GABAergic neurons containing somatostatin-like immunoreactivity in the rat hippocampus and dentate gyrus

Summary The distribution of somatostatin-like immunoreactive (SS-LI) material and its colocalization with glutamic acid decarboxylase (GAD)-like immunoreactivity were studied in the rat hippocampus and dentate gyrus neurons using immunohistochemistry. In the dentate gyrus and CA1 region, SS-LI perikarya were concentrated in the hilus and in the stratum oriens, respectively, whereas immunoreactive cell bodies were rarely seen in other layers. Approximately half of the SS-LI neurons of the CA3 region were situated in the stratum oriens, the other half being scattered in strata pyramidale, lucidum and radiatum. About 90% of SS-LI neurons were also GAD-like immunoreactive, whereas about 14% of GAD-like immunoreactive (GAD-LI) neurons were SS-like immunoreactive. The percentage of GAD-LI neurons which were also immunoreactive for SS varied from one layer to the other. This percentage was about 30% in the hilus of the dentate gyrus and in the stratum oriens of the CA1 and CA3 regions; it was 5–10% in the strata pyramidale, lucidum and radiatum of the CA3 region and reached only 2% in the granule cell layer and molecular layer of the dentate gyrus and in the stratum pyramidale and stratum radiatum in the CA1 region. These observations indicate that the majority of SS-LI neurons in the rat hippocampal formation are a subpopulation of GABAergic neurons.     

Late appearance of parvalbumin-immunoreactivity in the development of GABAergic neurons in the rat hippocampus

The calcium-binding protein parvalbumin (PARV) is supposed to have a protective function under conditions of experimental seizure and hypoxia in a subgroup of GABAergic inhibitory neurons in the adult rat hippocampus. Here we studied the appearance of PARV immunoreactivity in rat hippocampal non-pyramidal cells during postnatal development in comparison to glutamate decarboxylase (GAD) immunoreactivity. PARV-immunoreactive neurons were not observed before postnatal day 7 whereas GAD-positive neurons and terminal-like puncta were present at postnatal day 2 (P2) and were frequent around P5. From other studies it is known that all GABAergic neurons are formed prenatally. Our data thus indicate that in the early postnatal period GABAergic non-pyramidal cells are poorly protected by calcium-binding proteins against a pathological calcium influx.     

APPswe转基因小鼠海马结构中谷氨酸和γ-氨基丁酸能神经元的数量变化

目的:探讨谷氨酸和γ-氨基丁酸在阿尔茨海默病(AD)发生、发展中的作用.方法:利用硫磺素S染色技术检测APPswe转基因小鼠脑内沉积的老年斑;免疫荧光技术标记正常及APPswe转基因小鼠海马发育过程中谷氨酸、γ-氨基丁酸(GABA)阳性细胞. 结果: 模型组小鼠海马CA1、CA3区以及齿状回谷氨酸阳性细胞的数量明显少于正常对照组,而海马各区的GABA阳性细胞在两者之间变化不明显.结论: 谷氨酸与GABA能神经元数量的改变与Aβ诱导的神经元凋亡以及AD的病理发生有关.     

Innervation of substantia nigra neurons by cholinergic afferents from pedunculopontine nucleus in the rat: neuroanatomical and electrophysiological evidence

Dopaminergic neurons of the substantia nigra pars compacta are excited by nicotine and acetylcholine, and possess both high-affinity nicotine binding sites and intense acetylcholinesterase activity, consistent with a cholinoceptive role. A probable source of cholinergic afferents is the pedunculopontine nucleus, which forms part of a prominent group of cholinergic perikarya located caudal to the substantia nigra in the tegmentum. Although pedunculopontine efferents, many of them cholinergic, project to the substantia nigra pars compacta, it has not been established whether they terminate in this structure. In the first experiment, which combined retrograde tracing with immunohistochemical visualization of cholinergic neurons, cholinergic cells in and around the pedunculopontine nucleus were found to send projections to the substantia nigra. This projection was almost completely ipsilateral. Subsequent experiments employed anaesthetized rats; kainate was microinfused into tegmental sites in order to stimulate local cholinergic perikarya, and concurrently, extracellular recordings were made of single dopaminergic neurons in the substantia nigra. Consistent with our anatomical findings, unilateral microinfusion of kainic acid in or near the pedunculopontine nucleus increased the firing rate of dopaminergic neurons situated remotely in the ipsilateral substantia nigra. The kainate-induced excitation of nigral dopaminergic neurons was dose-related and was prevented by intravenous administration of the centrally-acting nicotinic cholinergic antagonist mecamylamine. These results suggest that cholinergic perikarya in the vicinity of the pedunculopontine tegmental nucleus innervate dopaminergic neurons in the substantia nigra pars compacta via nicotinic receptors.     

Septal GABAergic neurons innervate inhibitory interneurons in the hippocampus of the macaque monkey.

The septohippocampal projection was visualized in three Macaca mulatta monkeys by anterograde transport of Phaseolus vulgaris leucoagglutinin. Following injections of the lectin into the medial septal nucleus, P. vulgaris leucoagglutinin-labelled fibres were found in the hippocampal complex, mainly in stratum oriens of the CA1 subfield, throughout the CA3 subfield, and in the hilus and stratum moleculare of the dentate gyrus. The majority of labelled axons were varicose, and formed multiple contacts with cell bodies and dendrites of calbindin D28k- and parvalbumin-immunoreactive non-pyramidal cells. GABA immunoreactivity of P. vulgaris leucoagglutinin-labelled axons and their postsynaptic targets was investigated by sectioning varicose axon segments for correlated light and electron microscopy, and processing alternate ultrathin sections for postembedding immunogold staining for GABA. All P. vulgaris leucoagglutinin-labelled boutons examined were GABA-immunoreactive and the majority of them formed symmetrical synapses with GABA-immunoreactive cell bodies and dendrites. The results demonstrate that a GABAergic septohippocampal pathway exists in the monkey, and, similar to the rat, terminates on different types of GABAergic neurons, including the parvalbumin- and calbindin D28k-containing non-pyramidal cells.     

Commissural afferents innervate glutamate decarboxylase immunoreactive non-pyramidal neurons in the guinea pig hippocampus

The innervation of GABAergic hippocampal neurons by commissural fibers was investigated in the guinea pig by a combined anterograde degeneration - immunocytochemical technique. Presumed GABAergic neurons were identified by immunocytochemistry for glutamate decarboxylase (GAD) and the commissural fibers by electron-dense degeneration following contralateral transection of the fimbria. Commissural afferents were found to establish asymmetric synaptic contacts with non-pyramidal GAD-immunoreactive neurons located in subpyramidal and suprapyramidal zones of region CA1. The observed connection suggests that inhibition of pyramidal cells may occur in a feed-forward manner as postulated by electrophysiological studies.     

Quantitative ultrastructural differences between local and medial septal GABAergic axon terminals in the rat hippocampus

Functionally distinct subsets of hippocampal inhibitory neurons exhibit large differences in the frequency, pattern and short-term plasticity of GABA release from their terminals. Heterogeneity is also evident in the ultrastructural features of GABAergic axon terminals examined in the electron microscope, but it is not known if or how this corresponds to interneuron subtypes. We investigated the feasibility of separating morphologically distinct clusters of terminal types, using the approach of measuring several ultrastructural parameters of GABAergic terminals in the CA1 area of the rat hippocampus. Septo-hippocampal axon terminals were anterogradely labeled by biotinylated dextran amine and visualized by pre-embedding immunogold staining to delineate one homogeneous terminal population. Long series (100-150) of ultrathin sections were cut from stratum oriens and stratum radiatum of the CA1 area, and GABAergic terminals were identified by post-embedding immunogold staining. Stereologically unbiased samples of the total GABAergic axon terminal population and a random sample of the septal axon terminals were reconstructed in 3D, and several of their parameters were measured (e.g. bouton volume, synapse surface, volume occupied by vesicles, mitochondria volume). Septal terminals demonstrated significantly larger mean values for most parameters than the total population of local GABAergic terminals. There was no significant difference between terminals reconstructed in the basal and apical dendritic regions of pyramidal cells, neither for the septal nor for the local population. Importantly, almost all parameters were highly correlated, precluding the possibility of clustering the local terminals into non-overlapping subsets. Factor and cluster analysis confirmed these findings. Our results suggest that similarly to excitatory terminals, inhibitory terminals follow an "ultrastructural size principle," and that the terminals of different interneuron subtypes cannot be distinguished by ultrastructure alone.     

Immunocytochemical study of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus

Summary The structural features of PV-immunoreactive (PV-I) neurons, a particular subpopulation of GABAergic neurons, in the hippocampus were studied by immunocytochemistry. The PV-I cell bodies were concentrated within the stratum pyramidale (SP) and stratum oriens (SO) in the hippocampus. PV-I puncta were frequent in SP, while they were rarely seen in other layers. The dendritic arborization of PV-I neurons resembled that of some of the nonpyramidal cells observed after Golgi-impregnation. The most commonly observed PV-I neurons had their perikarya located in SP with dendrites extending into SO and the stratum radiatum (SR). Most of the dendrites in SR had typical beaded or varicose segments. The dendrites extending into SO had few beaded parts. There were many bipolar and multipolar neurons with smooth dendrites in SO, but only a small number of such multipolar neurons in SR. An electron microscopic analysis revealed that PV-I products were located to perikarya, dendrites, myelinated axons and synaptic boutons. The perikarya of PV-I neurons exhibited several ultrastructural features of nonpyramidal cells, e.g., abundant cisternae of endoplasmic reticulum, mitochondria and other perikaryal organelles, an infolded nuclear envelope and intranuclear inclusions. They received many asymmetric synapses with round presynaptic vesicles. There were numerous PV-I boutons, presumably axonal endings, covering the pyramidal cell bodies. The PV-I boutons also contacted the axon initial segments and proximal dendrites of the pyramidal cells. In addition PV-I terminals were found on somata and dendrites of both PV-I or PV-negative nonpyramidal cells. The results suggest that PV-containing neurons include basket and axo-axonic 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.     

GABAergic basal forebrain afferents innervate selectively GABAergic targets in the main olfactory bulb

In this work we have analyzed the targets of the GABAergic afferents to the main olfactory bulb originating in the basal forebrain of the rat. We combined anterograde tracing of 10 kD biotinylated dextran amine (BDA) injected in the region of the horizontal limb of the diagonal band of Broca that projects to the main olfactory bulb, with immunocytochemical detection of GABA under electron microscopy or vesicular GABA transporter (vGABAt) under confocal fluorescent microscopy. GABAergic afferents were identified as double labeled BDA-GABA boutons. Their targets were identified by their ultrastructure and GABA content. We found that GABAergic afferents from the basal forebrain were distributed all over the bulbar lamination, but were more abundant in the glomerular and inframitral layers (i.e. internal plexiform layer and granule cell layer). The fibers had thick varicosities with abundant mitochondria and large perforated synaptic specializations. They contacted exclusively GABAergic cells, corresponding to type 1 periglomerular cells in the glomerular layer, and to granule cells in inframitral layers. This innervation will synchronize the bulbar inhibition and consequently the response of the principal cells to the olfactory input. The effect of the activation of this pathway will produce a disinhibition of the bulbar principal cells. This facilitation might occur at two separate levels: first in the terminal tufts of mitral and tufted cells via inhibition of type 1 periglomerular cells; second at the level of the firing of the principal cells via inhibition of granule cells. The GABAergic projection from the basal forebrain ends selectively on interneurons, specifically on type 1 periglomerular cells and granule cells, and is likely to control the activity of the olfactory bulb via disinhibition of principal cells. Possible similarities of this pathway with the septo-hippocampal loop are discussed.     

芍药甙对体外培养的大鼠海马和隔区神经元的影响

目的研究芍药甙对海马和隔区神经元的影响。方法用新生SD大鼠的海马和隔区神经元进行体外培养,加入不同浓度的芍药甙,用四唑盐比色法(MTT)测量法测量培养神经元的光密度(OD值)。结果芍药甙的浓度高于30μg/ml时,各组的海马和隔区神经元MTT OD值均低于对照组的OD值(P<0.01);芍药甙的浓度在1.875～7.5μg/ml时各组的OD值均高于对照组(P<0.01或P<0.05)。结论在适当的浓度范围内的芍药甙对海马和隔区神经元的存活和生长具有促进作用。     

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.     

GABAergic neurons of different morphological classes are cogenerated in the mouse barrel cortex

Summary In the barrel cortex of the mouse, GAD-immunoreactive cells are positioned following an inside-out gradient. Each cortical layer is populated by a variety of classes of GABAergic neurons. The period of neuronal generation for each cortical layer lasts for several days, but it is not known whether the different classes of GABAergic neurons are generated synchronously. In order to correlate the time of generation with cell morphology, we combined [³H]thymidine autoradiography with glutamic acid decarboxylase immunocytochemistry, and used a recently developed method to morphologically classify GAD-positive neurons. We found that all classes of GAD-positive neurons destined to populate each cortical layer are generated concurrently. Therefore, the forms shown by the GAD-positive neurons in the adult animal are not related to the times of their neurogenesis.     

Glutamate-receptor-induced modulation of GABAergic synaptic transmission in the hippocampus

There is a large body of evidence about the short- and long-term changes in GABAergic transmission in the hippocampus produced by the action of different endogenous neuromodulators and in particular neurotransmitters. Both intrinsic hippocampal cells and afferent fibres coming into the hippocampus from various parts of the CNS release substances that are capable of changing inhibitory transmission. This review surveys current understanding of the action of glutamate on the inhibitory transmission mediated in the hippocampus via GABA(A) receptors. Here we pay special attention to the molecular and cellular mechanisms leading to spatio-temporal changes of the glutamate concentration in the extracellular space and to the localization and identity of glutamate receptors involved in this direct modulation of inhibition.     

Activation of bulbar reticular neurons by visceral afferents

Conclusion Synaptic activation of reticular neurons of the bulbar gigantocellular nucleus has been investigated with stimulation of the left greater splanchnic nerve, Synaptic activation of these neurons has been shown to be produced by ascending pathways formed by somatic and visceral afferents, running in the ventro-lateral part of the lateral, and the ventral, columns of the spinal cord. Synaptic processes arising in reticular neurons are uniform and differ only temporally. The pattern of development of synaptic processes in reticular neurons suggests that transmission of visceral impulses into the bulbar reticular formation takes place along two pathways. One of these, formed by the spinoreticular fibers of the lateral columns, activates neurons after a short latency (8–10 msec). Besides this there are also polysynaptic, nonspecific fibers in the ventrolateral and ventral columns, organized on the principle of long sensory tracts and including an intermediate link in the segmental interneurons. Synaptic processes in reticular neurons evoked by the polysynaptic pathways develop after 10–20 msec and originate from excitation of somatic and visceral afferents possessing lower excitability and a lower conduction velocity.Division of afferent fibers of the splanchnic nerve running in the dorsal columns and dorsolateral part of the lateral columns of the spinal cord has no significant influence on synaptic processes in reticular neurons.     

Age-related alterations of GABAergic input to CA1 pyramidal neurons and its control by nicotinic acetylcholine receptors in rat hippocampus

The aim of this study was to determine whether age-associated alterations in the GABAergic input to pyramidal neurons in the hippocampus are due to a dysfunction of GABAergic interneurons, and/or a decrease in their cholinergic control via nicotinic receptors (nAChRs). Electrophysiological recordings were obtained from pyramidal cells in the CA1 area of hippocampal slices from young (3-4 months old) and aged (25-30 months old) Sprague-Dawley rats. Synaptic GABA(A) receptor-mediated inhibitory postsynaptic currents and inhibitory postsynaptic potentials induced by stimulation of the stratum oriens were significantly smaller in aged rats. The frequency (but not amplitude) of spontaneous and miniature GABA inhibitory postsynaptic currents (IPSCs) was reduced in aged rats, suggesting a presynaptic alteration. Tetanic stimulation of cholinergic afferents to release endogenous acetylcholine, or an exogenous application of the nAChR agonist cytisine, increased the frequency of spontaneous IPSCs in young rats; however these effects were not evident in aged rats, indicating that the nicotinic control of GABA release is lowered during aging. None of these age-related alterations were reversed by a chronic treatment with donepezil, a cholinesterase inhibitor. Immunofluorescent labeling of GABA interneurons with somatostatin (SOM), parvalbumin (PV) or calbindin (CB), together with the vesicular acetylcholine transporter VAChT, revealed a selective loss of subpopulations of SOM and CB positive interneurons. This loss was associated with a general decrease in density of the cholinergic network in aged rats. Thus, the lower GABAergic inhibition observed in the aged rat hippocampus is due to a selective loss/dysfunction of subpopulations of GABAergic interneurons, associated with a widespread cholinergic deficit.     

Cellular architecture of the mouse hippocampus: a quantitative aspect of chemically defined GABAergic neurons with stereology

The structural organization of the central nervous system is an infrastructure of the modern neuroscience. Especially, anatomical research at the cellular level can serve as a fundamental resource to understand various novel findings from the molecular level to the system level. Of the decade, the importance of rigorous quantitative neuroanatomy is gradually realized, but earlier anatomical reports are usually presented in qualitative terms or quantified with biased methods. This article quantitatively describes the spatial distributions of chemically defined GABAergic neurons in the mouse hippocampus by using unbiased stereological techniques. We focus on the expression of nine major neurochemical markers: glutamic acid decarboxylase 67, three calcium binding proteins (parvalbumin, calretinin, calbindin D28K), four neuropeptides (somatostatin, neuropeptide Y, cholecystokinin, vasoactive intestinal protein) and neuronal nitric oxide synthase. Here we deal with their laminar distributions, numerical densities and the relative ratio to the total GABAergic neurons, with special reference to their differentiation along the dorsoventral axis of the mouse hippocampus. Furthermore, we estimate the absolute numbers of GABAergic neurons contained in a 300-mum-thick hypothetical slice. The present data outline the quantitative aspects of the cellular architecture of hippocampal GABAergic system and also give complementary information to the recent multidisciplinary analyses at the single cell level.     

Gap junctions on GABAergic neurons containing the calcium-binding protein parvalbumin in the rat hippocampus (CA1 region)

Summary Gap junctions were identified for the first time on chemically defined neurons in the central nervous system. Gap junctions were thus demonstrated on GABAergic neurons containing the calcium-binding protein parvalbumin (PV) in the rat hippocampus. Thin and semithin (0.5 m thick) sections were cut alternately and consecutively from osmium-fixed tissue which was embedded in epoxy resin and usable for conventional electron microscopic studies. The semithin sections were processed for postembedding immunocytochemistry using an anti-PV serum. Structures corresponding to the PV-immunoreactive (PV-I) profiles on the semithin sections were easily identified on electron micrographs from the adjacent thin sections. Using this technique gap junctions were found (1) between PV-I dendrites, (2) between PV-I dendrites and PV-I somata and (3) between PV-I dendrites and small processes whose origin could not be identified. Despite a systematic search, we did not find gap junction between PV-negative processes.     

Neuroendocrine bag cells of Aplysia are activated by bag cell peptide-containing neurons in the pleural ganglion

1. The generation of egg-laying behavior in the marine mollusk Aplysia involves a prolonged burst discharge in the neuroendocrine bag cells, which secrete neuropeptides derived from the egg-laying hormone/bag cell peptide (ELH/BCP) precursor protein. 2. Besides the bag cells, which are located in the abdominal ganglion, small clusters of neurons in the cerebral and pleural ganglia also express the ELH/BCP neuropeptides. We made intracellular recordings from 32 of these ELH/BCP cells in right pleural ganglia, in 18 preparations, to characterize their physiological properties and their functional relationship to the bag cells. 3. The identification of these ELH/BCP cells was confirmed by pressure injection of Lucifer yellow and subsequent immunocytochemical processing for alpha-BCP immunoreactivity. 4. The basic electrophysiological properties of the pleural ELH/BCP cells were similar to those of the bag cells. These pleural cells were directly demonstrated to be electrically coupled, and direct intracellular stimulation of individual pleural ELH/BCP cells initiated prolonged, synchronous burst discharges in the entire cluster through a positive feedback mechanism. 5. Burst discharges elicited in the pleural ELH/BCP cells consistently initiated burst discharges in the bag cells. Bag cell burst discharges were less effective in initiating burst discharges in the pleural ELH/BCP cells, indicating that there were reciprocal but asymmetrical connections. 6. The results show that the pleural ELH/BCP cells are functionally coupled to the bag cells. They support the hypothesis that the pleural ELH/BCP cells are part of the descending pathway that initiates bag cell activity and egg-laying behavior, in vivo.