Literature context: RRID:AB_90755
The present study has taken advantage of publicly available cell type specific mRNA expression databases in order to identify potential genes participating in the development of retinal AII amacrine cells. We profile two such genes, Delta/Notch-like EGF repeat containing (Dner) and nuclear factor I/A (Nfia), that are each heavily expressed in AII amacrine cells in the mature mouse retina, and which conjointly identify this retinal cell population in its entirety when using antibodies to DNER and NFIA. DNER is present on the plasma membrane, while NFIA is confined to the nucleus, consistent with known functions of each of these two proteins. DNER also identifies some other subsets of retinal ganglion and amacrine cell types, along with horizontal cells, while NFIA identifies a subset of bipolar cells as well as Muller glia and astrocytes. During early postnatal development, NFIA labels astrocytes on the day of birth, AII amacrine cells at postnatal (P) day 5, and Muller glia by P10, when horizontal cells also transiently exhibit NFIA immunofluorescence. DNER, by contrast, is present in ganglion and amacrine cells on P1, also labeling the horizontal cells by P10. Developing AII amacrine cells exhibit accumulating DNER labeling at the dendritic stalk, labeling that becomes progressively conspicuous by P10, as it is in maturity. This developmental time course is consistent with a prospective role for each gene in the differentiation of AII amacrine cells.
Literature context: ipore RRID:AB_90755 â€ƒâ€ƒâ€ƒâ€ƒGoat anti-TrkA (1:400) R&D
Glycoprotein-deleted rabies virus-mediated monosynaptic tracing has become a standard method for neuronal circuit mapping, and is applied to virtually all parts of the rodent nervous system, including the spinal cord and primary sensory neurons. Here we identified two classes of unmyelinated sensory neurons (nonpeptidergic and C-fiber low-threshold mechanoreceptor neurons) resistant to direct and trans-synaptic infection from the spinal cord with rabies viruses that carry glycoproteins in their envelopes and that are routinely used for infection of CNS neurons (SAD-G and N2C-G). However, the same neurons were susceptible to infection with EnvA-pseudotyped rabies virus in tumor virus A receptor transgenic mice, indicating that resistance to retrograde infection was due to impaired virus adsorption rather than to deficits in subsequent steps of infection. These results demonstrate an important limitation of rabies virus-based retrograde tracing of sensory neurons in adult mice, and may help to better understand the molecular machinery required for rabies virus spread in the nervous system. In this study, mice of both sexes were used.SIGNIFICANCE STATEMENT To understand the neuronal bases of behavior, it is important to identify the underlying neural circuitry. Rabies virus-based monosynaptic tracing has been used to identify neuronal circuits in various parts of the nervous system. This has included connections between peripheral sensory neurons and their spinal targets. These connections form the first synapse in the somatosensory pathway. Here we demonstrate that two classes of unmyelinated sensory neurons, which account for >40% of dorsal root ganglia neurons, display resistance to rabies infection. Our results are therefore critical for interpreting monosynaptic rabies-based tracing in the sensory system. In addition, identification of rabies-resistant neurons might provide a means for future studies addressing rabies pathobiology.
Literature context: ore RRID:AB_90755 Viruses
Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a > 20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.
Literature context: amCat# AB76442; RRID: AB_1524535Sheep polyclonal anti-tyrosine hydroxylaseMilliporeCat# AB1542(CH)Chicken polyclonal anti-GABAAbca
Understanding how dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) govern movements requires a detailed knowledge of how different neurotransmitter systems modulate DA neuronal excitability. We report a heterogeneity of electrophysiological properties between medial and lateral SNc neurons modulated by cholinergic neurotransmission. Lateral DA neurons received mainly excitatory (nicotinic or glutamatergic) mediated cholinergic neurotransmission. Medial DA neurons received predominantly GABAergic currents mediated by presynaptic nicotinic receptors or biphasic GABAergic and nicotinic neurotransmission conveyed by GABA and ACh corelease, which inhibited DA neurons. To examine whether cholinergic signaling in the SNc controls mouse behavior, we used optogenetics in awake behaving mice and found that activation of cholinergic terminals in the medial SNc decreased locomotion, whereas activation in the lateral SNc increased locomotion. Our findings provide novel insights on how cholinergic inputs to subregions of the SNc regulate the excitability of DA neurons differentially, resulting in different patterns of motor behavior.
Literature context: e AB1542, RRID:AB_90755) labeled a
Müller glia, the most abundant glia of vertebrate retina, have an elaborate morphology characterized by a vertical stalk that spans the retina and branches in each retinal layer. Müller glia play diverse, critical roles in retinal homeostasis, which are presumably enabled by their complex anatomy. However, much remains unknown, particularly in mouse, about the anatomical arrangement of Müller cells and their arbors, and how these features arise in development. Here we use membrane-targeted fluorescent proteins to reveal the fine structure of mouse Müller arbors. We find sublayer-specific arbor specializations within the inner plexiform layer (IPL) that occur consistently at defined laminar locations. We then characterize Müller glia spatial patterning, revealing how individual cells collaborate to form a pan-retinal network. Müller cells, unlike neurons, are spread across the retina with homogenous density, and their arbor sizes change little with eccentricity. Using Brainbow methods to label neighboring cells in different colors, we find that Müller glia tile retinal space with minimal overlap. The shape of their arbors is irregular but nonrandom, suggesting that local interactions between neighboring cells determine their territories. Finally, we identify a developmental window at postnatal Days 6 to 9 when Müller arbors first colonize the synaptic layers beginning in stereotyped inner plexiform layer sublaminae. Together, our study defines the anatomical arrangement of mouse Müller glia and their network in the radial and tangential planes of the retina, in development and adulthood. The local precision of Müller glia organization suggests that their morphology is sculpted by specific cell to cell interactions with neurons and each other.
Literature context: y (Chemicon; Cat# AB1542; RRID: AB_90755) was generated against native T
Dopamine- and tyrosine hydroxylase-immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse-related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD-95, and PSD-93), that TH cell somata and tapering neurites are also immunopositive for a γ-aminobutyric acid (GABA) receptor subunit (GABAA Rα1 ), and that a synaptic ribbon-specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707-1730, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: # AB1542, RRID:AB_90755) 1:400, ra
Characterizing the distinct functions of the T-type ion channel subunits Cav3.1, 3.2 or 3.3 has proven difficult due to their highly conserved amino-acid sequences and the lack of pharmacological blockers specific for each subunit. To precisely determine the expression pattern of the Cav3.2 channel in the nervous system we generated two knock-in mouse strains that express EGFP or Cre recombinase under the control of the Cav3.2 gene promoter. We show that in the brains of these animals, the Cav3.2 channel is predominantly expressed in the dentate gyrus of the hippocampus. In the peripheral nervous system, the activation of the promoter starts at E9.5 in neural crest cells that will give rise to dorsal root ganglia (DRG) neurons, but not sympathetic neurons. As development progresses the number of DRG cells expressing the Cav3.2 channel reaches around 7% of the DRG at E16.5, and remains constant until E18.5. Characterization of sensory neuron subpopulations at E18.5 showed that EGFP+ cells are a heterogeneous population consisting mainly of TrkB+ and TrkC+ cells, while only a small percentage of DRG cells were TrkA+. Genetic tracing of the sensory nerve end-organ innervation of the skin showed that the activity of the Cav3.2 channel promoter in sensory progenitors marks many mechanoreceptor and nociceptor endings, but spares slowly adapting mechanoreceptors with endings associated with Merkel cells. Our genetic analysis reveals for the first time that progenitors that express the Cav3.2 T-type calcium channel, defines a sensory specific lineage that populates a large proportion of the DRG. Using our Cav3.2-Cre mice together with AAV viruses containing a conditional fluorescent reporter (tdTomato) we could also show that Cre expression is largely restricted to two functionally distinct sensory neuron types in the adult ganglia. Cav3.2 positive neurons innervating the skin were found to only form lanceolate endings on hair follicles and are probably identical to D-hair receptors. A second population of nociceptive sensory neurons expressing the Cav3.2 gene was found to be positive for the calcitonin-gene related peptide but these neurons are deep tissue nociceptors that do not innervate the skin.
Literature context: a spaces (AB_90755 vs. RRID:
A central tenet in support of research reproducibility is the ability to uniquely identify research resources, i.e., reagents, tools, and materials that are used to perform experiments. However, current reporting practices for research resources are insufficient to identify the exact resources that are reported or to answer basic questions such as "How did other studies use resource X?" To address this issue, the Resource Identification Initiative was launched as a pilot project to improve the reporting standards for research resources in the Methods sections of articles and thereby improve identifiability and scientific reproducibility. The pilot engaged over 25 biomedical journal editors from most major publishers, as well as scientists and funding officials. Authors were asked to include Research Resource Identifiers (RRIDs) in their articles prior to publication for three resource types: antibodies, model organisms, and tools (i.e., software and databases). RRIDs are assigned by an authoritative database, for example, a model organism database for each type of resource. To make it easier for authors to obtain RRIDs, resources were aggregated from the appropriate databases and their RRIDs made available in a central Web portal (http://scicrunch.org/resources). RRIDs meet three key criteria: they are machine-readable, free to generate and access, and are consistent across publishers and journals. The pilot was launched in February of 2014 and over 300 articles have appeared that report RRIDs. The number of journals participating has expanded from the original 25 to more than 40, with RRIDs appearing in 62 different journals to date. Here we present an overview of the pilot project and its outcomes to date. We show that authors are able to identify resources and are supportive of the goals of the project. Identifiability of the resources post-pilot showed a dramatic improvement for all three resource types, suggesting that the project has had a significant impact on identifiability of research resources.
Literature context: . AB1542, RRID:AB_90755).
A central tenet in support of research reproducibility is the ability to uniquely identify research resources, i.e., reagents, tools, and materials that are used to perform experiments. However, current reporting practices for research resources are insufficient to allow humans and algorithms to identify the exact resources that are reported or answer basic questions such as "What other studies used resource X?" To address this issue, the Resource Identification Initiative was launched as a pilot project to improve the reporting standards for research resources in the methods sections of papers and thereby improve identifiability and reproducibility. The pilot engaged over 25 biomedical journal editors from most major publishers, as well as scientists and funding officials. Authors were asked to include Research Resource Identifiers (RRIDs) in their manuscripts prior to publication for three resource types: antibodies, model organisms, and tools (including software and databases). RRIDs represent accession numbers assigned by an authoritative database, e.g., the model organism databases, for each type of resource. To make it easier for authors to obtain RRIDs, resources were aggregated from the appropriate databases and their RRIDs made available in a central web portal ( www.scicrunch.org/resources). RRIDs meet three key criteria: they are machine readable, free to generate and access, and are consistent across publishers and journals. The pilot was launched in February of 2014 and over 300 papers have appeared that report RRIDs. The number of journals participating has expanded from the original 25 to more than 40. Here, we present an overview of the pilot project and its outcomes to date. We show that authors are generally accurate in performing the task of identifying resources and supportive of the goals of the project. We also show that identifiability of the resources pre- and post-pilot showed a dramatic improvement for all three resource types, suggesting that the project has had a significant impact on reproducibility relating to research resources.
Literature context: # AB1542, RRID:AB_90755) for 36 ho
Serotonergic (5-HT) and noradrenergic (NA) input to spinal motoneurons is essential for generating plateau potentials and self-sustained discharges. Extensor motoneurons are densely innervated by 5-HT and NA synapses and have robust plateau potentials and self-sustained discharges. Conversely, plateau potentials and self-sustained discharges are very rare in flexor motoneurons. The most likely reasons for this difference are that flexor motoneurons have few 5-HT and NA synapses and/or they are distributed distant to the channels responsible for plateau potentials and self-sustained discharges. However, the distribution of 5-HT and NA synapses on flexor motoneurons is unknown. Here we describe the distribution and density of 5-HT and NA synapses on motoneurons that innervate the flexor neck muscle, rectus capitis anterior (RCA), in the adult cat. Using a combination of intracellular staining, fluorescent immunohistochemistry, and 3D reconstruction techniques, we found that 5-HT and NA synapses are widely distributed throughout the dendritic trees of RCA motoneurons, albeit with a strong bias to small-diameter dendrites and to medial dendrites in the case of NA contacts. The number of 5-HT and NA contacts per motoneuron ranged, respectively, from 381 to 1,430 and from 642 to 1,382, which is 2.3- and 1.4-fold less than neck extensor motoneurons (Montague et al., J Comp Neurol 2013;521:638-656). These results suggest that 5-HT and NA synapses on flexor motoneurons may provide a powerful means of amplifying synaptic currents without incurring plateau potentials or self-sustained discharges. This feature is well suited to meet the biomechanical demands imposed on flexor muscles during different motor tasks.
Literature context: #AB1542, RRID:AB_90755), ubiquiti
Accumulating evidence from genetic and biochemical studies implicates dysfunction of the autophagic-lysosomal pathway as a key feature in the pathogenesis of Parkinson's disease (PD). Most studies have focused on accumulation of neurotoxic α-synuclein secondary to defects in autophagy as the cause of neurodegeneration, but abnormalities of the autophagic-lysosomal system likely mediate toxicity through multiple mechanisms. To further explore how endolysosomal dysfunction causes PD-related neurodegeneration, we generated a murine model of Kufor-Rakeb syndrome (KRS), characterized by early-onset Parkinsonism with additional neurological features. KRS is caused by recessive loss-of-function mutations in the ATP13A2 gene encoding the endolysosomal ATPase ATP13A2. We show that loss of ATP13A2 causes a specific protein trafficking defect, and that Atp13a2 null mice develop age-related motor dysfunction that is preceded by neuropathological changes, including gliosis, accumulation of ubiquitinated protein aggregates, lipofuscinosis, and endolysosomal abnormalities. Contrary to predictions from in vitro data, in vivo mouse genetic studies demonstrate that these phenotypes are α-synuclein independent. Our findings indicate that endolysosomal dysfunction and abnormalities of α-synuclein homeostasis are not synonymous, even in the context of an endolysosomal genetic defect linked to Parkinsonism, and highlight the presence of α-synuclein-independent neurotoxicity consequent to endolysosomal dysfunction.
Literature context: rica, MA; RRID:AB_90755) was raise
The C1 cells, located in the rostral ventrolateral medulla (RVLM), are activated by pain, hypoxia, hypoglycemia, infection, and hypotension and elicit cardiorespiratory stimulation, adrenaline and adrenocorticotropic hormone (ACTH) release, and arousal. The orexin neurons contribute to the autonomic responses to acute psychological stress. Here, using an anatomical approach, we consider whether the orexin neurons could also be contributing to the autonomic effects elicited by C1 neuron activation. Phenylethanolamine N-methyl transferase-immunoreactive (PNMT-ir) axons were detected among orexin-ir somata, and close appositions between PNMT-ir axonal varicosities and orexin-ir profiles were observed. The existence of synapses between PNMT-ir boutons labeled with diaminobenzidine and orexinergic neurons labeled with immunogold was confirmed by electron microscopy. We labeled RVLM neurons with a lentiviral vector that expresses the fusion protein ChR2-mCherry under the control of the catecholaminergic neuron-selective promoter PRSx8 and obtained light and ultrastructural evidence that these neurons innervate the orexin cells. By using a Cre-dependent adeno-associated vector and TH-Cre rats, we confirmed that the projection from RVLM catecholaminergic neurons to the orexinergic neurons originates predominantly from PNMT-ir catecholaminergic (i.e., C1 cells). The C1 neurons were found to establish predominantly asymmetric synapses with orexin-ir cell bodies or dendrites. These synapses were packed with small clear vesicles and also contained dense-core vesicles. In summary, the orexin neurons are among the hypothalamic neurons contacted and presumably excited by the C1 cells. The C1-orexin neuronal connection is probably one of several suprabulbar pathways through which the C1 neurons activate breathing and the circulation, raise blood glucose, and facilitate arousal from sleep.
Literature context: n AB1542, RRID:AB_90755) was used
Dendritic arbors of retinal ganglion cells (RGCs) collect information over a certain area of the visual scene. The coverage territory and the arbor density of dendrites determine what fraction of the visual field is sampled by a single cell and at what resolution. However, it is not clear whether visual stimulation is required for the establishment of branching patterns of RGCs, and whether a general principle directs the dendritic patterning of diverse RGCs. By analyzing the geometric structures of RGC dendrites, we found that dendritic arbors of RGCs underwent a substantial spatial rearrangement after eye-opening. Light deprivation blocked both the dendritic growth and the branch patterning, suggesting that visual stimulation is required for the acquisition of specific branching patterns of RGCs. We further showed that vision-dependent dendritic growth and arbor refinement occurred mainly in the middle portion of the dendritic tree. This nonproportional growth and selective refinement suggest that the late-stage dendritic development of RGCs is not a passive stretching with the growth of eyes, but rather an active process of selective growth/elimination of dendritic arbors of RGCs driven by visual activity. Finally, our data showed that there was a power law relationship between the coverage territory and dendritic arbor density of RGCs on a cell-by-cell basis. RGCs were systematically less dense when they cover larger territories regardless of their cell type, retinal location, or developmental stage. These results suggest that a general structural design principle directs the vision-dependent patterning of RGC dendrites.
Literature context: . AB1542, RRID:AB_90755), the prim
The role of dopaminergic (DA) projections from the ventral tegmental area (VTA) in appetitive and rewarding behavior has been widely studied, but the VTA also has documented DA-independent functions. Several drugs of abuse, act on VTA GABAergic neurons, and most studies have focused on local inhibitory connections. Relatively little is known about VTA GABA projection neurons and their connections to brain sites outside the VTA. This study employed viral-vector-mediated cell-type-specific anterograde tracing, classical retrograde tracing, and immunohistochemistry to characterize VTA GABA efferents throughout the brain. We found that VTA GABA neurons project widely to forebrain and brainstem targets, including the ventral pallidum, lateral and magnocellular preoptic nuclei, lateral hypothalamus, and lateral habenula. Minor projections also go to central amygdala, mediodorsal thalamus, dorsal raphe, and deep mesencephalic nuclei, and sparse projections go to prefrontal cortical regions and to nucleus accumbens shell and core. These projections differ from the major VTA DA target regions. Retrograde tracing studies confirmed results from the anterograde experiments and differences in projections from VTA subnuclei. Retrogradely labeled GABA neurons were not numerous, and most non-tyrosine hydroxylase/retrogradely labeled cells lacked GABAergic markers. Many non-TH/retrogradely labeled cells projecting to several areas expressed VGluT2. VTA GABA and glutamate neurons project throughout the brain, most prominently to regions with reciprocal connections to the VTA. These data indicate that VTA GABA and glutamate neurons may have more DA-independent functions than previously recognized.
Literature context: n AB1542, RRID:AB_90755), and mous
Although the neuroanatomical distribution of catecholaminergic (CA) neurons has been well documented across all vertebrate classes, few studies have examined CA connectivity to physiologically and anatomically identified neural circuitry that controls behavior. The goal of this study was to characterize CA distribution in the brain and inner ear of the plainfin midshipman fish (Porichthys notatus) with particular emphasis on their relationship with anatomically labeled circuitry that both produces and encodes social acoustic signals in this species. Neurobiotin labeling of the main auditory end organ, the saccule, combined with tyrosine hydroxylase immunofluorescence (TH-ir) revealed a strong CA innervation of both the peripheral and central auditory system. Diencephalic TH-ir neurons in the periventricular posterior tuberculum, known to be dopaminergic, send ascending projections to the ventral telencephalon and prominent descending projections to vocal-acoustic integration sites, notably the hindbrain octavolateralis efferent nucleus, as well as onto the base of hair cells in the saccule via nerve VIII. Neurobiotin backfills of the vocal nerve in combination with TH-ir revealed CA terminals on all components of the vocal pattern generator, which appears to largely originate from local TH-ir neurons but may include input from diencephalic projections as well. This study provides strong neuroanatomical evidence that catecholamines are important modulators of both auditory and vocal circuitry and acoustic-driven social behavior in midshipman fish. This demonstration of TH-ir terminals in the main end organ of hearing in a nonmammalian vertebrate suggests a conserved and important anatomical and functional role for dopamine in normal audition.
Literature context: #AB1542, RRID:AB_90755). Subseque
Phasic increases in dopamine (DA) are involved in the detection and selection of relevant sensory stimuli. The DAergic and cholinergic system dynamically interact to gate and potentiate sensory inputs to striatum. Striatal cholinergic interneurons (CINs) respond to relevant sensory stimuli with an initial burst, a firing pause, or a late burst, or a combination of these three components. CIN responses coincide with phasic firing of DAergic neurons in vivo. In particular, the late burst of CINs codes for the anticipated reward. To examine whether DAergic midbrain afferents can evoke the different CIN responses, we recorded from adult olfactory tubercle slices in the mouse ventral striatum. Olfactory inputs to striatal projection neurons were gated by the cholinergic tone. Phasic optogenetic activation of DAergic terminals evoked combinations of initial bursts, pauses, and late bursts in subsets of CINs by distinct receptor pathways. Glutamate release from midbrain afferents evoked an NMDAR-dependent initial burst followed by an afterhyperpolarization-induced pause. Phasic release of DA itself evoked acute changes in CIN firing. In particular, in CINs without an initial burst, phasic DA release evoked a pause through D2-type DA receptor activation. Independently, phasic DA activated a slow depolarizing conductance and the late burst through a D1-type DA receptor pathway. In summary, DAergic neurons elicit transient subsecond firing responses in CINs by sequential activation of NMDA, D2-type, and D1-type receptors. This fast control of striatal cholinergic tone by phasic DA provides a novel dynamic link of two transmitter systems central to the detection and selection of relevant stimuli.
Excitation of neurons in the ventromedial hypothalamus (VMH), especially those residing in the dorsomedial part of the nucleus (VMHdm), evokes sympathetic nervous system (SNS) outflow, modulating a number of physiological functions including feeding and blood glucose homeostasis. However, the anatomical basis of VMH-mediated SNS activation has thus far proved elusive. To understand how VMH neurons exercise output functions and describe an anatomical link between these neurons and the SNS, we identified downstream neural targets of the VMHdm by injecting an adenoviral vector encoding Cre recombinase (Cre)-regulated farnesylated green fluorescent protein (GFPf ) into the VMHdm of mice that express Cre in neurons expressing the VMH-specific transcription factor steroidogenic factor 1 (SF1). We confirm previously described projection patterns of the VMHdm and report the existence of a formerly unidentified projection pathway to a number of autonomic centers in the brainstem. These VMH efferents travel caudally through the periaqueductal gray (PAG) and then ventrally through the lateral lemniscus to the ventral surface of the brain, where they eventually reach caudal autonomic centers including the C1 catecholamine cell group of the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS), where VMH efferents make close contacts with catecholaminergic neurons. We also found that VMHdm fibers reach a number of brainstem areas, including the retrotrapezoid nucleus (RTN), which are important in regulating respiration. Thus, the present study indicates that the VMH may modulate sympathetic and autonomic activity via synaptic contacts in the RTN, NTS, and RVLM and provides significant anatomical evidence to support a role of the VMH in respiratory regulation.
Elevating levels of nerve growth factor (NGF) can have pronounced effects on the survival and maintenance of distinct populations of neurons. We have generated a line of transgenic mice in which NGF is expressed under the control of the smooth muscle α-actin promoter. These transgenic mice have augmented levels of NGF protein in the descending colon and urinary bladder, so these tissues display increased densities of NGF-sensitive sympathetic efferents and sensory afferents. Here we provide a thorough examination of sympathetic and sensory axonal densities in the descending colon and urinary bladder of NGF transgenic mice with and without the expression of the p75 neurotrophin receptor (p75NTR). In response to elevated NGF levels, sympathetic axons (immunostained for tyrosine hydroxylase) undergo robust collateral sprouting in the descending colon and urinary bladder of adult transgenic mice (i.e., those tissues having smooth muscle cells); this sprouting is not augmented in the absence of p75NTR expression. As for sensory axons (immunostained for calcitonin gene-related peptide) in the urinary bladders of transgenic mice, fibers undergo sprouting that is further increased in the absence of p75NTR expression. Sympathetic axons are also seen invading the sensory ganglia of transgenic mice; these fibers form perineuronal plexi around a subpopulation of sensory somata. Our results reveal that elevated levels of NGF in target tissues stimulate sympathetic and sensory axonal sprouting and that an absence of p75NTR by sensory afferents (but not by sympathetic efferents) leads to a further increase of terminal arborization in certain NGF-rich peripheral tissues.
Brain structures related to reproduction are thought to depend on the action of gonadal steroids acting either during early life (organizing irreversible effects) or adulthood (activating transient effects). More recently puberty has become a focus of attention and it was demonstrated that action of sex steroid hormones at this time plays a critical role in the final organization of brain and behavior. We studied by BrdU immunohistochemistry the ontogeny from hatching to sexual maturity of a previously identified cell population in the preoptic area labeled by a BrdU injection at the end of embryonic period (E12) of sexual differentiation in male and female Japanese quail. After an initial increase between E12 and hatching, the density of BrdU-immunoreactive cells decreased until the beginning of puberty but then increased again during sexual maturation in the caudal preoptic area specifically. Divisions of these cells took place in the brain parenchyma as indicated by the large numbers of pairs of labeled cells. No sex difference affecting these processes could be detected at any stage of development. Large numbers of new cells thus arise around puberty in the caudal preoptic area and presumably contribute to the reorganization of this structure that precedes the emergence of adult reproductive behaviors.
The input-output properties of motoneurons are dynamically regulated. This regulation depends, in part, on the relative location of excitatory and inhibitory synapses, voltage-dependent and -independent channels, and neuromodulatory synapses on the dendritic tree. The goal of the present study was to quantify the number and distribution of synapses from two powerful neuromodulatory systems that originate from noradrenergic (NA) and serotonergic (5-HT) neurons. Here we show that the dendritic trees of motoneurons innervating a dorsal neck extensor muscle, splenius, in the adult cat are densely, but not uniformly innervated by both NA and 5-HT boutons. Identified splenius motoneurons were intracellularly stained with Neurobiotin. Using 3D reconstruction techniques we mapped the distributions of contacts formed by NA and 5-HT boutons on the reconstructed dendritic trees of these motoneurons. Splenius motoneurons received an average of 1,230 NA contacts (range = 647-1,507) and 1,582 5-HT contacts (range = 1,234-2,143). The densities of these contacts were 10 (NA) to 6 (5-HT)-fold higher on small compared to large-diameter dendrites. This relationship largely accounts for the bias of NA and 5-HT contacts on distal dendrites and is partially responsible for the higher density of NA contacts on dendrites located more than 200 μm dorsal to the soma. These results suggest that the neuromodulatory actions of NA and 5-HT are compartmentalized and regulate the input-output properties of motoneurons according to precisely arranged interactions with voltage-dependent and -independent channels that are primarily located on small-diameter dendrites.
To determine the role of homotypic interactions between neighboring dopaminergic amacrine (DA) cells upon dendritic morphogenesis, the morphology of single cells was examined relative to the positioning of all neighboring homotypic cells. For each labeled cell, the dendritic field was reconstructed, its Voronoi domain was calculated, and the two were related. The dendritic fields of DA cells were observed to be large, sparse, and highly irregular. Dendrites readily overlapped those of neighboring cells, showing no evidence for dendritic tiling or inter-digitation consistent with homotypic repulsion or avoidance. Furthermore, a direct comparison of dendritic field area with the Voronoi domain area of the same cell showed no evidence for dendritic growth being constrained or biased by the local distribution of homotypic neighbors in wild-type retinas. A comparison of the processes of adjacent filled cells confirmed their immediate proximity to one another within the inner plexiform layer, indicating that they do not engage in mutual avoidance by coursing at different depths. Together, these results suggest that the morphogenesis of DA cells is independent of homotypic interactions. However, in the absence of the pro-apoptotic Bax gene, which yields a fourfold increase in DA cell number, a small but significant reduction in dendritic field size was obtained, although not so great as would be predicted by the increase in density. The present results are considered in light of recent studies on the role of cell adhesion molecules expressed by developing DA cells.
Hemoglobin is the oxygen carrier in vertebrate blood erythrocytes. Here we report that hemoglobin chains are expressed in mammalian brain neurons and are regulated by a mitochondrial toxin. Transcriptome analyses of laser-capture microdissected nigral dopaminergic neurons in rats and striatal neurons in mice revealed the presence of hemoglobin alpha, adult chain 2 (Hba-a2) and hemoglobin beta (Hbb) transcripts, whereas other erythroid markers were not detected. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed the expression of Hba-a2 and Hbb in nigral dopaminergic neurons, striatal gamma-aminobutyric acid (GABA)ergic neurons, and cortical pyramidal neurons in rats. Combined in situ hybridization histochemistry and immunohistochemistry with the neuronal marker neuronal nuclear antigen (NeuN) in rat brain further confirmed the presence of hemoglobin mRNAs in neurons. Immunohistochemistry identified hemoglobin alpha- and beta-chains in both rat and human brains, and hemoglobin proteins were detected by Western blotting in whole rat brain tissue as well as in cultures of mesencephalic neurons, further excluding the possibility of blood contamination. Systemic administration of the mitochondrial inhibitor rotenone (2 mg/kg/d, 7d, s.c.) induced a marked decrease in Hba-a2 and Hbb but not neuroglobin or cytoglobin mRNA in transcriptome analyses of nigral dopaminergic neurons. Quantitative RT-PCR confirmed the transcriptional downregulation of Hba-a2 and Hbb in nigral, striatal, and cortical neurons. Thus, hemoglobin chains are expressed in neurons and are regulated by treatments that affect mitochondria, opening up the possibility that they may play a novel role in neuronal function and response to injury.
The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin's cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRb-IRES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor.
We previously showed that chronic psychostimulant exposure induces the transcription factor DeltaFosB in gamma-aminobutyric acid (GABA)ergic neurons of the caudal tier of the ventral tegmental area (VTA). This subregion was defined as the tail of the VTA (tVTA). In the present study, we showed that tVTA can also be visualized by analyzing FosB/DeltaFosB response following acute cocaine injection. This induction occurs in GABAergic neurons, as identified by glutamic acid decarboxylase (GAD) expression. To characterize tVTA further, we mapped its inputs by using the retrograde tracers Fluoro-Gold or cholera toxin B subunit. Retrogradely labeled neurons were observed in the medial prefrontal cortex, the lateral septum, the ventral pallidum, the bed nucleus of the stria terminalis, the substantia innominata, the medial and lateral preoptic areas, the lateral and dorsal hypothalamic areas, the lateral habenula, the intermediate layers of the superior colliculus, the dorsal raphe, the periaqueductal gray, and the mesencephalic and pontine reticular formation. Projections from the prefrontal cortex, the hypothalamus, and the lateral habenula to the tVTA were also shown by using the anterograde tracer biotinylated dextran amine (BDA). We showed that the central nucleus of the amygdala innervates the anterior extent of the VTA but not the tVTA. Moreover, the tVTA mainly receives non-aminergic inputs from the dorsal raphe and the locus coeruleus. Although the tVTA has a low density of dopaminergic neurons, its afferents are mostly similar to those targeting the rest of the VTA. This suggests that the tVTA can be considered as a VTA subregion despite its caudal location.
Spinal cord injury commonly causes chronic, neuropathic pain. The mechanisms are poorly understood but may include structural plasticity within spinal and supraspinal circuits. Our aim was to determine whether structural remodeling within the dorsal horn rostral to an incomplete injury differs from a complete spinal cord transection. Four immunohistochemical populations of primary afferent C-fibers, and descending catecholamine and serotonergic projections, were examined in segments T9-T12 at 2 and 12 weeks after a T13 clip-compression injury in adult male rats. Dorsal root ganglia were also examined. Two weeks after injury, fibers immunoreactive for calcitonin gene-related peptide (CGRP) or GDNF-family receptors (GFRalpha1, GFRalpha2, GFRalpha3) showed distinct injury responses within the superficial dorsal horn. CGRP fibers decreased, but GFRalpha1, GFRalpha2 and GFRalpha3 fibers did not change. In contrast, all groups were decreased by 12 weeks after injury. Catecholamine fibers showed a decrease at 2 weeks followed by an increase in density at 12 weeks, whereas serotonergic fibers showed a decrease (restricted to deep dorsal horn) at 12 weeks. These results show that the dorsal horn of the spinal cord undergoes substantial structural plasticity rostral to a compression injury, with the most profound effect being a prolonged and possibly permanent loss of primary afferent fibers. This loss was more extensive and more prolonged than the loss that follows spinal cord transection. Our results provide further evidence that anatomical reorganization of sensory and nociceptive dorsal horn circuits rostral to an injury could factor in the development or maintenance of spinal cord injury pain.
The ventrolateral bed nucleus of the stria terminalis (BSTvl) receives direct input from two specific subpopulations of neurons in the nucleus tractus solitarius (NTS). It is heavily innervated by aldosterone-sensitive NTS neurons, which are selectively activated by sodium depletion, and by the A2 noradrenergic neurons, which are activated by visceral and immune- and stress-related stimuli. Here, we used a retrograde neuronal tracer to identify other brain sites that innervate the BSTvl. Five general brain regions contained retrogradely labeled neurons: cerebral cortex (infralimbic and insular regions), rostral forebrain structures (subfornical organ, organum vasculosum of the lamina terminalis, taenia tecta, nucleus accumbens, lateral septum, endopiriform nucleus, dorsal BST, substantia innominata, and, most prominently the amygdala--primarily its basomedial and central subnuclei), thalamus (central medial, intermediodorsal, reuniens, and, most prominently the paraventricular thalamic nucleus), hypothalamus (medial preoptic area, perifornical, arcuate, dorsomedial, parasubthalamic, and posterior hypothalamic nuclei), and brainstem (periaqueductal gray matter, dorsal and central superior raphe nuclei, parabrachial nucleus, pre-locus coeruleus region, NTS, and A1 noradrenergic neurons in the caudal ventrolateral medulla). In the arcuate hypothalamic nucleus, some retrogradely labeled neurons contained either agouti-related peptide or cocaine/amphetamine-regulated transcript. Of the numerous retrogradely labeled neurons in the perifornical hypothalamic area, few contained melanin-concentrating hormone or orexin. In the brainstem, many retrogradely labeled neurons were either serotoninergic or catecholaminergic. In summary, the BSTvl receives inputs from a variety of brain sites implicated in hunger, salt and water intake, stress, arousal, and reward.
A fundamental organizational principle of the central nervous system is that gray matter is the province of neuronal somata, white matter their processes. However, the rat and primate dorsal columns (archetypal spinal "white matter" tracts) are actually of intermediate character, insofar as they contain a surprisingly prominent neuropil of unknown function. Here I report on the morphology, inputs, projections, and functional properties of these neurons. Small fusiform and larger lentiform neurons are most abundant in the gracile fasciculus of the cervical and lumbar enlargements and are absent from the cuneate fasciculus and corticospinal tract. Many have dendrites that run along the dorsal pia, and, although in transverse sections these neurons appear isolated from the gray matter, they are also connected to area X by varicose and sometimes loosely fasciculated dendrites. These neurons receive neurochemically diverse, compartmentalized synaptic inputs (primary afferent, intrinsic and descending), half express the substance P receptor, and some project supraspinally. Unlike substantia gelatinosa neurons, they do not express protein kinase C gamma. Functionally, they have small receptive fields, which are somatotopically appropriate with respect to their anterior-posterior position along the neuraxis. They respond to innocuous and/or noxious mechanical stimulation of the distal extremities, and some are prone to central sensitization or "windup." Morphologically, neurochemically, and functionally, therefore, these cells most closely resemble neurons in laminae III-VI in the dorsal horn. The proximity of their dorsal dendrites to the pia mater may also reflect an ability to integrate internal (e.g., changes in cerebrospinal fluid compostition) and external (e.g., somatic) stimuli.
It is well documented that neuronal activity is required for the developmental segregation of retinal ganglion cell (RGC) synaptic connectivity with ON and OFF bipolar cells in mammalian retina. Our recent study showed that light deprivation preferentially blocked the developmental RGC dendritic redistribution from the center to sublamina a of the inner plexiform layer (IPL). To determine whether OFF signals in visual stimulation are required for OFF RGC dendritic development, the light-evoked responses and dendritic stratification patterns of RGCs in Spastic mutant mice, in which the OFF signal transmission in the rod pathway is largely blocked due to a reduction of glycine receptor (GlyR) expression, were quantitatively studied at different ages and rearing conditions. The dendritic distribution in the IPL of these mice was indistinguishable from wildtype controls at the age of postnatal day (P)12. However, the adult Spastic mutants had altered RGC light-evoked synaptic inputs from ON and OFF pathways, which could not be mimicked by pharmacologically blocking of glycinergic synaptic transmission on age-matched wildtype animals. Spastic mutation also blocked the developmental redistribution of RGC dendrites from the center to sublamina a of the IPL, which mimicked the effects induced by light deprivation on wildtype animals. Moreover, light deprivation of the Spastic mutants had no additional impact on the RGC dendritic distribution and light response patterns. We interpret these results as that visual stimulation regulates the maturation of RGC synaptic activity and connectivity primarily through GlyR-mediated synaptic transmission.
We have investigated the development of autonomic nerves in the urogenital tract of male mice and the effect of neurturin gene deletion on this process. At birth, autonomic innervation of the reproductive organs was sparse, but urinary bladder smooth muscle was well innervated. Further innervation of reproductive tissues occurred until P21, but noradrenergic axons established their complete terminal field later than nitrergic cholinergic axons: in adults the former are more prevalent, yet this became apparent only at P7 (vas deferens, seminal vesicles), P14 (prostate) or after P14 (penis). Neurturin was essential for initial projection of axons (mucosa of vas deferens), maintenance of terminal fields (prostate and seminal vesicles), or both functions (cavernosum of penis). In contrast, some targets (e.g., bladder muscle and suburothelium, vas deferens smooth muscle) were unaffected by neurturin gene deletion. Pelvic ganglion neurons more than doubled between birth and adulthood, probably as aresult of continued maturation of p75-positive undifferentiated neuronal precursors rather than cell division. The adult number of neurons was achieved by P7 (sympathetic) or P21 (parasympathetic). In adult neurturin knockout mice, there were approximately 25% fewer parasympathetic neurons compared with wild types, because of failure of differentiation after P14. This study revealed the complexity of postnatal maturation of urogenital innervation, with each organ showing a distinct chronology of innervation and different requirement for neurturin. Our results also indicate that in adults there will be distinct differences in neurturin dependence between organs, such that proregenerative therapies may have to be tailored specifically for the nerve pathway of interest.
The central nucleus of the amygdala (CeA) orchestrates autonomic and other behavioral and physiological responses to conditioned stimuli that are aversive or elicit fear. As a related CeA function is the expression of hypoalgesia induced by conditioned stimuli or systemic morphine administration, we examined postsynaptic opioid modulation of neurons in each major CeA subdivision. Following electrophysiological recording, biocytin-filled neurons were precisely located in CeA regions identified by chemoarchitecture (enkephalin-immunoreactivity) and cytoarchitecture (DAPI nuclear staining) in fixed adult rat brain slices. This revealed a striking distribution of physiological types, as 92% of neurons in capsular CeA were classified as late-firing, whereas no neurons in the medial CeA were of this class. In contrast, 60% or more of neurons in the lateral and medial CeA were low-threshold bursting neurons. Mu-opioid receptor (MOPR) agonists induced postsynaptic inhibitory potassium currents in 61% of CeA cells, and this ratio was maintained in each subdivision and for each physiological class of neuron. However, MOPR agonists more frequently inhibited bipolar/fusiform cells than triangular or multipolar neurons. A subpopulation of MOPR-expressing neurons were also inhibited by delta opioid receptor agonists, whereas a separate population were inhibited kappa opioid receptors (KOPR). The MOPR agonist DAMGO inhibited 9/9 CeM neurons with projections to the parabrachial nucleus identified by retrograde tracer injection. These data support models of striatopallidal organization that have identified striatal-like and pallidal-like CeA regions. Opioids can directly inhibit output from each subdivision by activating postsynaptic MOPRs or KOPRs on distinct subpopulations of opioid-sensitive neurons.
Acetylcholine, acting on presynaptic nicotinic receptors (nAChRs), modulates the release of neurotransmitters in the brain. The rat dorsal raphe nucleus (DR) and the locus coeruleus (LC) receive cholinergic input and express the alpha7nAChR. In previous reports, we demonstrated that estradiol (E) administration stimulates DR serotonergic and LC noradrenergic function in the macaque. In addition, it has been reported that E induces the expression of the alpha7nAChR in rats. We questioned whether E increased the expression of the alpha7nAChR in the macaque DR and LC. We utilized double immunostaining to study the effect of a simulated preovulatory surge of E on the expression of the alpha7nAChR in the DR and the LC and to determine whether alpha7nAChR colocalizes with serotonin and tyrosine hydroxylase (TH) in macaques. There was no difference in the number of alpha7nAChR-positive neurons between ovariectomized (OVX) controls and OVX animals treated with a silastic capsule containing E (Ecap). However, supplemental infusion of E for 5-30 hours to Ecap animals (Ecap + inf) significantly increased the number of alpha7nAChR-positive neurons in DR and LC. In addition, supplemental E infusion significantly increased the number of neurons in which alpha7nAChR colocalized with serotonin and TH. These results constitute an important antecedent for study of the effects of nicotine and ovarian steroid hormones in the physiological functions regulated by the DR and the LC in women.