Subunit composition of α5-containing nicotinic receptors in the rodent habenula

Gene association studies in humans have linked the α5 subunit gene CHRNA5 to an increased risk for nicotine dependence. In the CNS, nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit are expressed at relatively high levels in the habenulo-interpeduncular system. Recent experimental evidence furthermore suggests that α5-containing receptors in the habenula play a key role in controlling the intake of nicotine in rodents. We have now analysed the subunit composition of hetero-oligomeric nAChRs in the habenula of postnatal day 18 (P18) C57Bl/6J control mice and of mice with deletions of the α5, the β2, or the β4 subunit genes. Receptors consisting of α3β4* clearly outnumbered α4β2*-containing receptors not only in P18 but also in adult mice. We found low levels of α5-containing receptors in both mice (6%) and rats (2.5% of overall nAChRs). Observations in β2 and β4 null mice indicate that although α5 requires the presence of the β4 subunit for assembling (but not of β2), α5 in wild-type mice assembles into receptors that also contain the subunits α3, β2, and β4.     

Transgenic technology for visualization and manipulation of the neural circuits controlling behavior in zebrafish

The vertebrate brain is innately equipped with neural circuits that make quick behavioral decisions possible. Elucidating these neural circuits, determining how their master plans are encoded in the genome, and revealing how they can be modified by postnatal experiences will facilitate our understanding of how nature and nurture interact to establish an animal's behavior. In this review, we explain how transgenic zebrafish can cast insights into the developmental mechanisms and functional roles of the neural circuits that directly and indirectly control visuomotor behavior, by taking as an example a transgenic line Tg( brn3a-hsp70:GFP ) enabling visualization of the tectobulbar and habenulo-interpeduncular tracts. These insights emphasize the benefits of applying advanced transgenic technology in zebrafish to future research into this area.     

Structural plasticity of axon terminals in the adult

There is now conclusive evidence for widespread ongoing structural plasticity of presynaptic boutons and axon side-branches in the adult brain. The plasticity complements that of postsynaptic spines, but axonal plasticity samples larger volumes of neuropil, and has a larger impact on circuit remodeling. Axons from distinct neurons exhibit unique ratios of stable (t1/2>9 months) and dynamic (t1/2 5-20 days) boutons, which persist as spatially intermingled subgroups along terminal arbors. In addition, phases of side-branch dynamics mediate larger scale remodeling guided by synaptogenesis. The plasticity is most pronounced during critical periods; its patterns and outcome are controlled by Hebbian mechanisms and intrinsic neuronal factors. Novel experience, skill learning, life-style, and age can persistently modify local circuit structure through axonal structural plasticity.     

Differential regulation of neuronal excitability by nicotine and substance P in subdivisions of the medial habenula

The medial habenula (MHb) plays an important role in nicotine-related behaviors, such as aversion and withdrawal. The MHb is composed of distinct subregions with unique neurotransmitter expression and neuronal connectivity. Here, we showed that nicotine and substance P (SP) differentially regulate neuronal excitability in subdivisions of the MHb (ventrolateral division, MHbVL; dorsal division; MHbD and superior division: MHbS). Nicotine remarkably increased spontaneous neuronal firing in the MHbVL and MHbD, but not in the MHbS, which was consistent with different magnitudes of whole-cell inward currents evoked by nicotine in each subdivision. Meanwhile, SP enhanced neuronal excitability in the MHbVL and MHbS. Although the MHbD is composed of SP-expressing neurons, they did not respond to SP. Neurons in the MHbVL increased their firing in response to bath-applied nicotine, which was attenuated by neurokinin receptor antagonists. Furthermore, nicotine addiction and withdrawal attenuated and augmented excitatory SP effects in the MHbVL, respectively. On the whole, we suggest that MHb-involving nicotine-related behaviors might be associated with SP signaling in MHb subdivisions.     

Acetylcholine potentiates glutamate transmission from the habenula to the interpeduncular nucleus in losers of social conflict

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Ongoing habenular activity is driven by forebrain networks and modulated by olfactory stimuli

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Psychiatric disorder‐related abnormal behavior and habenulointerpeduncular pathway defects in Wnt1‐cre and Wnt1‐GAL4 double transgenic mice

The neural crest is a unique structure in vertebrates. Wnt1‐cre and Wnt1‐GAL4 double transgenic (dTg) mice have been used in a variety of studies concerning neural crest cell lineages in which the Cre/loxP or GAL4/UAS system was applied. Here, we show psychiatric disorder‐related behavioral abnormalities and histologic alterations in a neural crest‐derived brain region in dTg mice. The dTg mice exhibited increased locomotor activity, decreased social interaction, and impaired short‐term spatial memory and nesting behavior. The choline acetyltransferase‐ and vesicular glutamate transporter 2‐immunoreactive habenulointerpeduncular fiber tracts that project from the medial habenular nucleus of the epithalamus to the interpeduncular nucleus of the midbrain tegmentum appeared irregular in the dTg mice. Both the medial habenula nucleus and the interpeduncular nucleus were confirmed to be derived from the neural crest. The findings of this study suggest that neural crest‐derived cells have pathogenic roles in the development of psychiatric disorders and that the dTg mouse could be a useful animal model for studying the pathophysiology of mental illness such as autism and schizophrenia. Scientists that use the dTg mice as a cre‐transgenic deleter line should be cautious in its possible toxicity, especially if behavioral analyses are to be performed.     

Use of transgenic zebrafish reporter lines to study calcium signalling in development.

Calcium signals are associated with many of the events common to animal development. Understanding the role of these calcium signals requires the ability to visualise and manipulate calcium levels in the developing embryo. Recent work has led to the development of sensitive protein-based probes that can be used to generate transgenic animals for the analysis of calcium signalling in vivo. This paper focuses on the use of genetically encoded calcium probes to follow calcium signals in zebrafish. It reviews progress and speculates on the potential for use in the future.     

Heterogeneity of axon terminals expressing VGLUT1 in the cerebral neocortex

Using immunocytochemical techniques and confocal microscopy we have studied the localization of the vesicular glutamate transporters (VGLUTs) 1 and 2 in the mammalian cerebral cortex. The cardinal observations gathered to date can be summarized as follows: 1) Many VGLUT1-positive puncta coexpressing synaptophysin-1 outline pyramidal cell somata and proximal dendrites; of these, a sizeable fraction coexpress VGAT, the vesicular transporter for GABA; 2) VGLUT2-positive puncta are also present in layers II-III and some of them coexpress VGLUTI. These findings suggest that in the cerebral cortex of adult rats axon terminals expressing VGLUT1 are heterogeneous.     

Electron-microscopic characterization of adrenergic axon terminals in the diencephalon of the rat

Summary The fine structure of adrenergic axon terminals was examined in the paraventricular nucleus of the thalamus (PNT) and in the hypothalamic arcuate nucleus-median eminence (ARC-ME) complex by use of phenylethanolamine-N-methyl transferase (PNMT) immunocytochemistry. In the PNT, immunoreactive terminals formed a dense and well-circumscribed plexus. In the ARC, labeled varicosities were less numerous and more evenly distributed. In the ME, they were scarce and confined to the inner zone. In all these areas, the diameter of immunoreactive varicosities ranged between 0.2 and 1.3 m; in the ME and in the transitional zone between the ARC and the ME, a population of larger boutons (>2 m) was also visible. All immunoreactive varicosities exhibited densely packed small, clear vesicles associated with a few large granular vesicles. In the PNT and the ARC, but not in the ME, they formed synaptic contacts with dendritic elements and were occasionally apposed to neuronal cell bodies. These axo-somatic appositions showed no junctional specializations. In the ME and transitional zone, immunoreactive terminals were frequently juxtaposed to, and occasionally established differentiated synaptic contacts with, tanycytes. These data support a transmitter role for adrenaline in the diencephalon and suggest that adrenaline plays a role in hypothalamo-hypophysiotropic regulation through interactions with neural and glial elements.     

nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells

In Drosophila, nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory synaptic transmission in mushroom body Kenyon cells, a neuronal population involved in generation of complex behaviors, including responses to drugs of abuse. To determine whether activation of nAChRs can induce cellular changes that contribute to functional plasticity in these neurons, we examined nicotine-evoked responses in cells cultured from brains of late stage OK107-GAL4 pupae. Kenyon cells can be identified by expression of green fluorescent protein (GFP+). Nicotine activates alpha-bungarotoxin-sensitive nAChRs, causing a rapid increase in intracellular calcium levels in over 95% of the Kenyon cells. The nicotine-evoked calcium increase has a voltage-gated calcium channel (VGCC) dependent component and a VGCC-independent component that involves calcium influx directly through nAChRs. Thapsigargin treatment reduces the nicotine response consistent with amplification by calcium release from intracellular stores. The response to nicotine is experience-dependent: a short conditioning pulse of nicotine causes a transient 50% reduction in the magnitude of the response to a test pulse of nicotine when the interpulse interval is 4 h. This cellular plasticity is dependent on activation of the VGCC-component of the nicotine response and on cAMP-signaling, but not on protein synthesis. These data demonstrate that activation of nAChRs induces a calcium-dependent plasticity in Kenyon cells that could contribute to adult behaviors involving information processing in the mushroom bodies including responses to nicotine.     

Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord

Summary The noradrenergic terminals in the substantia gelatinosa of the dorsal horn of the cervical spinal cord of the rat were investigated by means of the histofluorescence technique and electron-microscopic cytochemistry using the glyoxylic acid-KMnO₄ fixation technique. In accordance with the topographical distribution of fluorescent catecholaminergic fibers, noradrenergic terminals containing small granular vesicles were frequently observed electron microscopically in the outer layer of the substantia gelatinosa. These terminals were most frequently found to appose without showing typical synaptic features, small-caliber dendrites, spine apparatus, and rarely, large caliber dendrites. Only in a few cases, the noradrenergic terminals exhibited typical synaptic contacts with dendritic elements of small size. In addition, noradrenergic terminals apposed non-noradrenergic terminals containing small agranular vesicles. In rats bearing surgical lesions of the dorsal roots, no noradrenergic terminal were found in contact with the degenerated axon terminals in the substantia gelatinosa. These findings suggest that the noradrenergic afferents to the substantia gelatinosa may exert their influence on sensory transmission via dorsal horn cells.     

Evidence for putative photoreceptor axon terminals in the medulla externa of the crayfish

Summary Within the medulla externa of the crayfish compound eye a class of axonal endings with similar characteristics to the photoreceptor terminals of the lamina ganglionaris were studied with light and electron microscopic techniques. These terminals are restricted to the superficial layers of the medulla externa and each is marked by a rod-shaped inclusion selectively impregnated with reduced silver methods.Electron microscopy of the medullary terminals confirms the presence of a rod inclusion composed of fine regularly arranged filaments. These inclusions are often closely associated with mitochondria and glycogen deposits within the endings. Synaptic contacts made by these terminals are characterized by a presynaptic ribbon density which is in contact with two or three postsynaptic elements. Often one postsynaptic element participates in more than one synaptic complex. Numerous invaginated processes, microtubules, synaptic vesicles, and ER cisternae are also present in the medullary terminals.The eighth retinula cell in the retina of the crayfish studied here resembles that previously observed by Nässel (1976). The similarity of the medullary terminals to the photoreceptor endings in the lamina suggest that they may belong to one of the eight photoreceptor cells forming an ommatidium.This work was supported in part by grants from the National Science Foundation (BNS77-15803) and National Institute of Health (NS08964)The authors wish to acknowledge the technical assistance of Ms. Georgia Hammond-Soltis     

Nicotine inhibits the VTA-to-amygdala dopamine pathway to promote anxiety

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Changes in synaptic vesicles of axon terminals of the cat motor cortex after stimulation

The relationship between the size of synaptic vesicles and their distance from the active zone of the synapse was investigated quantitatively in axon terminals on dendritic spines and branches of neurons of the cat motor cortex in a resting state (moderate barbiturate anesthesia) and after prolonged repetitive stimulation of somatosensory area SII. The dimensions of the vesicles belonging to each of the three layers distinguishable in transverse section through the terminals on electron micrographs were recorded as a diminishing variance series. They were characterized by the value of a special rank statistic. Predominance of vesicles of the smallest sizes in layer I, next to the active zone of the synapse, in both the control and the experimental material was established by statistical analysis (using, in particular, the criterion of signs and ²). After stimulation of cortico-cortical projections a significant gradient of decrease in the mean size of the vesicles from peripheral layer III to layer I developed in the terminals studied. Compared with the control, the size of the vesicles decreased both in layer I and in the intermediate layer II. The functional significance of the phenomenon is discussed.A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 7, No. 6, pp. 639–646, November–December, 1975.     

Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord

A subset of primary sensory neurons produces BDNF, which is implicated in control of nociceptive neurotransmission. We previously localized full-length trkB receptors on their terminals within lamina II. To functionally study these receptors, we here employed patch-clamp recordings, calcium imaging and immunocytochemistry on slices from 8-12 days post-natal rats. In this preparation, BDNF (100-500 ng/mL) enhances the release of sensory neurotransmitters (glutamate, substance P, CGRP) in lamina II by acting on trkB receptors expressed by primary afferent fibers of the peptidergic nociceptive type (PN-PAFs). Effect was blocked by trk antagonist K252a or anti-trkB antibody clone 47. A pre-synaptic mechanism was demonstrated after (i) patch-clamp recordings where the neurotrophin induced a significant increase in frequency, but not amplitude, of AMPA-mediated mEPSCs, (ii) real time calcium imaging, where sustained application of BDNF evoked an intense response in up to 57% lamina II neurons with a significant frequency rise. Antagonists of ionotropic glutamate receptors and NK(1) receptors completely inhibited the calcium response to BDNF. Reduction of CGRP (a specific marker of PN-PAFs) and substance P content in dorsal horn following BDNF preincubation, and analysis of the calcium response after depletion with capsaicin, confirmed that the neurotrophin presynaptically enhanced neurotransmitter release from PN-PAFs. This is the first demonstration that trkB receptors expressed by PN-PAF terminals in lamina II are functional during postnatal development. Implications of this finding are discussed considering that BDNF can be released by these same terminals and microglia, a fraction of which (as shown here) contains BDNF also in unactivated state.     

Transcriptional and functional divergence in lateral hypothalamic glutamate neurons projecting to the lateral habenula and ventral tegmental area

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在体神经元胞外记录用于大脑皮层可塑性研究

神经元网络可塑性是大脑学习和记忆功能的基础,可塑性的变化也是某些脑功能疾病的成因。研究大脑皮层可塑性不仅可以为认识可塑性机制提供基本方法,也可对自然衰老过程和神经退行性疾病的病理过程进行观测,进而可以为评价抗衰老药物和治疗神经退行性疾病提供新方法。本文基于经典的大鼠胡须配对模型建立了一套实验方案,通过在体细胞外记录实验的数据分析,比较修剪胡须后相同时间内神经元感受野不对称变化程度的差异,衡量不同生理条件下大鼠体感皮层神经元网络可塑性。本文以中年和青年大鼠体感皮层神经元网络可塑性比较为例,详细介绍了实验方法中的关键技术和操作,如皮层D2功能柱的定位和D2功能柱内不同层神经元的定位等,结果和我室以前相关研究证明了此实验方案的可行性。