Characterization of two neuronal subclasses through constellation pharmacology

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."     

Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.     

Secretagogin is a Ca2+-binding protein specifying subpopulations of telencephalic neurons

The Ca²⁺-binding proteins (CBPs) parvalbumin, calbindin, and calretinin are phenotypic markers of terminally differentiated neurons in the adult brain. Although subtle phylogenetic variations in the neuronal distribution of these CBPs may occur, morphologically and functionally diverse subclasses of interneurons harbor these proteins in olfactory and corticolimbic areas. Secretagogin (scgn) is a recently cloned CBP from pancreatic β and neuroendocrine cells. We hypothesized that scgn is expressed in the mammalian brain. We find that scgn is a marker of neuroblasts commuting in the rostral migratory stream. Terminally differentiated neurons in the olfactory bulb retain scgn expression, with scgn being present in periglomerular cells and granular layer interneurons. In the corticolimbic system, scgn identifies granule cells distributed along the dentate gyrus, indusium griseum, and anterior hippocampal continuation emphasizing the shared developmental origins, and cytoarchitectural and functional similarities of these neurons. We also uncover unexpected phylogenetic differences in scgn expression, since this CBP is restricted to primate cholinergic basal forebrain neurons. Overall, we characterize scgn as a neuron-specific CBP whose distribution identifies neuronal subtypes and hierarchical organizing principles in the mammalian brain.     

Role of P(1) purinergic receptors in myocardial ischemia sensory transduction

OBJECTIVES: To characterize the role that cardiac sensory P(1) purinergic (adenosine A(1) or A(2)) receptors play in transducing myocardial ischemia. METHODS: Porcine nodose ganglion cardiac sensory neuron adenosine A(1) or A(2) receptor function was studied in situ during control states as well as in the presence of the peptides bradykinin and substance P or focal ventricular ischemia. The responses of porcine nodose ganglion cardiac and non-cardiac afferent neuronal somata to adenosine were also studied in vitro. RESULTS: Local application of A(1) or A(2) adenosine receptor agonists modified the activity generated by ventricular sensory neurites associated with 70 and 74% of identified nodose ganglion cardiac afferent somata in situ, respectively, exciting most neurons. In contrast, adenosine reduced the excitability of nodose ganglion cardiac afferent neuronal somata in vitro. Bradykinin and substance P affected 56 and 63%, respectively, of tested afferent neurons. The capacity of ventricular sensory neurites to transduce signals relating to these peptides was virtually eliminated by the presence of P(1) purinergic receptor antagonists. So was their capacity to transduce focal ventricular ischemia. Since most cardiac sensory neurites responded differently to adenosine in vivo than did cardiac afferent neuronal somata in vitro, it appears that the transduction properties of cardiac afferent neurons need to be characterized in situ. CONCLUSIONS: Most ventricular sensory neurites associated with nodose ganglion afferent neurons possess adenosine A(1) and/or A(2) receptors that play a primary role in transducing myocardial ischemic events to central neurons. These data support clinical observations implicating cardiac sensory purinoceptors in transducing myocardial ischemic events.     

Ethanol Modulation of Gaba-Activated Current Responses in Acutely Dissociated Retinal Bipolar Cells and Ganglion Cells

This study examined the effect of acute ethanol exposure on GABA-activated whole-cell current responses elicited in bipolar cells and ganglion cells of the rat retina. Acute exposure to ethanol potentiated GABA responses in 86% of the bipolar cells and in 52% of the ganglion cells tested. As determined in bipolar cells, ethanol was maximally effective at a concentration of 50 mM. In bipolar cells, a GABA_c receptor-mediated component of the whole-cell response to GABA could be uncovered which was also potentiated by ethanol. However, ethanol was ineffective in enhancing bipolar cell responses to glycine. GABA-activated current responses monitored in ganglion cells that were insensitive to modulation by ethanol were sensitive to potentiation by diazepam. At higher concentrations (100–175 mM), ethanol by itself occasionally induced a chloride-mediated current but this occurred independent of an ethanol-induced potentiation of GABA responses. These data establish that ethanol can modulate the sensitivity of retinal neurons to GABA. Overall, the results presented in this study set the stage for future studies to examine the cellular and molecular bases for a differential neuronal sensitivity to an ethanol-induced modulation of GABA responses.     

Continuous Exposure of Cultured Rat Cerebellar Macroneurons to Ethanol-Depressed NMDA and KCl-Stimulated Elevations of Intracellular Calcium

This series of experiments measured ethanol-induced changes In levels of tree Intracelular calcium. Cerebellar macroneurons, harvested from rat embryos on embryonic day 17, were cultured In the presence of 75 mM ethanol for 24,48, or 96 hr. Intracellular calcium concentrations in control and ethanol-exposed neurons did not differ after 24 hr, but they were significantly elevated In the neurons exposed to ethanol for 48 or 96 hr. Similarly, Increases In intracellular calcium elicited by stimulation wk 50 μM NMDA were not significantly different In control and ethanol-exposed neurons after 24 hr. After 48 and 96 hr, however, NMDA-stimulated Increases in Intracellular calcium levels in control neurons were significantly greater than in the ethanol-exposed neurons. These results showed that, when calcium levels were elevated by prolonged exposure to ethanol, the neurons were significantly less responsive to NMDA stimulation. Increases in intracelular calcium elicited by stimulation with 30 mM KCI were not significantly different in the control and treated neurons after 24 and 48 hr of ethanol exposure. After 96 hr of exposure to ethanol, however, them was a significant Increase In intracellular calcium levels In control neurons following KCI stimulation, but not in the ethanol-exposed neurons. The fact that neuronal responses to KCI stimulation were depressed only following 96 hr of exposure to ethanol makes it unlikely that voltage-regulated channels were the primary mediators of the ethanol-induced elevations in intracellular calcium in chronically exposed neurons.     

The visual response of retinal ganglion cells is not altered by optic nerve transection in transgenic mice overexpressing Bcl-2

Attempts to rescue retinal ganglion cells from retrograde degeneration have had limited success, and the residual function of surviving neurons is not known. Recently, it has been found that axotomized retinal ganglion cells die by apoptotic mechanisms. We have used adult transgenic mice overexpressing the Bcl-2 protein, a powerful inhibitor of apoptosis, as a model for preventing injury-induced cell death in vivo. Several months after axotomy, the majority of retinal ganglion cells survived and exhibited normal visual responses. In control wild-type mice, the vast majority of axotomized retinal ganglion cells degenerated, and the physiological responses were abolished. These results suggest that strategies aimed at increasing Bcl-2 expression, or mimicking its function, might effectively counteract trauma-induced cell death in the central nervous system. Neuronal survival is a necessary condition in the challenge for promoting regeneration and eventually restoring neuronal function.     

Expression of catecholaminergic characteristics by primary sensory neurons in the normal adult rat in vivo.

Expression of catecholaminergic characteristics by primary sensory neurons was examined in the vagal nodose and glossopharyngeal petrosal ganglia of the normal adult rat in vivo. Catecholaminergic phenotypic expression was documented by immunocytochemical localization of tyrosine hydroxylase (TyrOHase; EC 1.14.16.2), radiochemical assay of specific TyrOHase catalytic activity, and cytochemical localization of formaldehyde-induced catecholamine fluorescence (FIF) within principal ganglion cells. The TyrOHase-containing cells exhibited morphologic features typical of primary sensory neurons, such as an initial axon glomerulus and a single, bifurcating neurite process. These cells were distinguished from TyrOHase- and FIF-positive small intensely fluorescent cells by size, morphology, and staining intensity. TyrOHase-containing neurons appeared to be insensitive to neonatal treatment with 6-hydroxydopamine, thereby distinguishing them from sympathetic neurons. Nodose and petrosal ganglia of adult rats exhibited TyrOHase catalytic activity, linear with respect to tissue concentration over a 10-fold range, indicating that the immunoreactive enzyme was functional. Transection of specific ganglionic nerve roots depleted TyrOHase catalytic activity and neuronal immunoreactivity within the petrosal ganglion, suggesting that target organ innervation regulates enzyme levels within ganglion perikarya. Our study indicates that primary sensory neurons express catecholaminergic transmitter traits in the normal adult rat. Consequently, in the periphery, catecholaminergic characters are not restricted to the sympathoadrenal axis but are expressed by functionally and embryologically diverse populations of autonomic neurons.     

Replacement of Nerve-Growth Factor by Ganglionic Non-Neuronal Cells for the Survival In Vitro of Dissociated Ganglionic Neurons

Nerve-growth factor is known to cause a considerable increase in the number of neurons putting out processes and surviving in cell cultures of dissociated dorsal-root and sympathetic ganglia from embryonic chicks. Similar effects of nerve-growth factor have now been noted with cultures of dissociated dorsal-root ganglia from newborn mice or rats. In all three sensory ganglionic systems, the effects of the nerve-growth factor on fiber production and neuronal survival could be mimicked, in the absence of the factor, by adequate increase of the non-neuronal cells in the cultures. The results suggest a hypothesis that views the role of the nerve-growth factor as subordinate to that of the non-neuronal cells.     

Radial stretch reveals distinct populations of mechanosensitive mammalian somatosensory neurons

Primary afferent somatosensory neurons mediate our sense of touch in response to changes in ambient pressure. Molecules that detect and transduce thermal stimuli have been recently identified, but mechanisms underlying mechanosensation, particularly in vertebrate organisms, remain enigmatic. Traditionally, mechanically evoked responses in somatosensory neurons have been assessed one cell at a time by recording membrane currents in response to application of focal pressure, suction, or osmotic challenge. Here, we used radial stretch in combination with live-cell calcium imaging to gain a broad overview of mechanosensitive neuronal subpopulations. We found that different stretch intensities activate distinct subsets of sensory neurons as defined by size, molecular markers, or pharmacological attributes. In all subsets, stretch-evoked responses required extracellular calcium, indicating that mechanical force triggers calcium influx. This approach extends the repertoire of stimulus paradigms that can be used to examine mechanotransduction in mammalian sensory neurons, facilitating future physiological and pharmacological studies.     

OFF ganglion cells cannot drive the optokinetic reflex in zebrafish

Whereas the zebrafish retina has long been an important model system for developmental and genetic studies, little is known about the responses of the inner retinal neurons. Here we report single-unit ganglion cell recordings from 5- to 6-day-old zebrafish larvae. In wild-type larvae we identify at least five subtypes of ganglion cell responses to full-field illumination, with ON-OFF and ON-type cells predominating. In the nrc mutant retina, in which the photoreceptor terminals develop abnormally, we observe normal OFF responses but abnormal ON-OFF responses and no ON responses. Previously characterized as blind, these mutants lack an optokinetic reflex (OKR), but in another behavioral assay nrc mutant fish have near-normal responses to the offset of light and slow and sluggish responses to the onset of light. Pharmacological block of the ON pathway mimics most of the nrc visual defects. We conclude that the abnormal photoreceptor terminals in nrc mutants predominantly perturb the ON pathway and that the ON pathway is necessary to drive the OKR in larval zebrafish.     

Effects of Captopril on Sympathetic Control of the Heart and Vasculature in Dogs

ABSTRACT

Experiments were conducted in pentobarbital anesthetized dogs to investigate the effects of captopril on sympathetic neuronal control of the heart and hindlimb vasculature. Captopril, 3.1 mg/kg, i. v. produced marked reductions in blood pressure and hindlimb perfusion pressure, an observation consistent with the high plasma renin activity in the test animals. Increments in hindlimb perfusion pressure elicited by electrical stimulation of the lumbar sympathetic chain were also significantly reduced following Captopril administration (p <. 002). The subsequent administration of a ten fold higher dose of Captopril, 31. 0 mg/kg, produced no further attenuation of the neurally mediated responses. In contrast to the decreased vascular responses to nerve stimulation after captopril, the tachycardia produced by stimulation of pre- or post-ganglionic neurons to the stellate ganglion were not altered. The results of the present study suggest that captopril acts by inhibiting vascular sympathetic neuronal function when the activity of the renin-angiotensin system is elevated. The attenuation of neurally mediated vasoconstriction may be due to the interruption of angiotensin II formation, thereby, preventing the facilitatory effects of angiotensin on sympathetic neurons.     

Quantitative expression profiling of identified neurons reveals cell-specific constraints on highly variable levels of gene expression

The postdevelopmental basis of cellular identity and the unique cellular output of a particular neuron type are of particular interest in the nervous system because a detailed understanding of circuits responsible for complex processes in the brain is impeded by the often ambiguous classification of neurons in these circuits. Neurons have been classified by morphological, electrophysiological, and neurochemical techniques. More recently, molecular approaches, particularly microarray, have been applied to the question of neuronal identity. With the realization that proteins expressed exclusively in only one type of neuron are rare, expression profiles obtained from neuronal subtypes are analyzed to search for diagnostic patterns of gene expression. However, this expression profiling hinges on one critical and implicit assumption: that neurons of the same type in different animals achieve their conserved functional output via conserved levels and quantitative relationships of gene expression. Here we exploit the unambiguously identifiable neurons in the crab stomatogastric ganglion to investigate the precise quantitative expression profiling of neurons at the level of single-cell ion channel expression. By measuring absolute mRNA levels of six different channels in the same individually identified neurons, we demonstrate that not only do individual cell types possess highly variable levels of channel expression but that this variability is constrained by unique patterns of correlated channel expression.     

Recent advances in basic aging research on the nervous system in Japan

Recent studies on aging of the nervous system are reviewed with special reference to neuronal cell death, compensatory reaction, trophic factors, brain cholinergic systems and the autonomic nervous system. Studies on spinal motoneurons labeled with a tracer substance transported retrogradely demonstrated differential age effect on different types. Compensatory reactions were also seen among surviving motoneurons. Motoneuronal survival appears to be correlated with the amount of activity. However, the causal relationship between them is not yet conclusive. The effects of nerve growth factor on sympathetic and dorsal-root ganglion cells seem to be well preserved in the aged, although there are some controversial findings on the ratio of NGF-dependent neurons versus NGF-independent neurons. It has been shown that acidic fibroblast growth factor or other substances may prevent degeneration of the basal forebrain cholinergic neurons and improve memory and learning performance in aged animals. The cholinergic system also regulates the regional cerebral blood flow, and this function seems to be well maintained in aged rats. Microneurography techniques have revealed increased activity of postganglionic sympathetic nerves innervating muscles in aged human subjects. The activity of preganglionic sympathetic nerves innervating the adrenal gland and the section rate of noradrenaline have been shown to increase in the aged rat. These changes might cause high blood pressure in the aged, although some species differences have been noted between humans and rats.     

Two types of calcium response limited to single spines in cerebellar Purkinje cells.

Of fundamental importance in understanding neuronal function is the unambiguous determination of the smallest unit of neuronal integration. It was recently suggested that a whole dendritic branchlet, including tens of spines, acts as the fundamental unit in terms of dendritic calcium dynamics in Purkinje cells. By contrast, we demonstrate that the smallest such unit is the single spine. The results show, by two-photon excited fluorescence laser scanning microscopy, that individual spines are capable of independent calcium activation. Moreover, two distinct spine populations were distinguished by their opposite response to membrane hyperpolarization. Indeed, in a subpopulation of spines calcium entry can also occur through a pathway other than voltage-gated channels. These findings challenge the assumption of a unique parallel fiber activation mode and prompt a reevaluation of the level of functional complexity ascribed to single neurons.     

Brainstem norepinephrine neurons mediate ethanol-evoked pressor response but not baroreflex dysfunction

BACKGROUND: Ethanol elicits strain-dependent blood pressure and baroreflex sensitivity responses in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats; the mechanisms underlying these divergent effects are not clear. The authors tested the hypothesis that differential neuronal actions of ethanol may account for these strain-dependent responses. To this end, the authors investigated the direct effects of ethanol on norepinephrine (NE)-containing neurons in the rostral ventrolateral medulla (RVLM), which modulate sympathetic neuronal activity, and on c-Jun-expressing neurons in the nucleus tractus solitarius (NTS), whose activity is inversely correlated with baroreflex sensitivity. METHODS: In a newly developed model system in conscious, freely moving rats, the effect of intra-RVLM or intra-NTS ethanol was investigated on neuronal NE at the microinjection site (in vivo electrochemistry), blood pressure, heart rate, spontaneous baroreflex sensitivity, and c-Jun expression in the NTS. RESULTS: Ethanol (1, 5, or 10 microg) microinjection into the RVLM elicited dose-dependent increases in RVLM NE and blood pressure in SHRs but not in WKY rats. Ethanol had no effect on the activity of the NE-containing neurons in the NTS of either strain. However, baroreflex dysfunction elicited by intra-NTS ethanol in conscious WKY rats was associated with enhanced expression of c-Jun in the NTS. CONCLUSIONS: (1) Ethanol activation of the NE-containing neurons in the RVLM of SHRs contributes to the centrally mediated pressor response, (2) the NE-containing neurons in the NTS are not involved in ethanol-induced baroreflex dysfunction, and (3) direct activation of the c-Jun-containing neurons in the NTS is implicated in baroreflex dysfunction elicited by ethanol in normotensive rats.     

From the Cover: Neurons in the anterior olfactory nucleus pars externa detect right or left localization of odor sources

Rodents can localize odor sources by comparing odor inputs to the right and left nostrils. However, the neuronal circuits underlying such odor localization are not known. We recorded neurons in the anterior olfactory nucleus (AON) while administering odors to the ipsilateral or contralateral (ipsi- or contra-) nostril. Neurons in the AON pars externa (AONpE) showed respiration phase-locked excitatory spike responses to ipsinostril-only stimulation with a category of odorants, and inhibitory responses to contranostril-only stimulation with the same odorants. Simultaneous odor stimulation of the ipsi- and contranostrils elicited significantly smaller responses than ipsinostril-only stimulation, indicating that AONpE neurons subtract the contranostril odor inputs from ipsinostril odor inputs. An ipsilateral odor source induced larger responses than a centrally located source, whereas an odor source at the contralateral position elicited inhibitory responses. These results indicate that individual AONpE neurons can distinguish the right or left position of an odor source by referencing signals from the two nostrils.     

支气管哮喘持续状态下豚鼠气道感觉神经生长相关蛋白43的表达

目的研究支气管哮喘(简称哮喘)过敏性刺激诱发气道感觉神经敏化机制。方法成年雄性豚鼠39只,按随机数字表法分为生理盐水致敏/激发对照组(A组,9只)、卵白蛋白(OVA)致敏/生理盐水激发对照组(B组,9只)、OVA致敏/激发实验组(C组,21只)。A组以生理盐水(0.5ml/只)致敏,B、C组以10%OVA(0.5ml/只)致敏,第10天开始雾化吸入生理盐水(A、B组)或1%OVA(C组)进行激发,每天1次,每次30min,根据实验需要又将C组21只豚鼠分为激发1d组(C1组,6只)、连续激发3d组(C2组,6只)、连续激发5d组(C3组,9只)。利用免疫荧光双标技术结合激光共聚焦扫描显微观察与Westernblot技术,研究生长相关蛋白43(GAP43)在气道神经以及结状神经节、颈静脉神经节内分布与水平及与P物质(SP)和胶质源神经生长因子(GDNF)受体c RET表达神经元关系。结果免疫荧光结果显示,C3组豚鼠气道内GAP43免疫反应阳性神经呈网状分布于大、中支气管内,以黏膜下层为主,部分GAP43阳性神经纤维向黏膜层内延伸;在结状神经节和颈静脉神经节内有大量GAP43免疫阳性神经胞体,在结状神经节内主要与SP免疫阳性胞体共存,在颈静脉神经节内主要与c RET免疫阳性胞体共存。Westernblot结果显示,A、B、C1、C2、C3组GAP43蛋白表达水平吸光度(A)值分别为0.38±0.04、0.41±0.03、0.49±0.05、0.79±0.08、0.76±0.04。C1、C2、C3组分别与A、B组比较差异均有统计学意义(P均0.05);C2组GAP43蛋白表达与C1组比较差异有统计学意义(P<0.01),但与C3组GAP43蛋白表达比较差异无统计学意义(P>0.05)。结论哮喘过敏性刺激能诱发气道感觉神经———SP肽能神经、GDNF敏感性神经纤维与胞体表达GAP43蛋白。     

谷氨酸对神经元钙离子内流和凋亡的影响及谷氨酸受体拮抗剂保护作用的研究

目的　探讨兴奋性氨基酸谷氨酸介导神经元钙离子内流与诱导神经元凋亡间的关系。方法　体外培养新生大鼠皮质神经元 ,用原位末端标记技术显示凋亡细胞 ;用激光共聚焦方法来观察细胞内钙离子浓度变化 ;谷氨酸处理神经元造成兴奋性氨基酸毒性 ;用MK 80 1做保护研究。结果　体外培养 9d的大多数神经细胞 ,用 1mmol/L谷氨酸处理后 ,细胞内钙离子快速增加 ;谷氨酸处理后细胞死亡的形式主要是凋亡。预先用MK 80 1(10 μmol/L)可明显的减少由谷氨酸介导的钙离子反应 ,并且MK 80 1可同时减少谷氨酸诱导的细胞凋亡。结论　谷氨酸诱导的神经元钙离子内流与细胞凋亡有明显的关系 ,钙离子内流在与谷氨酸毒性导致的细胞凋亡中起重要作用。