Distribution of monoaminergic, cholinergic, and GABAergic markers in the human cerebral cortex

Mapping of a number of biochemical markers for noradrenergic, dopaminergic, serotoninergic, cholinergic and GABAergic systems was undertaken in 93 samples removed from the human cerebral cortex. The right hemisphere of brains from two subjects with no known history of neurological and psychiatric diseases was examined. Neurotransmitter markers were present in all cortical samples analysed, suggesting a widespread distribution of the corresponding neurons throughout the cerebral cortex. Each marker distributed heterogeneously in a distinct pattern. Noradrenaline concentrations were highest in the frontoparietal region and lowest in prefrontal and occipital areas. Markers for dopaminergic neurons (dopamine levels, dopamine/noradrenaline ratio and homovanillic acid levels) seemed denser in the prefrontal and temporal regions. 5-Hydroxyindolacetic acid levels were particularly high in the occipital area and decreased along the caudorostral axis. Choline acetyltransferase activity was highest in temporal and frontal lobes, at variance with muscarinic receptor distribution, which was highest in occipital cortex. Glutamate decarboxylase activity, an index of GABAergic innervation, did not vary markedly among the different areas of the cerebral cortex. The different biochemical markers investigated were detected in all cerebral cortical regions; their distribution was not homogeneous. A mismatch was observed between the distribution of cholinergic neuronal systems and receptors.     

The axo-axonic interneuron in the cerebral cortex of the rat,cat and monkey

The synaptic connections of a specific type of identified cortical interneuron, the axo-axonic cell, were studied using Golgi methods. In the light-microscope axo-axonic cells were demonstrated in certain layers of the primary and secondary visual cortex of rat, cat and monkey, in the motor cortex of cat and in the subiculum and pyriform cortex of rat. The dendrites originating from the oval soma were oriented radially in a lower and upper spray within a cylinder about 100–150 μm wide. Electronmicroscopy of Golgi impregnated, gold-toned axo-axonic cells showed predominantly but not exclusively asymmetrical synaptic contacts on their dendrites and spines, few synaptic contacts on the perikarya some of which were asymmetrical, and no synaptic contacts on the axon initial segment. The axon usually arborized within the vicinity of the cell's own dendritic field in an area 100–200 μm in diameter. In the kitten motor cortex the axon of a neuron in layer III descended to layer VI, providing a columnar arborization.The axon formed specialized, 10–50 μm long terminal segments invariably oriented parallel with the axon initial segment of pyramidal cells. All 85 identified symmetrical-type synaptic contacts, deriving from 31 specialized terminal segments, were found exclusively on the axon initial segment of pyramidal neurons. Rare, lone boutons of axo-axonic cells also made synaptic contact only with axon initial segments, confirming the exclusive target specificity of these cells. In identified gold-toned boutons, flattened pleomorphic vesicles were present. Electron-microscopy showed that axons ending in specialized terminal segments may originate from myelinated fibres, indicating that Golgi impregnation has revealed only part of the axon. Counting of axon terminal segments, each of which was in contact with the axon initial segment of a pyramidal neuron, revealed 166 pyramidal neurons receiving input from a partially reconstructed axo-axonic cell in the motor cortex of the kitten, and 67 from another cell in the visual cortex of the cat. The convergence of five axo-axonic cells onto one pyramidal cell was demonstrated in the striate cortex of the cat by counting all synaptic contacts on three initial segments. Cells from a one-month-old kitten were compared with those of the adult. The axon of the developing neurons was more diverse, having many growth cones and filopodia which made no specialized membrane contacts. However, the developing specific terminal segments formed synapses only with axon initial segments.It is concluded that the presence of axo-axonic cells in all the species and cortical areas we have examined suggests their association with the structural design of pyramidal cells, wherever the latter occur, and with their participation in the information processing of pyramidal cells. Axo-axonic cells are uniquely endowed with the means of simultaneously influencing the action potential at the site of origin in groups of pyramidal cells. This strategic location may enable them to synchronise the activity of pyramidal neurons, either through inhibitory gating or through changing the threshold of pyramidal cells to certain inputs.     

Calcium binding proteins and neuropeptides as molecular markers of GABAergic interneurons in the cat visual cortex

Summary In the cat visual cortex, almost all parvalbumin-positive cells are GABAergic, and about 80% of the calbindin D-28K-positive neurons are also GABA-immunoreactive. About 37% of the GABAergic neurons contain parvalbumin and a smaller fraction (about 18%) contains calbindin. Furthermore, parvalbumin and calbindin are localized in two separate neuronal populations in the cat visual cortex, suggesting that two GABAergic populations can be distinguished, one containing parvalbumin and one containing calbindin. Double staining for parvalbumin and neuropeptides (CCK, SRIF and NPY), revealed no double-labeled cells, with the exception of a few SRIF- and parvalbumin-positive neurons. These results show that cortical GABAergic cells can be differentiated on basis of their calcium binding protein and neuropeptide immunoreactivity.     

Extracellular concentrations of aspartate and glutamate in rat neostriatum following chemical stimulation of frontal cortex

Summary Extracellular concentrations of excitatory amino acids in the neostriatum of anaesthetized rats were studied by in vivo microdialysis and the influence of chemical stimulation of the neocortex assessed. Administration of gamma-aminobutyric acid (GABA) antagonists to the neocortex evoked an increase in the extracellular concentration of aspartate (to 315 and 210% of basal values for microinfusion and topical application, respectively), but not of extracellular glutamate. Such selectivity was also found in response to N-methyl-D-aspartate (NMDA, 348% increase in extracellular aspartate concentration). Yet co-administration of NMDA with GABA antagonists increased the extracellular concentration of glutamate (to 278% of basal values) as well as aspartate (to 611% of basal values). The results suggest that either the activated neurons are hypoglycemic or hypoxic, or that aspartate is the major neurotransmitter of corticostriatal neurons.     

Local connections to specific types of layer 6 neurons in the rat visual cortex

Because layer 6 of the cerebral cortex receives direct thalamic input and provides projections back to the thalamus, it is in a unique position to influence thalamocortical interactions. Different types of layer 6 pyramidal neurons provide output to different thalamic nuclei, and it is therefore of interest to understand the sources of functional input to these neurons. We studied the morphologies and local excitatory input to individual layer 6 neurons in rat visual cortex by combining intracellular labeling and recording with laser-scanning photostimulation. As in previous photostimulation studies, we found significant differences in the sources of local excitatory input to different cell types. Most notably, there were differences in local input to neurons that, based on analogy to barrel cortex, are likely to project only to the lateral geniculate nucleus of the thalamus versus those that are likely to also project to the lateral posterior nucleus. The more striking finding, however, was the paucity of superficial layer input to layer 6 neurons in the rat visual cortex, contrasting sharply with layer 6 neurons in the primate visual cortex. These observations provide insight into differences in function between cortical projections to first-order versus higher-order thalamic nuclei and also show that these circuits can be organized differently in different species.     

Differential effect of kainic acid on somatostatin, GABAergic and cholinergic neurons in the rat striatum

Kainic acid was injected into the rat striatum and its effects on presumptive striatal cholinergic, GABAergic and somatostatin-containing neurons were examined with three histochemical staining methods. Presumptive cholinergic and GABAergic neurons are damaged to a similar extent, but somatostatin neurons are more sensitive to the neurotoxic effect of kainic acid and are more severely affected.     

Action of peripherally administered cholecystokinin on monoaminergic and GABAergic neurons in the rat brain

In an acute study, cholecystokinin octapeptide sulfate (CCK) in doses of 1, 10 or 100 micrograms/kg body weight was injected intraperitoneally into rats just prior to the dark cycle. Rats were sacrificed two hours following the CCK injection. Norepinephrine levels were elevated in the dorsal amygdala of rats injected with 10 micrograms of CCK as well as in the septum of rats injected with 1 and 10 micrograms of CCK. The dopamine level in the septum of rats injected with 1 microgram of CCK as well as the gamma-aminobutyric acid (GABA) level in the lateral hypothalamus of rats injected with 10 micrograms of CCK were also elevated. In a chronic study, CCK (1 microgram/kg body weight/h) was subcutaneously infused into rats with Alzet osmotic minipump for seven consecutive days. The daily food consumption did not change during the 7 days of CCK infusion. The dopamine turnover in the striatum accelerated and the GABA level increased. On the contrary, dopamine metabolism in the substantia nigra and locus coeruleus decreased. Furthermore, the serotonin level in the substantia nigra decreased. Norepinephrine levels decreased in the nucleus paraventricularis, the locus coeruleus and the substantia nigra. The results suggest that peripherally administered CCK may act on the monoaminergic neurons and GABAergic neurons in the brain.     

GABAergic neuronal subtypes in the human frontal cortex — development and deficits in schizophrenia

Recent studies have provided evidence for a deficit of GABA-containing interneurons in the frontal cortex in schizophrenia. That this deficit might be brought about during early foetal or neonatal life is a hypothesis consistent with the substantial indications for a neurodevelopmental aetiology of the disease. GABAergic neurons can be defined by the presence of one of three types of calcium binding proteins, which are thought to have neuroprotective properties. We have undertaken an investigation into the postnatal ontology of these neuronal subtypes and find that calretinin expression is relatively constant and present from before birth, calbindin expression is also present early but redistributes in the cortex over the first months of life, while parvalbumin-immunoreactivity is not observed until between 3 and 6 months of age. Investigation of frontal cortical tissue taken post mortem from a series of schizophrenic patients and matched control subjects revealed that parvalbumin-, but not calretinin-immunoreactive cells are significantly diminished in schizophrenia. These observations support the hypothesis that GABAergic deficits in schizophrenia may stem from toxic events occurring during cortical development which selectively target immature neurons before protection by parvalbumin is conferred.     

Immunohistochemical localizations of secretin, cholecystokinin, and somatostatin in the rat small intestine after acute cisplatin treatment

Cisplatin (CDDP) is an antitumor platinum complex that causes the well-studied side effect of renal tubular failure. In the present study, the acute effects of CDDP treatment on the localization of gut hormones in the rat small intestine were examined by immunohistochemistry. Male Sprague-Dawley rats were used for these experiments. Rats were injected intravenously with CDDP (3 mg/kg) in saline or were left untreated (control). After the rats were euthanized at 1, 3, 5, or 10 days after CDDP treatment, the small intestines (duodenum, jejunum, and ileum) were quickly removed, fixed, embedded in paraffin, and cut. No mucosal toxicity was detected by histopathological observation in any of the intestines of CDDP-treated rats. The immunohistochemical detection was performed using anti-secretin, anti-cholecystokinin (CCK), and anti-somatostatin with the avidin-biotin-immuno-peroxidase procedure. The total number of immunoreactive cells per complete cross-section was counted. In the duodenum, the numbers of secretin-immunoreactive cells and somatostatin-immunoreactive cells were dramatically increased 5 days after CDDP treatment. In the jejunum, the number of CCK-immunoreactive cells was increased 1 day after CDDP treatment and those of secretin-immunoreactive cells and CCK-immunoreactive cells were increased 5 days after CDDP treatment. In the ileum, the number of CCK-immunoreactive cells was increased 1 day after CDDP treatment. The change in the secretin-immunoreactive cell count may be caused by metabolic inhibition of gastrin following CDDP-induced nephrotoxicity. The change in the CCK-immunoreactive cell count may promote the excretion of bile. Therefore, somatostatin may regulate secretin and CCK secretion. We conclude that the distribution of these hormone-immunoreactive cells in the rat small intestine might be controlled by CDDP-induced nephrotoxicity without gut mucosal toxicity.     

Specific changes of somatostatin mRNA expression in the frontal cortex and hippocampus of diabetic rats

Most current studies of diabetic encephalopathy have focused on brain blood flow and metabolism, but there has been little research on the influence of diabetes on brain tissue and the causes of chronic diabetic encephalopathy. The technique of molecular biology makes it possible to explore the mechanism of chronic diabetic encephalopathy by testing the distribution of somatostatin in the brain. We have therefore analysed, by in situ hybridization histochemistry, the changes in somatostatin (SST) mRNA in the frontal cortex and hippocampus of rats made diabetic by the injection of streptozotocin. Ten Sprague–Dawley control rats were compared with ten streptozotocin-induced diabetic rats. The weight, blood glucose and urine glucose did not differ between the two groups before the injection of streptozotocin. Four weeks after the injection of streptozotocin the weight, blood glucose and urine glucose of the diabetic rats were, respectively, 199.1 ± 15.6 g, 23.7 ± 3.25 mmol L⁻¹ and (++) to (+++) whereas those of the control group were 265.5 ± 30.3 g, 4.84 ± 0.63 mmol L⁻¹ and (–). Somatostatin mRNA was reduced in the diabetic rats. The number of SST mRNA-positive neurons and the optical density of positive cells in the hippocampus and frontal cortex of the diabetic rats were 80.6 ± 17.5 mm⁻² and 76.5 ± 17.6 compared with 150.5 ± 21.1 mm⁻² and 115.1 ± 18.5 in the control rats. The induction of diabetes is thus associated with a decreased expression of SST mRNA in the hippocampus and frontal cortex, which might be an important component of chronic diabetic encephalopathy.     

Development of GABAergic neurons in rat visual cortex as identified by glutamate decarboxylase-like immunoreactivity

The development of glutamate decarboxylase (GAD)-like immunoreactivity was studied in albino rats. GAD-positive structures appeared and accumulated in a characteristic temporo-spatial sequence: from embryonic day 18 onwards in lamina (L) I and subplate; after birth in L I-II, L IV-V and deep L VI including white matter; after two weeks in superficial L I, L II to L III-IV border, L V and L VI-white matter. GAD-positive axon varicosities formed perisomatic 'baskets' on L V pyramids on postnatal day 8 and a few days later on those L II-III. During the third week GAD-positive structures attained adult densities and established different laminar patterns in the primary and secondary visual areas.     

Serial lesion effect in rat medial frontal cortex as a function of age

—This experiment examined the potential for behavioral recovery in juvenile, adult, and senescent rats following serial lesions of medial frontal cortex. The subjects were trained on spatial delayed alternation in a T-maze under conditions designed to enhance the probability of a a serial lesion effect. All subjects were given extensive handling and adaptation to the maze, interoperative training, and long interoperative and postoperative intervals. There were several major behavioral findings: (a) the aged intact subjects were not impaired in their ability to learn spatial delayed alternation, (b) one-stage bilateral lesions of frontal cortex produced equivalent deficits on spatial delayed alternation at all ages, (c) subjects in all of the ages, (c) subjects in all of the age categories demonstrated a serial lesion effect, but (d) the 150 day and 570 day serial lesion groups demonstrated significantly better performances than the 35 day serial lesions group on several measures of performance.     

大鼠纹状体边缘区内神经降压肽、生长抑素、P物质及胆囊收缩素样免疫阳性反应的老年变化

目的　研究大鼠纹状体边缘区中和学习记忆有关的神经肽的年龄变化。方法　用免疫组化及显微图象分析方法研究了老年大鼠和成年大鼠纹状体边缘区中神经降压肽 (NT)、生长抑素 (SOM)、P物质 (SP)及胆囊收缩素 (CCK)样免疫阳性反应的年龄变化。结果　成年大鼠的神经降压肽、生长抑素、P物质及胆囊收缩素样免疫阳性纤维和胞体在纹状体边缘区中分布密集 ,形成一条明显的带状 ,带的宽度和位置与边缘区一致。老年大鼠的纹状体边缘区中也可见神经降压肽、生长抑素、P物质及胆囊收缩素样免疫阳性纤维终末 ,但和成年大鼠相比 ,阳性反应分布的范围较小 ,染色的深度较浅 ,未见阳性胞体。结论　边缘区中存在较多的NT、SOM、SP和CCK免疫阳性纤维终末和胞体 ,老年大鼠的边缘区中上述免疫阳性纤维终末的分布显著减少 ,未见阳性胞体。推测老年大鼠的学习记忆能力的下降可能和老年大鼠纹状体边缘区中NT、SOM、SP及CCK含量减少有关。     

Electroconvulsive shock increases serotonin transporter in the rat frontal cortex

The antidepressive action of electroconvulsive shock (ECS) is thought to involve the alteration in serotonin (5-HT) neurotransmission, including the increase in 5-HT release and uptake. In our previous study, 5-HT transporter (5-HTT) mRNA expression was decreased after single and repetitive ECS in rat raphe nucleus. In the present study, we investigated the effects of single and repetitive ECS on the protein levels of 5-HTT in the frontal cortex, hippocampus and raphe nucleus of rat brain using quantitative Western blot analysis. Single ECS did not alter 5-HTT protein expression in any brain regions examined. Repetitive ECS stably increased 5-HTT protein in the frontal cortex, but not in the hippocampus and raphe nucleus. Because ECS is known to facilitate the release of neurotransmitters, our results suggest that the increased 5-HTT protein expression in the frontal cortex might be a compensatory change against the enhanced 5-HT release by ECS in presynaptic terminals.     

Patterns of spontaneous activity and morphology of interneuron types in organotypic cortex and thalamus-cortex cultures.

The physiological and morphological properties of interneurons in infragranular layers of rat visual cortex have been studied in organotypic cortex monocultures and thalamus-cortex co-cultures using intracellular recordings and biocytin injections. Cultures were prepared at the day of birth and maintained for up to 20 weeks. Twenty-nine interneurons of different types were characterized, in addition to 170 pyramidal neurons. The cultures developed a considerable degree of synaptically driven "spontaneous" bioelectric activity without epileptiform activity. Interneurons in cortex monocultures and thalamus-cortex co-cultures had the same physiological and morphological properties, and also pyramidal cell properties were not different in the two culture conditions. All interneurons and the majority of pyramidal cells displayed synaptically driven action potentials. The physiological group of fast-spiking interneurons included large basket cells, columnar basket cells (two cells with an arcade axon) and horizontally bitufted cells. The physiological group of slow-spiking interneurons included Martinotti cells and a "long-axon" cell. Analyses of the temporal patterns of activity revealed that fast-spiking interneurons have higher rates of spontaneous activity than slow-spiking interneurons and pyramidal cells. Furthermore, fast-spiking interneurons fired spontaneous bursts of action potentials in the gamma frequency range. We conclude from these findings that physiological and morphological properties of interneurons in organotypic mono- and co-cultures match those of interneurons characterized in vivo or in acute slice preparations, and they maintain in long-term cultures a well-balanced state of excitation and inhibition. This suggests that cortex-intrinsic or cell-autonomous mechanisms are sufficient for the expression of cell type-specific electrophysiological properties in the absence of afferents or sensory input.     

Parvalbumin immunoreactivity: a reliable marker for the effects of monocular deprivation in the rat visual cortex.

In mammals, monocular deprivation performed during the early stages of postnatal development (critical period) dramatically affects the functional organization of the visual cortex. Since the early work of Hubel and Wiesel, the effects of monocular deprivation are accounted for by the fibers driven by the two eyes competing for the control of cortical territories. In cat and monkey striking structural changes accompany the functional effects of monocular deprivation. Also, in the rat, monocular deprivation causes functional alteration at the level of visual cortex; no structural correlates of these effects, however, have so far been described. Parvalbumin is a calcium binding protein that in the neocortex colocalizes with a subpopulation of GABAergic neurons. Here we report that in the rat monocular deprivation results in a dramatic reduction of parvalbumin-like immunoreactivity in the visual cortex contralateral to the deprived eye. This effect is due to competitive phenomena and not to visual deprivation itself, it is restricted to the binocular portion of the visual cortex and neither binocular deprivation, nor dark rearing can induce it. We conclude that parvalbumin-like immunoreactivity is a useful immunohistochemical marker for the effects of monocular deprivation in the rat visual cortex.     

Concomitant increase of somatostatin, neuropeptide Y and glutamate decarboxylase in the frontal cortex of rats with decreased seizure threshold

The neuropeptides somatostatin and neuropeptide Y and the activity of glutamate decarboxylase were determined in the frontal cortex of rats subjected to experimental epilepsy. Two different animal models, (1) rats kindled for 4 weeks by daily injection of pentylenetetrazole, and (2) rats which had undergone strong limbic seizures induced by kainic acid, were used. Decreased seizure threshold, as shown by injection of a subconvulsive dose of pentylenetetrazole, was observed 10 days after the last kindling session and 1 month after injection of kainic acid, respectively. Significantly increased levels of somatostatin (by 60%), neuropeptide Y (135%) and increased activity of glutamate decarboxylase (22%) were found in the frontal cortex of rats previously treated with kainic acid. Separation of somatostatin-like immunoreactivity by size exclusion high-performance liquid chromatography showed a marked increase of immunoreactivity in fractions containing the somatostatin precursor (by 200%) and less prominently of somatostatin-14 and somatostatin-28 (by 60 and 80%, respectively). Michaelis-Menten kinetics of glutamate decarboxylase revealed an increased maximal velocity (Vmax) in the frontal cortex of kainic acid-treated rats, but no change in the Km value was found. Similar results were also obtained in pentylenetetrazole-kindled rats. Injection of cysteamine (100 mg/kg, i.p.) resulting in a 30% decrease of cortical somatostatin in kainic acid-pretreated rats markedly suppressed seizures induced by an otherwise subconvulsive dose of pentylenetetrazole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pilocarpine-induced status epilepticus causes acute interneuron loss and hyper-excitatory propagation in rat insular cortex

Recent clinical studies have shown that the insular cortex (IC) is involved in temporal lobe epilepsy and suggested that the IC mediates spreading of epileptic activity from the temporal lobe, including the hippocampus and amygdala, to the frontal cortex. However, little is known about anatomical and physiological features of the IC in models of temporal lobe epilepsy. The present study evaluated the distribution pattern of GABAergic interneurons, especially parvalbumin (PV)- and somatostatin (SS)-immunopositive neurons, and excitatory propagation pattern in the IC of rats 4–7 days and 2 months after pilocarpine-induced status epilepticus (4–7 d and 2 m post-SE rats, respectively). The number of PV-immunopositive neuron profiles in the agranular IC (AI) significantly decreased by 24.6% and 41.5% in 7 d and 2 m post-SE rats, respectively. The dysgranular and granular IC (DI+GI) exhibited only 5.2% loss of PV-immunopositive neurons in 7 d post-SE rats, while 2 m post-SE rats showed 30.4% loss of PV-immunopositive neurons. There was no significant change of the SS-immunopositive neuron profile numbers in the AI and DI+GI of 7 d and 2 m post-SE rats. The regions with decreased numbers of PV-immunopositive neuron profiles overlapped with those where many degenerating cells were detected by Fluoro-Jade B staining. The area of excitatory propagation responding to electrical stimulation of the caudal AI was expanded in 4–7 d post-SE rats, and excitation frequently propagated to the frontal cortex including the motor cortex. Optical signals in the AI of 4–7 d post-SE rats were larger in amplitude than those of controls. In contrast to the AI, the DI of 4–7 d post-SE rats showed similar excitatory propagation pattern and amplitude to that of controls. These results suggest that the region-specific loss of PV-immunopositive neurons occurred in the AI 4–7 d after pilocarpine-induced status epilepticus, which may play an important role in facilitating excitatory propagation in the IC.     

Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition

Sponge immunocyte identification is of interest to comparative immunologists since characterizing these cells will allow investigations into the mechanisms of non-self recognition in the oldest animal phylum. Here, we report that polyclonal antibodies raised against the core protein of a proteoglycan involved in cell adhesion in the marine sponge Microciona prolifera are specific markers for archaeocytes, the totipotent sponge cells. Archaeocytes are mobilized upon allogeneic contact and they accumulate in the contact zone. A second type of cell, the gray cells, are specifically recognized by monoclonal antibodies raised against CD44, a hyaluronan receptor. Gray cells do also accumulate in the contact area. Specific staining of a third sponge cell type, the rhabdiferous cells, shows that these do not accumulate upon allografting. These specific cell markers allow tracking of archaeocytes and gray cells, and show that they play an active role in sponge allogeneic reactions.