First-order Neurons Research Articles

Effect of injury on S1 dorsal root ganglia in an experimental model of neuropathic faecal incontinence

An experimental model of neuropathic faecal incontinence has recently been established. This study aimed to quantify and compare the effect of crush and compression injury on first-order sensory neurones of the inferior rectal nerve (IRN) using a nuclear marker of axonal injury, activating transcription factor (ATF) 3. Eighteen Wistar rats were allocated to three groups: an unoperated control group, an IRN crush group (positive control) and a retrouterine balloon compression group. Five days after surgery, all animals were anaesthetized and perfused with fixative, and S1 dorsal root ganglia (DRG) were harvested. The tissue was sampled and neuronal nuclear ATF-3 expression calculated. Estimated total S1 DRG ATF-3 nuclear labelling was higher in the nerve crush (median (interquartile range) 171 (60-824) cells) and balloon compression (59 (20-274) cells) groups, compared with that in the unoperated control group (9 (3-24) cells) (P = 0.001 and P = 0.008 respectively). In all groups, most neurones displaying the marker of injury were of the C-fibre class. This study confirmed the presence of axonal injury in a pelvic compression model of obstetric injury. C-fibre afferent pathways appeared to be most vulnerable. Neuromodulation may function through augmentation of residual C-fibre pathways.

Activation of central trigeminovascular neurons by cortical spreading depression.

Cortical spreading depression (CSD) has long been implicated in migraine attacks that begin with visual aura. Having shown that a wave of CSD can trigger long-lasting activation of meningeal nociceptors--the first-order neurons of the trigeminovascular pathway thought to underlie migraine headache--we now report that CSD can activate central trigeminovascular neurons in the spinal trigeminal nucleus (C1-2). Stimulation of the cortex with pinprick or KCl granule was used to induce CSD in anesthetized rats. Neuronal activity was monitored in C1-2 using single-unit recording. In 25 trigeminovascular neurons activated by CSD, mean firing rate (spikes/s) increased from 3.6 ± 1.2 before CSD (baseline) to 6.1 ± 1.8 after CSD (p < 0.0001) for a period >13 minutes. Neuronal activity returned to baseline level after 30.0 ± 3.1 minutes in 14 units, and remained elevated for 66.0 ± 8.3 (22-108) minutes through the entire recording period in the other 11 units. Neuronal activation began within 0.9 ± 0.4 (0-2.5) minutes after CSD in 7 neurons located in laminae I-II, or after a latency of 25.1 ± 4.0 (7-75) minutes in 9 neurons located in laminae I-II, and 9 neurons located in laminae III-V. In 27 trigeminovascular neurons not activated by CSD, mean firing rate was 2.0 ± 0.7 at baseline and 1.8 ± 0.7 after CSD. We propose that CSD constitutes a nociceptive stimulus capable of activating peripheral and central trigeminovascular neurons that underlie the headache of migraine with aura.

Signal processing in first- and second-order vestibular neurons

This symposium was held in Anaheim California, on February 8, 2010, at the 33rd annual Midwinter Meeting of the Association for Research in Otolaryngology (ARO). The meeting program and abstracts are published online (http://aro.org/abstracts/abstracts.html). Organized by Jay Goldberg and Kenna Peusner, the symposium was directed toward clinicians who want to bridge the gap between basic science and treating patients for vestibular disorders, vestibular neuroscientists using structural and functional approaches to understand the labyrinth and its central pathways at the system, cellular, and molecular levels, and auditory neuroscientists intrigued by the similarities and differences in signal processing in the two systems. This special issue contains those presentations on signal processing in second-order vestibular neurons and their connections. The articles underwent peer-review for this special issue. We would like to thank the Journal of Vestibular Research for the production of this special issue devoted to the symposium. The vestibular system demonstrates a remarkable plasticity in response to changing environmental demands [9] and to recover function after pathologies affecting the peripheral vestibular receptors on one side [16]. Second-order vestibular neurons are characterized by a high degree of plasticity in their connections with first-order vestibular neurons and nonlabyrinthine inputs (for review, see [20]). As demonstrated in this symposium, recent research has been directed toward defining synaptic transmission and ionic conductances involved in signal processing from the vestibular periphery to the first brain centers located in the vestibular nuclei. The experiments utilize multiple structural and functional approaches performed on intact animals, isolated vestibular circuits in culture, or brain slice preparations. The article by Beraneck and Straka entitled “Vestibular Signal Processing by Separate Sets of Neuronal Filters” reviews recent research on detecting static head position and head motion by the peripheral vestibular receptors and the activation of specific receptor areas by high or low frequency movements during locomotion. Vestibular nerve afferents convey the different dynamics of head motion in a wide range of frequencies to second–order vestibular neurons, which are equipped dynamically to process the signals [18]. From the firing patterns generated by second-order vestibular nuclei neurons, the authors propose that there are two different populations of vestibular nuclei neurons that process high or low frequency signals within two separate pathways [19]. Tonic-firing vestibular nuclei neurons process low frequency signals, and are well-suited for synaptic integration, whereas phasic-firing vestibular nuclei neurons process high frequency signals and are designed to detect events. This dual system may underlie the ability of the brain to distinguish between tilt and linear translation, or self-induced motion and passive body movements (for review, see [1]). Finally, differences in the dynamic properties between frog and mammalian vestibular nuclei neurons are discussed in relation to species-specific locomotor patterns.

Multinodal regulation of the arcuate/paraventricular nucleus circuit by leptin.

Melanocortin-4 receptor (MC4R) is critical for energy homeostasis, and the paraventricular nucleus of the hypothalamus (PVN) is a key site of MC4R action. Most studies suggest that leptin regulates PVN neurons indirectly, by binding to receptors in the arcuate nucleus or ventromedial hypothalamus and regulating release of products like α-melanocyte-stimulating hormone (α-MSH), neuropeptide Y (NPY), glutamate, and GABA from first-order neurons onto the MC4R PVN cells. Here, we investigate mechanisms underlying regulation of activity of these neurons under various metabolic states by using hypothalamic slices from a transgenic MC4R-GFP mouse to record directly from MC4R neurons. First, we show that in vivo leptin levels regulate the tonic firing rate of second-order MC4R PVN neurons, with fasting increasing firing frequency in a leptin-dependent manner. We also show that, although leptin inhibits these neurons directly at the postsynaptic membrane, α-MSH and NPY potently stimulate and inhibit the cells, respectively. Thus, in contrast with the conventional model of leptin action, the primary control of MC4R PVN neurons is unlikely to be mediated by leptin action on arcuate NPY/agouti-related protein and proopiomelanocortin neurons. We also show that the activity of MC4R PVN neurons is controlled by the constitutive activity of the MC4R and that expression of the receptor mRNA and α-MSH sensitivity are both stimulated by leptin. Thus, leptin acts multinodally on arcuate nucleus/PVN circuits to regulate energy homeostasis, with prominent mechanisms involving direct control of both membrane conductances and gene expression in the MC4R PVN neuron.

TRIENNIAL GROWTH SYMPOSIUM: Neural regulation of feed intake: Modification by hormones, fasting, and disease1,2

Appetite is a complex process that results from the integration of multiple signals at the hypothalamus. The hypothalamus receives neural signals; hormonal signals such as leptin, cholecystokinin, and ghrelin; and nutrient signals such as glucose, FFA, AA, and VFA. This effect is processed by a specific sequence of neurotransmitters beginning with the arcuate nucleus and orexigenic cells containing neuropeptide Y or agouti-related protein and anorexigenic cells containing proopiomelanocortin (yielding the neurotransmitter α-melanocyte-stimulating hormone) or cells expressing cocaine amphetamine-related transcript. These so-called first-order neurons act on second-order orexigenic neurons (containing either melanin-concentrating hormone or orexin) or act on anorexigenic neurons (e.g., expressing corticotropin-releasing hormone) to alter feed intake. In addition, satiety signals from the liver and gastrointestinal tract signal through the vagus nerve to the nucleus tractus solitarius to cause meal termination, and in combination with the hypothalamus, integrate the various signals to determine the feeding response. The activities of these neuronal pathways are also influenced by numerous factors such as nutrients, fasting, and disease to modify appetite and hence affect growth and reproduction. This review will begin with the central nervous system pathways and then discuss the ways in which hormones and metabolites may alter the process to affect feed intake with emphasis on farm animals.

When does acute pain become chronic?

The transition from acute to chronic pain appears to occur in discrete pathophysiological and histopathological steps. Stimuli initiating a nociceptive response vary, but receptors and endogenous defence mechanisms in the periphery interact in a similar manner regardless of the insult. Chemical, mechanical, and thermal receptors, along with leucocytes and macrophages, determine the intensity, location, and duration of noxious events. Noxious stimuli are transduced to the dorsal horn of the spinal cord, where amino acid and peptide transmitters activate second-order neurones. Spinal neurones then transmit signals to the brain. The resultant actions by the individual involve sensory-discriminative, motivational-affective, and modulatory processes in an attempt to limit or stop the painful process. Under normal conditions, noxious stimuli diminish as healing progresses and pain sensation lessens until minimal or no pain is detected. Persistent, intense pain, however, activates secondary mechanisms both at the periphery and within the central nervous system that cause allodynia, hyperalgesia, and hyperpathia that can diminish normal functioning. These changes begin in the periphery with upregulation of cyclo-oxygenase-2 and interleukin-1β-sensitizing first-order neurones, which eventually sensitize second-order spinal neurones by activating N-methyl-d-aspartic acid channels and signalling microglia to alter neuronal cytoarchitecture. Throughout these processes, prostaglandins, endocannabinoids, ion-specific channels, and scavenger cells all play a key role in the transformation of acute to chronic pain. A better understanding of the interplay among these substances will assist in the development of agents designed to ameliorate or reverse chronic pain.

Leptin Induces Phosphorylation of Neuronal Nitric Oxide Synthase in Defined Hypothalamic Neurons

Studies have indicated that the neurotransmitter nitric oxide (NO) mediates leptin's effects in the neuroendocrine reproductive axis. However, the neurons involved in these effects and their regulation by leptin is still unknown. We aimed to determine whether NO neurons are direct targets of leptin and by which mechanisms leptin may influence neuronal NO synthase (nNOS) activity. Nicotinamide adenine dinucleotide phosphate diaphorase activity and leptin-induced phosphorylation of signal transducer and activator of transcription-3 immunoreactivity were coexpressed in subsets of neurons of the medial preoptic area, the paraventricular nucleus of the thalamus, the arcuate nucleus (Arc), the dorsomedial nucleus of the hypothalamus (DMH), the posterior hypothalamic area, the ventral premammillary nucleus (PMV), the parabrachial nucleus, and the dorsal motor nucleus of the vagus nerve. Fasting blunted nNOS mRNA expression in the medial preoptic area, Arc, DMH, PMV, and posterior hypothalamic area, and this effect was not restored by acute leptin administration. No difference in the number of neurons expressing nNOS immunoreactivity was noticed comparing hypothalamic sections of fed (wild type and ob/ob), fasted, and fasted leptin-treated mice. However, we found that in states of low leptin levels, as in fasting, or lack of leptin, as in ob/ob mice, the number of neurons expressing the phosphorylated form of nNOS is decreased in the Arc, DMH, and PMV. Notably, acute leptin administration to fasted wild-type mice restored the number of phosphorylated form of nNOS neurons to that observed in fed wild-type mice. Herein we identified the first-order neurons potentially involved in NO-mediated effects of leptin and demonstrate that leptin regulates nNOS activity predominantly through posttranslational mechanisms.

Olfactory system

The olfactory system has several unique features in terms of mechanisms of sensory transduction, relay, and central processing of information and provides a typical example of functional plasticity.1–5 Olfactory deficits are frequently found in neurodegenerative disorders, particularly Parkinson disease (PD) and Alzheimer disease (AD). The neuropathologic substrate of olfactory dysfunction in neurodegenerative disorders is incompletely elucidated and likely reflects degenerative changes at multiple levels of the olfactory system, including the olfactory epithelium, olfactory bulb, primary olfactory cortices, and their secondary targets.6,7 These issues have been extensively reviewed1–7 and only some salient points are emphasized here. ### Olfactory receptor cells and sensory transduction. The olfactory sensory neurons form clusters interposed among patches of respiratory neuroepithelium.1 These neurons are bipolar cells with a dendritic end containing 3–50 cilia that project into the overlying mucus; their unmyelinated axons form bundles that project through the cribriform plate and synapse in the olfactory bulb.1,2 Odorants bind to guanine nucleotide binding (G) protein–coupled receptors located in the cilia of the olfactory receptor neuron. There are about 400 types of receptors expressed in human olfactory neurons and an individual olfactory sensory neuron typically responds to more than one odorant type.1,5 The first step in olfactory signal transduction is the activation of the Gαolf, which stimulates adenylyl cyclase 3 and production of cyclic adenosyl monophosphate (figure). This triggers opening of a cyclic nucleotide–gated channel allowing calcium (Ca2+) influx and depolarization; subsequent opening of Ca2+ activated chloride (Cl−) channels elicits Cl− efflux that amplifies the depolarization of the sensory neuron.1 The olfactory receptor neurons have several unique features: they can regenerate; serve both as odorant-selective receptor cells and first-order neurons; and are directly exposed to the external environment, thus providing a primary route for invasion into the brain by viruses or toxins.5 …

A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits

We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions.

Polymorphisms in ion channel genes: emerging roles in pain

Why does one soldier, after sustaining a missile wound in the radial nerve on the battlefield, experience incapacitating neuropathic pain, while another, with a similar injury, complains of motor and sensory deficits in the absence of pain? Why is one patient with diabetic neuropathy disabled with neuropathic pain, while another patient with diabetic neuropathy of apparently similar severity experiences numbness or paraesthesias that are not painful? Pain is a complex phenomenon, mediated by transductive processes in the periphery, conduction to the spinal cord via first-order spinal sensory (dorsal root ganglion) neurons and processing at multiple higher levels that include the dorsal horn, thalamus and cortex. Multiple processes and molecules undoubtedly shape the perception of pain. Exciting progress, however, has recently been made in linking common variants of genes encoding two different ion channels within first-order sensory neurons along the pain pathway to inter-individual differences in pain. Discovery and characterization of these genetic variants, and of additional variants that will likely be identified in the future, open up the possibility of understanding more fully, at the molecular level, the processes that shape pain, and which may point the way to new therapeutic strategies. Ion channels play major roles in electrical signalling along the pain pathway; their central role in this process is underscored by the preferential expression within dorsal root ganglion neurons of three sodium channels, Nav1.7, Nav1.8 and Nav1.9, that are not expressed at high levels within the CNS (Cummins et al. , 2007). It is now well established that mutations of two ion channel genes can produce pain syndromes. The Nav1.7 sodium channel, which is preferentially expressed within dorsal root ganglion neurons including nocioceptors and sympathetic ganglion neurons (Djouhri et al. , 2003), displays slow inactivation in the closed-state near resting …

Neuropathic pain in diabetes—evidence for a central mechanism

Hyperexcitability of and aberrant spontaneous impulse generation by damaged first-order sensory neurons and their peripheral axons are well-established processes that strongly contribute to pain associated with diabetic neuropathy. Studies in the past 5 years, however, suggest that, as in many neuropathic pain disorders, central neuropathic mechanisms can also contribute to pain experienced with diabetes. These studies have demonstrated that thalamic dysfunction occurs in patients with diabetes mellitus, and that in experimental models of this disease neurons in the ventral posterolateral thalamus can become hyperexcitable, firing at abnormally high frequencies and generating aberrant spontaneous activity. In this article, we discuss these findings, which suggest that thalamic neurons can act as central generators or amplifiers of pain in diabetes.

Leptin: Primary Central Site of Action?

The discovery of the fat hormone leptin 16 yr ago (1) heralded an explosion in obesity research that continues to this day. The original premise that this hormone acted simply as an adipostat directing appropriate energy balance regulatory output in the brain has comprehensively unfurled to reveal a far more complex picture. Inevitably perhaps, as the complexity increases so does the controversy. In keeping with this trend, the paper by Qi et al. (2) published in the current issue of Endocrinology calls into question the primacy of the hypothalamic arcuate nucleus in the central leptin signaling pathway. The widely accepted model is that adipocyte-derived leptin acts on first-order neurons in the hypothalamic arcuate nucleus, and the message is relayed to second-order neurons in other hypothalamic regions such as the dorsomedial nucleus, ventromedial nucleus, paraventricularnucleus, and lateral hypothalamic area (3–5). From a multitude of animal experiments, it is unequivocal that the central actions of leptin are anorexigenic. Thus, hypoleptinemia is a powerful orexigenic signal during fasting, and leptin administration decreases appetite and body weight (3–5). Early experiments also reported the pivotal importance of the arcuate nucleus to this response because the anorectic effects of peripherally and centrally administered leptin were abolished in rats following monosodium glutamate lesion of the arcuate nucleus (6, 7). In addition, it has been demonstrated that reactivation of the leptin receptor specifically in the arcuate nucleus leads to decreased food intake and body weight in leptin receptordeficient mice (8) and rats (9). Clearly, receptor distribution provides a good indication of sites of action, and there is fairly universal agreement on the distribution of the signaling (long) form of the leptin receptor in the brain. Thus, it is localized in the arcuate, dorsomedial, ventromedial and paraventricular nuclei, and the lateral hypothalamic area of the adult hypothalamus, regions known to be involved in energy balance and appetite control but with the highest density in the arcuate nucleus (10). Primary targets for leptin in the arcuate nucleus have long been identified as proopiomelanocortin and neuropeptide Y neurons, which coexpress the leptin receptor and in which leptin induces phosphorylation of the signal transducer and activator of transcription 3, a marker of leptin receptor activation (3–5, 10). It follows that deletion of the leptin receptor specifically in arcuate proopiomelanocortin and neuropeptide Y neurons of mice would result in a hyperphagic and obese phenotype; however, the effects on food intake and adiposity are less extreme compared with global leptin receptor deletion, indicating that other leptin-regulated pathways are also involved (11, 12). As mentioned above, the leptin receptor shows a wider hypothalamic distribution besides the arcuate nucleus and leptin receptor deletion specifically in the ventromedial nucleus also results in increased body weight and adiposity in mice (13). Indeed, neuronal activation in many hypothalamic sites is reported in response to leptin administration in rodents (10, 14). However, it remains equivocal whether the responses of different hypothalamic nuclei to leptin are independent or orchestrated by the arcuate nucleus. Now Qi et al. (2) report novel evidence for direct action of leptin on cells in the dorsal, ventromedial, and paraventricular hypothalamic nuclei in sheep, in the presence or absence of an arcuatenucleus.Thus, they found immunostaining for fos, a marker of neuronal activation, in the dorsal, ventromedial and paraventricular hypothalamic nuclei following peripheral leptin administration in their model of hypothalamo-pituitary disconnection in which the arcuate is

The Action of Leptin on Appetite-Regulating Cells in the Ovine Hypothalamus: Demonstration of Direct Action in the Absence of the Arcuate Nucleus

It is widely accepted that leptin acts on first-order neurons in the arcuate nucleus (ARC) with information then relayed to other hypothalamic centers. However, the extent to which leptin mediates its central actions solely, or even primarily, via this route is unclear. We used a model of hypothalamo-pituitary disconnection (HPD) to determine whether leptin action on appetite-regulating systems requires the ARC. This surgical preparation eliminates the ARC. We measured effects of iv leptin to activate hypothalamic neurons (Fos labeling). In ARC-intact animals, leptin increased the percentage of Fos-positive melanocortin neurons and reduced percentages of Fos-positive neuropeptide Y neurons compared with saline-treated animals. HPD itself increased Fos labeling in the lateral hypothalamic area (LHA). Leptin influenced Fos labeling in the dorsomedial nucleus (DMH), ventromedial nucleus, and paraventricular nucleus (PVN) in HPD and normal animals, with effects on particular cell types varying. In the LHA and DMH, leptin decreased orexin cell activation in HPD and ARC-intact sheep. HPD abolished leptin-induced expression of Fos in melanin-concentrating hormone cells in the LHA and in CRH cells in the PVN. In contrast, HPD accentuated activation in oxytocin neurons. Our data from sheep with lesions encompassing the ARC do not suggest a primacy of action of leptin in this nucleus. We demonstrate that first order to second order signaling may not represent the predominant means by which leptin acts in the brain to generate integrated responses. We provide evidence that leptin exerts direct action on cells of the DMH, ventromedial nucleus, and PVN.

Tooth pulp inflammation increases brain-derived neurotrophic factor expression in rodent trigeminal ganglion neurons

Nociceptive pathways with first-order neurons located in the trigeminal ganglion (TG) provide sensory innervation to the head, and are responsible for a number of common chronic pain conditions, including migraines, temporomandibular disorders and trigeminal neuralgias. Many of those conditions are associated with inflammation. Yet, the mechanisms of chronic inflammatory pain remain poorly understood. Our previous studies show that the neurotrophin brain-derived neurotrophic factor (BDNF) is expressed by adult rat TG neurons, and released from cultured newborn rat TG neurons by electrical stimulation and calcitonin gene-related peptide (CGRP), a well-established mediator of trigeminal inflammatory pain. These data suggest that BDNF plays a role in activity-dependent plasticity at first-order trigeminal synapses, including functional changes that take place in trigeminal nociceptive pathways during chronic inflammation. The present study was designed to determine the effects of peripheral inflammation, using tooth pulp inflammation as a model, on regulation of BDNF expression in TG neurons of juvenile rats and mice. Cavities were prepared in right-side maxillary first and second molars of 4-week-old animals, and left open to oral microflora. BDNF expression in right TG was compared with contralateral TG of the same animal, and with right TG of sham-operated controls, 7 and 28 days after cavity preparation. Our ELISA data indicate that exposing the tooth pulp for 28 days, with confirmed inflammation, leads to a significant upregulation of BDNF in the TG ipsilateral to the affected teeth. Double-immunohistochemistry with antibodies against BDNF combined with one of nociceptor markers, CGRP or transient receptor potential vanilloid type 1 (TRPV1), revealed that BDNF is significantly upregulated in TRPV1-immunoreactive (IR) neurons in both rats and mice, and CGRP-IR neurons in mice, but not rats. Overall, the inflammation-induced upregulation of BDNF is stronger in mice compared to rats. Thus, mouse TG provides a suitable model to study molecular mechanisms of inflammation-dependent regulation of BDNF expression in vivo.

A new role for photoresponsive neurons called simple photoreceptors in the sea slug Onchidium verruculatum: Potentiation of synaptic transmission and motor response

The simple photoreceptors Ip-1 and Ip-2 are intrinsically light-sensitive neurons that exist in the abdominal ganglion of the sea slug Onchidium verruculatum. Using isolated ganglia and semi-intact or intact animal preparations, the present studies examined the light-sensing and physiological roles of Ip-1 and Ip-2 cells, which respond jointly to light by inducing a slow hyperpolarizing receptor potential. First, the synaptic inputs received by Ip-1 and Ip-2 and the axonal branches arising from their cell bodies were investigated. We found that these cells are not only first-order photosensory neurons, but also second-order neurons (interneurons), relaying inhibitory synaptic inputs such as water pressure and/or tactile senses. The amphibian Onchidium opens a pneumostome at low tide in order to aero-breathe. This pneumostome opening; i.e., aero-breathing behavior, was produced by spike discharges of Ip-1 and Ip-2 cells. Furthermore, the present results suggested that the hyperpolarizing photoresponse of Ip-1 and Ip-2 cells operates in the potentiation of inhibitory sensory synaptic transmission. Thus, we conclude that the simple photoreceptors of Onchidium play a role in the long-lasting potentiation of synaptic transmission and the subsequent behavioral response and so may be involved in a new photosensory modality, non-image-forming vision.

The added value of rabies virus as a retrograde tracer when combined with dual anterograde tract-tracing

Rabies virus (RV) has widely been used as a trans-synaptic retrograde tracer to analyze chains of connected neurons. The use of antibodies directed against the viral nucleoprotein enables viral nucleocapsids to be visualized within the cell soma, as well as within the thickest main dendrites. However, through this approach it is often difficult to accurately define post-synaptic elements (thin dendrites and/or dendritic spines). This limitation can now easily been circumvented by taking advantage of antibodies directed against a soluble viral phosphoprotein that spreads throughout the cytoplasm of the infected neuron, thereby producing Golgi-like immunofluorescent labeling of first-order projection neurons that are infected with RV. Furthermore, when combined with anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), this procedure to detect RV facilitates the accurate visualization of both the pre- and post-synaptic elements. Finally, this method of viral detection is sufficiently sensitive to detect weakly labeled second-order neurons, which can then be further characterized neurochemically. Several examples are provided to illustrate why retrograde trans-synaptic tracing using RV can be regarded as an important breakthrough in the analysis of brain circuits, providing an unprecedented level of resolution.

Role of the trigeminal mesencephalic nucleus in rat whisker pad proprioception

BackgroundTrigeminal proprioception related to rodent macrovibrissae movements is believed to involve skin receptors on the whisker pad because pad muscles operate without muscle spindles. This study was aimed to investigate in rats whether the trigeminal mesencephalic nucleus (TMnu), which provides proprioceptive feedback for chewing muscles, may be also involved in whisker pad proprioception.MethodsTwo retrograde tracers, Dil and True Blue Chloride, were injected into the mystacial pad and the masseter muscle on the same side of deeply anesthetized rats to label the respective projecting sensory neurons. This double-labeling technique was used to assess the co-innervation of both structures by the trigeminal mesencephalic nucleus (TMnu).In a separate group of anesthetized animals, the spontaneous electrical activities of TMnu neurons were analyzed by extracellular recordings during spontaneous movements of the macrovibrissae. Mesencephalic neurons (TMne) were previously identified by their responses to masseter muscle stretching. Changes in TMne spontaneous electrical activities, analyzed under baseline conditions and during whisking movements, were statistically evaluated using Student's t-test for paired observations.ResultsNeuroanatomical experiments revealed different subpopulations of trigeminal mesencephalic neurons: i) those innervating the neuromuscular spindles of the masseter muscle, ii) those innervating the mystacial pad, and iii) those innervating both structures. Extracellular recordings made during spontaneous movements of the macrovibrisae showed that whisking neurons similar to those observed in the trigeminal ganglion were located in the TMnu. These neurons had different patterns of activation, which were dependent on the type of spontaneous macrovibrissae movement. In particular, their spiking activity tonically increased during fan-like movements of the vibrissae and showed phasic bursting during rhythmic whisking. Furthermore, the same neurons may also respond to masseter muscle stretch.Conclusionsresults strongly support the hypothesis that the TMnu also contains first-order neurons specialized for relaying spatial information related to whisker movement and location to trigeminal-cortical pathways. In fact, the TMnu projects to second-order trigeminal neurons, thus allowing the rat brain to deduce higher-order information regarding executed movements of the vibrissae by combining touch information carried by trigeminal ganglion neurons with proprioceptive information carried by mesencephalic neurons.

Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map

Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at ∼50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a 3-dimensional discrete neural map are unclear. Here we show that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) is differentially expressed in different classes of PNs. Loss- and gain-of-function studies indicate that Caps instructs the segregation of Caps-positive and negative PN dendrites to discrete glomerular targets. Moreover, Caps does not mediate homophilic interactions and regulates PN dendrite targeting independent of pre-synaptic ORNs. The closely related protein Tartan plays a partially redundant function with Capricious. These LRR proteins are likely part of a combinatorial cell-surface code that instructs discrete olfactory map formation.

Peripheral inflammation regulates BDNF expression in rat trigeminal ganglion neurons

The trigeminal system, with first-order neurons in the trigeminal ganglion (TG), provides sensory innervation to the majority of craniofacial tissues. Many peripheral targets of TG neurons constitute the source of common chronic pain conditions, such as trigeminal neuralgias, migraine headaches, and temporomandibular disorders. Although it is well established that chronic trigeminal pain is frequently associated with inflammation of peripheral endings of TG neurons, the exact molecular mechanisms of trigeminal nociceptive transmission are poorly understood. The goal of the present study was to examine the effects of peripheral inflammation in the trigeminal system on regulation of the neurotrophin brain-derived neurotrophic factor (BDNF) in TG neurons in vivo, and to begin investigating the underlying molecular mechanisms using an in vitro model of trigeminal inflammation. In our studies in vivo, we used a rat model of tooth pulp inflammation. Cavities were prepared in rightside maxillary molars of 4-week-old animals. BDNF expression in right TG was compared with contralateral TG of the same animal and with right TG of shamoperated controls, 7 and 28 days after cavity preparation. Using ELISA, we determined that BDNF levels were significantly upregulated in right, compared to left, TG. Double BDNF/TrpV1 and BDNF/CGRP immunohistochemistry revealed that inflammation recruits new TrpV1but not CGRP-immunoreactive cells to express BDNF. In the in vitro portion of the project, newborn rat TG neurons were grown under neuron-enriched conditions for 3–5 days, followed by treatment with various pro-inflammatory cytokines, including tumor necrosis factor (TNF ), interleukin-1 (IL-1 ), and interleukin-6 (IL-6). The cytokine treatment (4 and 24 hours) was combined with patterned electrical field stimulation to mimic activity of trigeminal neurons in vivo. At the end of the treatment period, cultures were processed for either quantitative PCR or ELISA to evaluate changes in BDNF mRNA and protein, respectively. Our results indicate that TNF and IL-1 regulate BDNF expression in a dose-dependent manner, and the effects are dramatically enhanced in the presence of electrical stimulation. In view of the previous evidence that BDNF is expressed at trigeminal nociceptive synapses in the brainstem, our current data point to BDNF as a likely key player of trigeminal inflammatory pain.

Mechanisms of Tactile Information Transmission through Whisker Vibrations

In their natural environment, rodents use their whiskers to locate and distinguish between objects of different textures and shapes. They do so by moving their whiskers actively as well as passively, through body and head movements. To determine the mechanisms by which surface coarseness is translated into neuronal discharges through passive whisker movements, we monitored head movements of awake behaving rats while approaching objects. We then replayed these movements in anesthetized rats, monitored the whiskers' movements across various surfaces, and concurrently recorded the activity of first-order sensory neurons. We found that whiskers, being the first stage of sensory information translation, shape transduction by amplifying small-amplitude high-frequency signals. Thus, surface coarseness is transmitted through high-velocity micromotions. Consistent with this, we find that during surface contact, discrete high-velocity movements, or stick-slip events, evoke first-order neuronal discharge. Transient ringing in whiskers, which primarily represents resonance vibrations, follows these events, but seldom causes neurons to discharge. These sensory transformations are influenced by the whiskers' biomechanical properties. To determine the resemblance of these tactile transformations during passive whisker movements and active whisking, we induced artificial whisking across various surface textures. We found that the processes by which tactile information becomes available to the animal are similar for these different modes of behavior. Together, these findings indicate that the temporal bandpass properties for spike generation in first-order neurons are matched by the biomechanical characteristics of whiskers, which translate surface coarseness into high-frequency whisker micromotions. These properties enable rodents to acquire tactile information through passive and active movements of their whiskers.