Characterization of axo-axonic synapses in the piriform cortex of Mus musculus

Previous anatomical and physiological studies have established major glutamatergic and GABAergic neuronal subtypes within the piriform cortical circuits. However, quantitative information regarding axo-axonic inhibitory synapses mediated by chandelier cells across major cortical subdivisions of piriform cortex is lacking. Therefore, we examined the properties of these synapses across the entire piriform cortex. Our results show the following. 1) γ-Aminobutyric acid membrane transporter 1-positive varicosities, whose appearance resembles chandelier cartridges, are found around the initial segments of axons of glutamatergic cells across layers II and III. 2) Both the density of axo-axonic cartridges and the degree of γ-aminobutyric acid membrane transporter 1 innervation in each axo-axonic synapse are significantly higher in the piriform cortex than in the neocortex. 3) Glutamate decarboxylase 67, vesicular GABA transporter, and parvalbumin, but not calbindin, are colocalized with the presynaptic varicosities, whereas gephyrin, Na-K-2Cl cotransporter 1, and GABA(A) receptor α1 subunit, but not K-Cl cotransporter 2, are colocalized at the presumed postsynaptic sites. 4) The axo-axonic cartridges innervate the majority of excitatory neurons and are distributed more frequently in putative centrifugal cells and posterior piriform cortex. We further describe the morphology of chandelier cells by using parvalbumin-immunoreactivity and single-cell labeling. In summary, our results demonstrate that a small population of chandelier cells mediates abundant axo-axonic synapses across the entire piriform cortex. Because of the critical location of these inhibitory synapses in relation to action potential regulation, our results highlight a critical role of axo-axonic synapses in regulating information flow and olfactory-related oscillations within the piriform cortex in vivo.     

Inhibitory neurons in the anterior piriform cortex of the mouse: Classification using molecular markers

The primary olfactory cortex (or piriform cortex, PC) is attracting increasing attention as a model system for the study of cortical sensory processing, yet little is known about inhibitory neurons in the PC. Here we provide the first systematic classification of GABA‐releasing interneurons in the anterior PC of mice, based on the expression of molecular markers. Our experiments used GAD67‐GFP transgenic mice, in which gamma‐aminobutyric acid (GABA)‐containing cells are labeled with green fluorescent protein (GFP). We first confirmed, using paired whole‐cell recordings, that GFP⁺ neurons in the anterior PC of GAD67‐GFP mice are functionally GABAergic. Next, we performed immunolabeling of GFP⁺ cells to quantify their expression of every possible pairwise combination of seven molecular markers: calbindin, calretinin, parvalbumin, cholecystokinin, neuropeptide Y, somatostatin, and vasoactive intestinal peptide. We found that six main categories of interneurons could be clearly distinguished in the anterior PC, based on the size and laminar location of their somata, intensity of GFP fluorescence, patterns of axonal projections, and expression of one or more of the seven markers. A number of rarer categories of interneurons could also be identified. These data provide a road map for further work that examines the functional properties of the six main classes of interneurons. Together, this information elucidates the cellular architecture of the PC and provides clues about the roles of GABAergic interneurons in olfactory processing. J. Comp. Neurol. 518:1670–1687, 2010. © 2009 Wiley‐Liss, Inc.     

Diverse interneuron populations have highly specific interconnectivity in the rat piriform cortex

Previous studies have suggested that the patterns of innervation and high interconnectivity of the piriform cortex (PC) provide for strong olfactory hippocampal memory; however, these same attributes may create high seizurogenic tendencies. Thus, understanding this wiring is important from a physiological and pathophysiological perspective. Distinct interneurons expressing differing calcium binding proteins (CBPs), parvalbumin (PV), calbindin (CB), and calretinin (CR), have been shown to exist in PC. However, a comprehensive examination of the distribution and innervation patterns of these neurons has not been done. Thus the purpose of this study was to combine the analysis of the CBP cell localization with analysis of their innervation patterns. Each type was differentially localized in the three layers of the PC. Only CR‐positive neurons were found in layer 1. PV and CB are coexpressed in layers 2–3, most expressing both PV and CB. A morphological estimate of the dendritic extent for each subtype showed that PV and PV/CB cells demonstrated equally wide, horizontal and vertical arborizations, whereas CB cells had wide horizontal and restricted vertical arborizations. CR cells had restricted horizontal and very long vertical arborizations. Postsynaptic morphological targeting was also found to be specific, namely, PV⁺ and PV/CB⁺ nerve terminals (NTs) innervate perisomatic regions of principal cells. CR⁺ NTs innervate only dendrites of principal cells, and CB⁺ NTs innervate both somata and dendrites of principal cells. These data show highly complex innervation patterns for all of the CBP interneurons of the PC and form a basis for further studies in the plasticity of this region. J. Comp. Neurol. 518:1570–1588, 2010. © 2009 Wiley‐Liss, Inc.     

Localization of GABA-A receptor alpha subunits on neurochemically distinct cell types in the rat locus coeruleus

The locus coeruleus (LC) provides the major source of noradrenaline to the central nervous system and is modulated by neurochemically diverse afferents. LC function is central to arousal, memory, cognition and the stress response, with dysfunction of the LC-noradrenergic axis implicated in debilitating psychiatric disorders. The precise targeting of neurotransmitter receptors within the LC is essential for processing the information contained in diverse afferents and thus LC output. The inhibitory modulation of LC neurons is thought to be effected mainly through GABA-A receptors (GABA(A)Rs). Diverse GABA(A)Rs are pentameric complexes assembled from a repertoire of subunits resulting in substantial diversity in their molecular, functional and pharmacological properties throughout the brain. The precise location of distinct GABA(A) R subunits in subregions of the LC, and the neurochemical identity of the cells that express them, remains to be determined. Here, we show that the GABA(A)R alpha1 subunit is expressed exclusively in neurochemically and morphologically diverse non-noradrenergic cell types within the LC, which may innervate the principal noradrenergic cells. Thus, the GABA(A)R alpha1 subunit could provide a neurochemical signature for a pool of local circuit interneurons in the LC. In contrast, non-overlapping GABA(A)R alpha2 and alpha3 subunit-immunoreactive puncta were enriched on noradrenergic dendrites and, to a lesser extent, on somata. The study reveals a cell-type- and domain-specific expression pattern of distinct GABA(A)R subunits in the LC. These data will serve as a template for understanding inhibitory modulation of this region and facilitate more directed pharmacological strategies for disorders arising from the impairment of LC function.     

Paucity of glutaminase-immunoreactive nonpyramidal neurons in the rat cerebral cortex.

Glutaminase has been considered to be a synthesizing enzyme of transmitter glutamate in pyramidal neurons of the cerebral cortex. In the present study, an attempt was made to examine with a double immunofluorescence method whether or not nonpyramidal neurons of the cerebral cortex are immunoreactive for glutaminase. Glutaminase was stained with mouse anti-glutaminase IgM and FITC-labeled anti-[mouse IgM] antibody. In the same section, parvalbumin (PA), calbindin (CB), choline acetyltransferase (CAT), vasoactive intestinal polypeptide (VIP), corticotropin releasing factor (CRF), cholecystokinin (CCK), somatostatin (SS), or neuropeptide Y (NPY) was visualized as a marker for nonpyramidal neurons with an antibody to each substance, biotinylated secondary antibody and Texas Red-labeled avidin. Virtually no glutaminase immunoreactivity was seen in PA-, CB-, CAT-, VIP-, CRF-, CCK-, SS-, or NPY-immunoreactive neuronal perikarya in the neocortex and mesocortex (cingulate and retrosplenial cortices), although it was detected in a few PA-, CB-, VIP-, CCK-, SS-, or NPY-immunoreactive nonpyramidal neurons in the piriform, entorhinal, and hippocampal cortices. PA- and CB-positive neurons have been reported to constitute the major population of GABAergic neurons in the cerebral cortex. Thus, the present results, together with the previous reports, suggest that most GABAergic, cholinergic and peptidergic nonpyramidal neurons in the neo- and mesocortex do not contain glutaminase.     

Alterations of hippocampal GAbaergic system contribute to development of spontaneous recurrent seizures in the rat lithium-pilocarpine model of temporal lobe epilepsy.

Reorganization of excitatory and inhibitory circuits in the hippocampal formation following seizure-induced neuronal loss has been proposed to underlie the development of chronic seizures in temporal lobe epilepsy (TLE). Here, we investigated whether specific morphological alterations of the GABAergic system can be related to the onset of spontaneous recurrent seizures (SRS) in the rat lithium-pilocarpine model of TLE. Immunohistochemical staining for markers of interneurons and their projections, including parvalbumin (PV), calretinin (CR), calbindin (CB), glutamic acid decarboxylase (GAD), and type 1 GABA transporter (GAT1), was performed in brain sections of rats treated with lithium-pilocarpine and sacrificed after 24 h, during the silent phase (6 and 12 days), or after the onset of SRS (10-18 days after treatment). Semiquantitative analysis revealed a selective loss of interneurons in the stratum oriens of CA1, associated with a reduction of GAT1 staining in the stratum radiatum and stratum oriens. In contrast, interneurons in CA3 were largely preserved, although GAT1 staining was also reduced. These changes occurred within 6 days after treatment and were therefore insufficient to cause SRS. In the dentate gyrus, extensive cell loss occurred in the hilus. The pericellular innervation of granule cells by PV-positive axons was markedly reduced, although the loss of PV-interneurons was only partial. Most strikingly, the density of GABAergic axons, positive for both GAD and GAT1, was dramatically increased in the inner molecular layer. This change emerged during the silent period, but was most marked in animals with SRS. Finally, supernumerary CB-positive neurons were detected in the hilus, selectively in rats with SRS. These findings suggest that alterations of GABAergic circuits occur early after lithium-pilocarpine-induced status epilepticus and contribute to epileptogenesis. In particular, the reorganization of GABAergic axons in the dentate gyrus might contribute to synchronize hyperexcitability induced by the interneuron loss during the silent period, leading to the onset of chronic seizures.     

Differential gene expression in migrating cortical interneurons during mouse forebrain development

γ‐Aminobutyric acid (GABA)ergic interneurons play a vital role in modulating the activity of the cerebral cortex, and disruptions to their function have been linked to neurological disorders such as schizophrenia and epilepsy. These cells originate in the ganglionic eminences (GE) of the ventral telencephalon and undergo tangential migration to enter the cortex. Currently, little is known about the signaling mechanisms that regulate interneuron migration. We therefore performed a microarray analysis comparing the changes in gene expression between the GABAergic interneurons that are actively migrating into the cortex with those in the GE. We were able to isolate pure populations of GABAergic cells by fluorescence‐activated cell sorting of cortex and GE from embryonic brains of glutamate decarboxylase 67 (GAD67)‐green fluorescent protein (GFP) transgenic mice. Our microarray analysis identified a number of novel genes that were upregulated in migrating cortical interneurons at both E13.5 and E15.5. Many of these genes have previously been shown to play a role in cell migration of both neuronal and non‐neuronal cell types. In addition, several of the genes identified are involved in the regulation of migratory processes, such as neurite outgrowth, cell adhesion, and remodeling of the actin cytoskeleton and microtubule network. Moreover, quantitative polymerase chain reaction and in situ hybridization analyses confirmed that the expression of some of these genes is restricted to cortical interneurons. These data therefore provide a framework for future studies aimed at elucidating the complexities of interneuron migration and, in turn, may reveal important genes that are related to the development of specific neurological disorders. J. Comp. Neurol. 518:1232–1248, 2010. © 2009 Wiley‐Liss, Inc.     

The development of hippocampal interneurons in rodents

Interneurons are GABAergic neurons responsible for inhibitory activity in the adult hippocampus, thereby controlling the activity of principal excitatory cells through the activation of postsynaptic GABAA receptors. Subgroups of GABAergic neurons innervate specific parts of excitatory neurons. This specificity indicates that particular interneuron subgroups are able to recognize molecules segregated on the membrane of the pyramidal neuron. Once these specific connections are established, a quantitative regulation of their strength must be performed to achieve the proper balance of excitation and inhibition. We will review when and where interneurons are generated. We will then detail their migration toward and within the hippocampus, and the maturation of their morphological and neurochemical characteristics. We will finally review potential mechanisms underlying the development of GABAergic interneurons.     

Co-expression of calretinin and gamma-aminobutyric acid in neurons of the entorhinal cortex of the common marmoset monkey

The gamma-aminobutyric acid (GABA)-containing interneuron population in the entorhinal cortex has been shown to consist of several subpopulations. In addition to GABA, these neurons contain another neurochemical substance, such as a neuropeptide or a calcium binding protein. In the present study, we examined the co-localization of calretinin and GABA in the entorhinal cortex of the common marmoset Callithrix jacchus, a New World monkey. Although the function of calretinin remains unclear, there are indications that it might have a protective role against cell death in a number of neuropathological diseases. Furthermore, it might have a regulatory role in the neurotransmission of GABAergic neurons. In contrast to the rat brain, sparse data exist regarding the degree of co-expression of these two markers in the monkey brain. Using immunofluorescence and confocal laser scanning microscopy, we found that an average of 56% of the calretinin-positive neurons in the monkey entorhinal cortex contained GABA, whereas about 27% of the GABA-positive neurons co-expressed calretinin. Interestingly, these numbers were higher in the superficial layers of the entorhinal cortex in comparison with the deep layers. However, no differences were found in co-localization percentages between the different entorhinal subfields. In general, the degree of co-localization was higher in comparison to findings in the rat entorhinal cortex. The higher amount of co-localization observed in the present study might reflect species differences between the primate and the non-primate brain.     

Neurons of the ascidian larval nervous system in Ciona intestinalis: I. Central nervous system

The tadpole larva of ascidians, basal living relatives of vertebrates, has a chordate body plan. The CNS has many homologies with that of vertebrates yet only about 100 neurons. These few, possibly fixed in number and composition, nevertheless govern a diverse repertoire of behaviors. To elucidate the circuits of the CNS first requires that we recognize each neuron type, for which we used electroporation to transfect precleavage embryos with a plasmid containing green fluorescent protein (GFP) driven by the promoter of the synaptotagmin gene. Hatched larvae were fixed and GFP 3-D reconstructions of confocal image stacks compiled into images of 31 whole or partial larvae, either with many GFP-labelled neurons or with few, each clearly visible. Neuron counts in the sensory vesicle (SV) and visceral ganglion (VG) indicated that between 75% (SV) and 69% (VG) of previously reported numbers of neurons were transfected. Based on their position, shape, and projections, the following neurons were identified in the SV: a prominent eminens neuron, possibly with direct input from papillar neurons, a large ventroposterior interneuron, photoreceptors of the ocellus, and putative antenna cells of the otolith. In the VG, we identified at least four subtypes of motor neuron, including an ovoid cell that may innervate distal tail muscle cells and contrapelo cells with ascending projections, unique among VG neurons. The caudal nerve cord contained the first reported neurons, the somata of planate neurons. These neurons are the first identified types, and will be used to construct a map of the nervous system for this model basal chordate.     

Calretinin immunoreactivity in the brain of the zebrafish, Danio rerio: distribution and comparison with some neuropeptides and neurotransmitter-synthesizing enzymes. II. Midbrain, hindbrain, and rostral spinal cord

The distribution of calretinin (CR) in the brainstem and rostral spinal cord of the adult zebrafish was studied by using immunocytochemical techniques. For analysis of some brainstem nuclei and regions, CR distribution was compared with that of complementary markers (choline acetyltransferase, glutamic acid decarboxylase, tyrosine hydroxylase, neuropeptide Y). The results reveal that CR is a marker of various neuronal populations distributed throughout the brainstem, including numerous cells in the optic tectum, torus semicircularis, secondary gustatory nucleus, reticular formation, somatomotor column, gustatory lobes, octavolateral area, and inferior olive, as well as of characteristic tracts of fibers and neuropil. These results indicate that CR may prove useful for characterizing a number of neuronal subpopulations in zebrafish. Comparison of the distribution of CR observed in the brainstem of zebrafish with that reported in an advanced teleost (the gray mullet) revealed a number of similarities, and also some interesting differences. Our results indicate that many brainstem neuronal populations have maintained the CR phenotype in widely divergent teleost lines, so CR studies may prove very useful for comparative analysis.     

Immunocytochemically defined interneuron populations in the hippocampus of mouse strains used in transgenic technology

Transgenic mice are overtaking the role of model animals in neuroscience. They are used in developmental, anatomical, and physiological as well as experimental neurology. However, most results on the organization of the nervous system derive from the rat. The rat hippocampus and its neuronal elements have been thoroughly investigated, revealing remarkable functional and morphological diversity and specificity among hippocampal interneurons. Our aim was to examine the properties of distinct hippocampal interneuron populations, i.e., those immunoreactive for calcium-binding proteins (parvalbumin, calbindin, and calretinin), neuropeptides (cholecystokinin, neuropeptide Y, somatostatin, vasoactive intestinal polypeptide), and certain receptors (metabotropic glutamate receptor 1alpha, cannabinoid receptor type 1) in four strains of mice widely used in transgenic technology, and to compare their properties to those in the rat. Our data indicate that the distribution as well as the dendritic and axonal arborization of mouse interneurons immunoreactive for the different markers was identical in the examined mouse strains, and in most respects are similar to the features found in the rat. The postsynaptic targets of neurons terminating in the perisomatic (parvalbumin), proximal (calbindin), and distal (somatostatin) dendritic region, as well as on other interneurons (calretinin), also matched those found in the rat. However, a few significant differences could also be observed between the two species in addition to the already described immunoreactivity of mossy cells for calretinin: the absence of spiny calretinin-immunoreactive interneurons in the CA3 region, sparse contacts between calretinin-immunoreactive interneurons, and the axon staining for somatostatin and neuropil labeling for cholecystokinin. We can conclude that the morphofunctional classification of interneurons established in the rat is largely valid for mouse strains used in transgenic procedures.     

Regulation of inhibitory neurotransmission by the scaffolding protein ankyrin repeat-rich membrane spanning/kinase D-interacting substrate of 220 kDa

Scaffolding proteins play a critical role in the proper development and function of neural circuits. In contrast to the case for excitatory circuits, in which the role of several scaffolding proteins has been characterized, less is known about the scaffolding proteins that regulate inhibitory neurotransmission. The ankyrin repeat-rich membrane spanning (ARMS)/kinase D-interacting substrate of 220 kDa (Kidins220) scaffolding protein is expressed during the establishment of γ-aminobutyric acid (GABA) neurotransmission and is highly regulated by activity. To evaluate whether ARMS/Kidins220 expression affects GABAergic neurotransmission, we modified the ARMS/Kidins220 levels during the period of its maximum expression in culture (DIV 1-10). Whereas a decrease in ARMS/Kidins220 levels suppressed GABAergic neurotransmission, overexpression of ARMS/Kidins220 produced an increase in GABAergic neurotransmission in hippocampal neurons. In addition, we found that ARMS/Kidins220 regulates GABAergic neurotransmission by a presynaptic mechanism. Our results suggest that the ARMS/Kidins220 scaffold protein plays a critical role in the regulation of inhibitory transmission in hippocampal neurons.     

Colocalization of GABA and glycine in the ventral nucleus of the lateral lemniscus in rat: an in situ hybridization and semiquantitative immunocytochemical study

We have studied by in situ hybridization for GAD65 mRNA in thick sections and by semiquantitative postembedding immunocytochemistry in consecutive semithin sections, the expression of gamma-aminobutyric acid (GABA) and glycine in cell bodies and axosomatic puncta of the rat ventral nucleus of the lateral lemniscus (VNLL), a prominent monaural brainstem auditory structure. The in situ hybridization and the densitometric analysis of the immunostaining suggest that the rat VNLL contains two main populations of neurons. Approximately one-third of neurons are unstained with either technique and are presumably excitatory; their cell bodies are enveloped by a large number of glycine-immunoreactive puncta. Most if not all of the remaining two-thirds colocalize GABA and glycine and are assumed to be inhibitory. These two populations show a complementary distribution within the VNLL, with inhibitory neurons located mainly ventrally and excitatory neurons dorsally. In scatterplots of gray values measured from cell bodies, the double-labeled cells appear to form a single cluster in terms of their staining intensities for the two transmitter candidates. However, this cluster may have to be further subdivided because cells with extreme GABA/glycine ratios differ from those with average ratios with respect to location or size. The VNLL seems unique among auditory structures by its large number of neurons that colocalize GABA and glycine. Although the functional significance of this colocalization remains unknown, our results suggest that the VNLL exerts convergent excitatory and inhibitory influences over the inferior colliculus, which may underlie the timing processing in the auditory midbrain.     

Gene expression analysis in the parvalbumin-immunoreactive PV1 nucleus of the mouse lateral hypothalamus

A solitary, elongated cluster of parvalbumin-immunoreactive neurons has been previously observed in the rodent ventrolateral hypothalamus. However, the function of this so-called PV1 nucleus is unknown. In this article, we report the results of an unbiased, broad and in-depth molecular characterization of this small, compact group of neurons. The Allen Brain Atlas database of in situ hybridization was screened in order to identify genes expressed in the PV1-nucleus-containing area of the hypothalamus, and those that might be co-expressed with parvalbumin. Although GABA is the principal neurotransmitter in parvalbumin-expressing cells in various other brain areas, we found that PV1 neurons express the vesicular glutamate transporter (VGlut) VGlut2-encoding gene Slc17a6 and are negative for the glutamic acid decarboxylase 1 (GAD1) gene. These cells also express the mRNA for the neuropeptides Adcyap1 and possibly Nxph4, express several types of potassium and sodium channels, are under the control of the neurotransmitter acetylcholine, bear receptors for the glial-derived neurotrophic factor, and produce an extracellular matrix rich in osteopontin. The PV1 nucleus is thus composed of glutamatergic nerve cells, expressing some typical markers of long-axon, projecting neurons (e.g. VGlut2), but also co-expressing genes typical of short-axon GABA neurons (e.g. a variety of potassium channels). Hence, neurons of the PV1 nucleus combine physiological characteristics of interneurons with those of projection neurons.     

绿色荧光蛋白报道基因在鼠C6胶质瘤细胞中的应用研究

目的 :探讨绿色荧光蛋白 (GFP)报道基因转染C6鼠胶质瘤细胞的体内外表达及对细胞生物学性状的影响。方法 :荧光相差显微镜筛选稳定表达GFP的克隆 ,流式细胞术分析细胞周期。将已转染和未转染瘤细胞植入SD大鼠脑内 ,建立动物模型 ,定期随机处死大鼠 ,鼠脑标本行病理学、增殖与凋亡的检测 ,激光共聚焦显微镜检查肿瘤细胞内荧光强度及其分布。结果 :GFP在C6瘤细胞的体外和体内均获得长期稳定表达 ,检测肿瘤细胞的敏感性及特异性优于HE染色。结论 :GFP对肿瘤细胞生物学性状无影响是比较理想的报道基因     

Continuous and intermittent transcranial magnetic theta burst stimulation modify tactile learning performance and cortical protein expression in the rat differently

Repetitive transcranial magnetic stimulation (rTMS) can modulate cortical excitability in a stimulus-frequency-dependent manner. Two kinds of theta burst stimulation (TBS) [intermittent TBS (iTBS) and continuous TBS (cTBS)] modulate human cortical excitability differently, with iTBS increasing it and cTBS decreasing it. In rats, we recently showed that this is accompanied by changes in the cortical expression of proteins related to the activity of inhibitory neurons. Expression levels of the calcium-binding protein parvalbumin (PV) and of the 67-kDa isoform of glutamic acid decarboxylase (GAD67) were strongly reduced following iTBS, but not cTBS, whereas both increased expression of the 65-kDa isoform of glutamic acid decarboxylase. In the present study, to investigate possible functional consequences, we applied iTBS and cTBS to rats learning a tactile discrimination task. Conscious rats received either verum or sham rTMS prior to the task. Finally, to investigate how rTMS and learning effects interact, protein expression was determined for cortical areas directly involved in the task and for those either not, or indirectly, involved. We found that iTBS, but not cTBS, improved learning and strongly reduced cortical PV and GAD67 expression. However, the combination of learning and iTBS prevented this effect in those cortical areas involved in the task, but not in unrelated areas. We conclude that the improved learning found following iTBS is a result of the interaction of two effects, possibly in a homeostatic manner: a general weakening of inhibition mediated by the fast-spiking interneurons, and re-established activity in those neurons specifically involved in the learning task, leading to enhanced contrast between learning-induced and background activity.