delayed cell migration in the developing rat brain following maternal omega 3 alpha linolenic acid dietary deficiency

Diminished levels of docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid (FA) synthesized from alpha linolenic acid (ALA, 18:3n-3), have been implicated in changes in neurotransmitter production, ion channel disruption and impairments of a variety of cognitive, behavioral and motor functions in the perinatal and adult mammal. Neuronal migration in the cortex and hippocampus of newborn and postnatal rats after ALA-deficiency, beginning on the 2nd day after conception and continuing for three weeks, was investigated. A marked decrease in the migration of bromodeoxyuridine⁽⁺⁾/neuronal nuclei⁽⁺⁾/neurofilament⁽⁺⁾ and glia fibrillary acidic protein^(–) neuronal cells to the dense cortical plate was accompanied by a corresponding abundance of non-migrating cells in several regions such as cortical layers IV–VI, corpus callosum and the sub-ventricular zone of ALA-deficient newborns. Similarly, a delayed migration of cells to CA1 and dentate gyrus areas was noticed while most cells were retained in the subicular area adjacent to the hippocampus. The reversibility of delay in migration in the hippocampus and cortex, after one and two weeks respectively, may be attributed to a temporary reelin disorganization or partial deficiency. Transient obstruction of neuronal cell migration may have long-lasting consequences on the organization of neuronal assemblies, on the connection between neurons (lateral connections) and acquisition of function in the adult brain.     

Age-related changes of neuron numbers in the frontal cortex of a transgenic mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is a neurodegenerative disorder, characterized by amyloid plaque accumulation, intracellular tangles and neuronal loss in selective brain regions. The frontal cortex, important for executive functioning, is one of the regions that are affected. Here, we investigated the neurodegenerative effects of mutant human amyloid precursor protein (APP) and presenilin 1 (PS1) on frontal cortex neurons in APP/PS1KI mice, a transgenic mouse model of AD, expressing two mutations in the human APP, as well as two human PS1 mutations knocked-in into the mouse PS1 gene in a homozygous (ho) manner. Although the hippocampus is significantly affected in these mice, very little is known about the effects of these mutations on selective neuronal populations and plaque load in the frontal cortex. In this study, cytoarchitectural changes were characterized using high precision design-based stereology to evaluate plaque load, total neuron numbers, as well as total numbers of parvalbumin- (PV) and calretinin- (CR) immunoreactive (ir) neurons in the frontal cortex of 2- and 10-month-old APP/PS1KI mice. The frontal cortex was divided into two subfields: layers II–IV and layers V–VI, the latter of which showed substantially more extracellular amyloid-beta aggregates. We found a 34% neuron loss in layers V–VI in the frontal cortex of 10-month-old APP/PS1KI mice compared to 2-month-old, while there was no change in PV- and CR-ir neurons in these mice. In addition, the plaque load in layers V–VI of 10-month-old APP/PS1KI mice was only 11% and did not fully account for the extent of neuronal loss. Interestingly, an increase was found in the total number of PV-ir neurons in all frontal cortical layers of single transgenic APP mice and in layers II–IV of single transgenic PS1ho mice between 2 and 10 months of age. In conclusion, the APP/PS1KI mice provide novel insights into the regional selective vulnerability in the frontal cortex during AD that, together with previous findings in the hippocampus, are remarkably similar to the human situation.     

The mode of synaptic activation of pyramidal neurons in the cat primary somatosensory cortex: an intracellular HRP study

Summary A total of 141 pyramidal neurons in the cat primary somatosensory cortex (SI) were recorded intracellularly under Nembutal anesthesia (7 in layer II, 43 in layer III, 8 in layer IV, 58 in layer V and 25 in layer VI). Most neurons were identified by intracellular staining with HRP, though some layer V pyramidal neurons were identified only electrophysiologically with antidromic activation of medullary pyramid (PT) or pontine nuclear (PN) stimulation. Excitatory synaptic potentials (EPSPs) were analyzed with stimulation of the superficial radial nerve (SR), the ventral posterolateral nucleus (VPL) in the thalamus and the thalamic radiation (WM). The pyramidal neurons in layers III and IV received EPSPs at the shortest latency: 9.1±2.1 ms (Mean+S.D.) for SR and 1.6±0.7 ms for VPL stimulation. Layer II pyramidal neurons also responded at a short latency to VPL stimulation (1.7±0.5 ms), though their mean latencies for SR-induced EPSPs were relatively longer (10.6±1.9 ms). The mean latencies were much longer in layers V and VI pyramidal neurons (10.2±2.4 ms and 2.9±1.5 ms in layer V pyramidal neurons and 9.9±2.5 ms and 2.8±1.6 ms in layer VI pyramidal ones, respectively for SR and VPL stimulation). The comparison of the latencies between VPL and WM stimulation indicates that most layer III–IV pyramidal neurons and some pyramidal cells in layers II, V and VI received monosynaptic inputs from VPL. These findings are consistent with morphological data on the laminar distribution of thalamocortical fibers, i.e., thalamocortical fibers terminate mainly in the deeper part of layers III and IV with some collaterals in layers V, VI and II-I. The time-sequences of the latencies of VPL-EPSPs indicate that corticocortical and/or transcallosal neurons (pyramidal neurons in layers II and III) fire first and are followed by firing of the output neurons projecting to the subcortical structures (pyramidal neurons in layers V and VI).     

Intrinsic circuitry and physiological properties of pyramidal neurons in rat barrel cortex

 Pyramidal neurons in the rat posteromedial barrel subfield (PMBSF) were characterized physiologically and filled with biocytin in in vitro brain slices. Intrinsic axons belonging to supragranular neurons pro-jected horizontally and vertically, arborizing in layers II/III and V, but had few or no projections to layers IV or VI. These axons projected horizontally for up to 2 mm, spanning two to seven barrel columns. Layer V neurons had more diffuse axon arbors that projected either vertically, arborizing in layers III to V, or horizontally, branching profusely in layers V and VI. The basal dendritic trees of neurons in layers II/III, V and VI spanned one or two barrel columns without being skewed toward particular barrel columns. Physiologically, regular- spiking neurons were classified as ”RS₁” or ”RS₂” according to their degree of late spike frequency adaptation. RS₁ neurons were found in superficial and deep layers, whereas RS₂ neurons were significantly more prevalent in the latter. Infragranular, but not supragranular neurons showed slow, inward rectification at hyperpolarized potentials. All neurons generated fast and medium afterhyperpolarizations following individual spikes; however, only infragranular pyramids had depolarizing afterpotentials interposed between the two afterhyperpolarizations. RS₁ neurons had larger cell bodies, longer total basal dendritic lengths, and more densely branched proximal dendritic trees than RS₂ neurons. These findings indicate that pyramidal neurons in the deep and superficial layers of the rat PMBSF have distinct patterns of intracortical axon arbors and distinct physiological properties. These features are probably involved in shaping and modulating the response properties of PMBSF neurons. Received: 24 May 1996 / Accepted: 7 November 1996     

Afferents to the seizure-sensitive neurons in layer III of the medial entorhinal area: a tracing study in the rat

Neurons in layer III of the medial entorhinal area (MEA) in the rat are extremely vulnerable to local injections of amino-oxyacetic acid and to exprimentally induced limbic seizures. A comparable specific pathology has been noted in surgical specimens from patients with temporal lobe epilepsy. Efforts to understand this preferential neuronal vulnerability led us to study the neural input to this layer in the rat. Iontophoretic injection of the retrograde tracer fast blue, aimed at layer III of the MEA, resulted in retrogradely labeled neurons in the presubiculum in all the injected hemispheres. The nucleus reuniens thalami, the anteromedial thalamic nucleus, the ventral portion of the claustrum (endopiriform nucleus), the dorsomedial parts of the anteroventral thalamic nucleus, and the septum-diagonal band complex were labeled less frequently. In only one experiment, retrogradely labeled neurons were observed in the ventrolateral hypothalamus and in the brainstem nucleus raphe dorsalis. Since projections from claustrum to the entorhinal cortex has not been studied in the rat with modern sensitive anterograde tracing techniques, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were placed into the ventral portion of the claustrum. Anterogradely labeled fibers in the entorhinal area proved not to be confined to the MEA, since a prominent projection distributed to the lateral entorhinal area as well. In both areas, the densest terminal labeling was present in layers IV–VI, whereas layer III appeared to be only sparsely labeled. The present data indicate that of all potential afferents only those from the presubiculum distribute preferentially to layer III of the MEA. This, in turn, suggests a potentially important role of the presubiculum in the seizure-related degeneration of neurons in layer III of the MEA.     

The laminar distribution and postnatal development of serotonin-immunoreactive axons in the cat primary visual cortex

Summary The laminar distribution and postnatal development of profiles immunoreactive to antibodies directed against serotonin (5-hydroxytryptamine, 5-HT) have been investigated in the primary visual cortex (striate cortex, area 17) of cats. In the adult cat, profiles with serotonin-like immunoreactivity consist exclusively of fibers which exhibit laminar differences in density and predominant orientation. Immunoreactive fibers are dense in layers I–III, less dense in layer V, and sparse in layers IV and VI. In layers I and VI the trajectories of these fibers are mainly tangential to the pial surface; in layers II–V they are predominantly radial and more irregular. The vast majority of immunoreactive fibers consists of fine axons with frequent small varicosities. In addition, there are a few thick axons. In 2-week-old cats, immunoreactive fibers are sparsely distributed through layers II–V. By 4 weeks, fiber density has decreased still further in layer IV and increased in layers I–III. By 6 weeks, the laminar pattern resembles that of adult cats except that fiber density is still lower than in adults. At three months of age, the mature pattern is established.     

Morphological analyses of NADPH-diaphorase/nitric oxide synthase positive structures in human visual cortex

Summary Human visual cortex was studied using NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry. Large, strongly stained, sparsely spined non-pyramidal cells (average soma diameter: 16 × 16 m) occur in layers H–VI, but are commonest in layers II–III. Small weakly stained multipolar cells (average soma diameter 3.6 × 4 m, stellate like cells) in layers II–VI are concentrated in layer IV of areas 17 and 18. The density of these cells, measured with a computer assisted microscopy system is less in area 18 than 17. Large, strongly stained, predominantly horizontal cells (average soma diameter 12 × 19 m) are localized in the underlying white matter. Axons of the large, strongly NADPH-diaphorase positive cells are thin and unbranched with fine boutons. These axons ascend to layer I. The large, strongly stained cells in layers II–VI we identify as Martinotti neurons. In layer I parallel unbranched positive fibres with some fine boutons run horizontally and build dense axonal plexuses together with the axons of Martinotti neurons. Axons of presumed extrinsic origin are morphologically different from NADPH-diaphorase positive intrinsic fibres. They show thick varicosities running in different directions and forming a network in layers III–VI. Basket like formations of these fibres were frequently observed in layers IV, V and VI. Other fibres seem to innervate blood vessels. Nitric oxide synthase was also demonstrated immunohistochemically by a polyclonal rabbit nitric oxide synthase antiserum. The morphology and distribution of the immunostained cells correspond with those seen with NADPH-diaphorase histochemistry. Double labelling experiments confirm the colocalization of NADPH-diaphorase and nitric oxide synthase in all demonstrated cells. Immunohistochemical demonstration of glial fibrillary acidic protein has shown that astrocytes are not involved in the NADPH-diaphorase/NOS system in the human visual cortex.     

Layer VII and the gray matter trajectories of corticocortical axons in rats

The trajectory of long distance intrahemispheric corticocortical axons has been investigated using the anterograde fluorescent axonal tracer fluororuby. Most axons of this kind were found to travel through the gray matter of layers VI and VII rather than in the white matter. The cell-sparse zone immediately superficial to layer VII contains a dense aggregate of longitudinally directed axons. Corticocortical axons traveling in the mediolateral plane also utilize the deep gray matter predominately. Layer VII neurons are persistent remnants of the subplate in rats. Based on our retrograde labeling results, they are involved in long distance as well as local corticocortical connections. Layer VII neurons are often labeled in a more continuous pattern after cortical injections of retrograde tracers than neurons of layers II, III and V, which are labeled in a patchy manner.