Retrograde Labeling Of Neurons Research Articles

Spontaneous development of full weight-supported stepping after complete spinal cord transection in the neonatal opossum, Monodelphis domestica.

Spinal cord trauma in the adult nervous system usually results in permanent loss of function below the injury level. The immature spinal cord has greater capacity for repair and can develop considerable functionality by adulthood. This study used the marsupial laboratory opossum Monodelphis domestica, which is born at a very early stage of neural development. Complete spinal cord transection was made in the lower-thoracic region of pups at postnatal-day 7 (P7) or P28, and the animals grew to adulthood. Injury at P7 resulted in a dense neuronal tissue bridge that connected the two ends of the cord; retrograde neuronal labelling indicated that supraspinal and propriospinal innervation spanned the injury site. This repair was associated with pronounced behavioural recovery, coordinated gait and an ability to use hindlimbs when swimming. Injury at P28 resulted in a cyst-like cavity encased in scar tissue forming at the injury site. Using retrograde labelling, no labelled brainstem or propriospinal neurons were found above the lesion, indicating that detectable neuronal connectivity had not spanned the injury site. However, these animals could use their hindlimbs to take weight-supporting steps but could not use their hindlimbs when swimming. White matter, demonstrated by Luxol Fast Blue staining, was present in the injury site of P7- but not P28-injured animals. Overall, these studies demonstrated that provided spinal injury occurs early in development, regrowth of supraspinal innervation is possible. This repair appears to lead to improved functional outcomes. At older ages, even without detectable axonal growth spanning the injury site, substantial development of locomotion was still possible. This outcome is discussed in conjunction with preliminary findings of differences in the local propriospinal circuits following spinal cord injury (demonstrated with fluororuby labelling), which may underlie the weight bearing locomotion observed in the apparent absence of axons bridging the lesion site in P28-injured Monodelphis.

A trigeminoreticular pathway: implications in pain.

Neurons in the caudalmost ventrolateral medulla (cmVLM) respond to noxious stimulation. We previously have shown most efferent projections from this locus project to areas implicated either in the processing or modulation of pain. Here we show the cmVLM of the rat receives projections from superficial laminae of the medullary dorsal horn (MDH) and has neurons activated with capsaicin injections into the temporalis muscle. Injections of either biotinylated dextran amine (BDA) into the MDH or fluorogold (FG)/fluorescent microbeads into the cmVLM showed projections from lamina I and II of the MDH to the cmVLM. Morphometric analysis showed the retrogradely-labeled neurons were small (area 88.7 µm2±3.4) and mostly fusiform in shape. Injections (20–50 µl) of 0.5% capsaicin into the temporalis muscle and subsequent immunohistochemistry for c-Fos showed nuclei labeled in the dorsomedial trigeminocervical complex (TCC), the cmVLM, the lateral medulla, and the internal lateral subnucleus of the parabrachial complex (PBil). Additional labeling with c-Fos was seen in the subnucleus interpolaris of the spinal trigeminal nucleus, the rostral ventrolateral medulla, the superior salivatory nucleus, the rostral ventromedial medulla, and the A1, A5, A7 and subcoeruleus catecholamine areas. Injections of FG into the PBil produced robust label in the lateral medulla and cmVLM while injections of BDA into the lateral medulla showed projections to the PBil. Immunohistochemical experiments to antibodies against substance P, the substance P receptor (NK1), calcitonin gene regulating peptide, leucine enkephalin, VRL1 (TPRV2) receptors and neuropeptide Y showed that these peptides/receptors densely stained the cmVLM. We suggest the MDH- cmVLM projection is important for pain from head and neck areas. We offer a potential new pathway for regulating deep pain via the neurons of the TCC, the cmVLM, the lateral medulla, and the PBil and propose these areas compose a trigeminoreticular pathway, possibly the trigeminal homologue of the spinoreticulothalamic pathway.

Systemic administration of fluorogold for anatomical pre‐labeling of autonomic and motor neurons in the rat spinal cord compromises urodynamic recordings in acute but not long‐term studies

The use of anatomical tracer injections into peripheral tissues for retrograde labeling of spinal cord neurons may compromise physiological experiments in combined functional and morphological studies. We investigated whether a systemic injection of a retrogradely transported tracer, fluorogold (FG), may provide an alternative to direct injections into end organs for combined anatomical and physiological studies of the lower urinary tract. Urodynamic studies including cystometrogram recordings and external urethral sphincter electromyography were used as functional outcome measures. Pre-labeling of spinal cord neurons by intraperitoneal (i.p.) administration of FG resulted in a transient decrease in voiding efficiency, increase in resting pressure as well as increase in bladder size and weight at 5-7 days after the tracer administration. In contrast, there were no urodynamic or end-organ effects detected at 6-8 weeks after the i.p. injection of FG. We suggest that pre-labeling of spinal autonomic and motor neurons using i.p. administration of FG may be a useful tool when combining anatomical and functional outcome measures in long-term but not acute studies.

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static or record extracellular neuronal activity during a movement. While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

Hindbrain neurovascular architecture in zebrafish and goldfish

Vascular dye injections and confocal microscopy, combined with transgenic or retrograde neuronal labeling, were used to document the hindbrain neurovascular architecture in larval and adult cyprinid fish (zebrafish & goldfish). The data provide initial assessments of the neurovascular territories of central arterial stems and the specific blood supplies to particular hindbrain nuclei. The hindbrain central arteries arose from the basilar artery as a segmental series of 8 to 11 unpaired vessels that penetrated the pial midline of each rhombomere (r). The arteries branched into vascular trees within the hindbrain neuroepithelium near the ventricular surface, continued branching towards the pial surface and ended as small, unbranched channels that traversed nuclei and fiber tracts. These capillaries then joined to form tributary vessels of a large pial venous plexus. Notably, the neurovascular territories supplied by central arterial stems in r3 and r8 were by far the largest. The r3 stem served trigeminal motor nuclei in r2–3, as well as migrated facial motoneurons in r6–7 via long descending branches. A single large stem in r8 served most of the vagal lobe, with stems in r4–7 and caudal r8 supplying smaller zones. The data showed that hindbrain vascular and CNS elements are patterned in relation to the early rhombomeric segmental framework, with aspects of the segmental plan being maintained in adult brains.Grant Funding Source: None

Advances in genomic study of cortical projection neurons

The mammalian neocortex gives rise to perception and initiates voluntary motor responses. The cortical laminae are comprised of six distinct cellular layers of local circuit neurons and projection neurons. To explore molecular identities of the distinct cortical projection neurons, discovery-orientated genomic approaches have been adopted. Microarray analysis of dissected cortical tissues has been applied to identify cortical layer markers. Early neuronal cells were sorted by FACS from GFPlabeled embryonic brains for gene expression profiling. Laser capture microdissection of retrograde-labeled projection neurons, when coupled with optimal RNA amplification technology, has become a valuable strategy for neuronal isolation and gene expression analysis in differentiated neurons. RNA sequencing technology is promising not only for the determination of gene expression, but also for discovery of posttranscriptional modifications of the complex neural system. There is no doubt that advances in genomic studies are opening up novel research avenues for our understanding of the cortical neuronal functions.

Efficacy of fluorescent tracers in retrograde labeling of cutaneous afferent neurons in the rat

Background In the present study, the labeling efficacy of tracers Fluoro-ruby (FR), Fluoro-emerald (FE), True Blue (TB), Fluoro-Gold (FG), Diamidino Yellow (DY) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) to retrogradely label the cutaneous afferent neurons in the rat was examined. Methods The proximal stump of the transected sural nerve was exposed for 1 hour either to one of the examined dyes (FR, FE, TB, FG, DY and DiI group) in single labeling experiments, or to mixtures of two dyes (TB-FG, FG-DiI, TB-DY and TB-DiI group) in double labeling experiments ( n = 5 for each group). After 10 days, dorsal root ganglia (DRGs) L3-S1 were harvested, cut to 20 μm thick longitudinal sections and all labeled neurons were counted. Results The average numbers of labeled DRG neurons in FR group (1063 ± 158; mean ± SD) and FE group (1067 ± 203) were statistically significantly lower than those in TB group (2831 ± 379), FG group (2802 ± 134), DY group (2888 ± 262) or DiI group (2900 ± 278) ( p < 0.05). In double labeling experiments, the average number of double labeled neurons in TB-DY group (2208 ± 207) was statistically significantly lower than those in TB-FG group (2775 ± 316), FG-DiI group (2921 ± 419), or TB-DiI group (2805 ± 179) ( p < 0.05). Conclusions Among examined tracers, TB, FG, DY and DiI, have the highest and similar labeling efficacy for retrograde labeling of cutaneous afferent neurons in the rat. The tracers TB, DiI and FG effectively label the same neuronal population in double labeling, therefore, their combinations are most suitable for double retrograde labeling studies of cutaneous afferent neurons in the rat.

Re-evaluation of spontaneous regeneration of the lateral olfactory tract

Spontaneous regeneration of the lateral olfactory tract (LOT) was re-evaluated in newborn rats using a fluorescent retrograde neuronal tracer as objective indicators of complete LOT transection. Complete LOT transection was evidenced by the loss of the white myelinated band characteristic for adult LOT and the total lack of retrograde neuronal labeling of mitral cells by Fast Blue that was injected during LOT transection. In completely LOT-transected young adult rats, mitral cells were retrogradely labeled consistently only by Fluoro-Gold that was injected into the olfactory cortex at the adult stage. Moreover, an anterograde neuronal tracer, biotinylated dextran amine (BDA), was demonstrated to pass from the neonatally LOT-transected bulb, through the transected retrobulbar site, towards the olfactory cortex, far caudally at a level near the optic chiasm. The regenerated structures lacked immunoreactivity for myelin basic protein and electron-dense myelinated axon bundles, and were also characterized by the thinness of the BDA(+) terminal zone within the olfactory cortex and the lack of its caudal extension. Young adult rats subjected to unilateral bulbectomy contralateral to the neonatally LOT-transected side showed perfect ability to discriminate cycloheximide solution by olfaction. From these findings, we conclude that the spontaneously regenerated olfactory system is functional despite structural incompleteness.

Ultrastructural analysis of the dorsal body gland of the terrestrial snail Megalobulimus abbreviatus (Becquaert, 1948)

The ultrastructure of the reproductive gland, dorsal body (DB), of Megalobulimus abbreviatus was analysed. Electron microscope immunohistochemistry was used to detect FMRFamide-like peptides in the nerve endings within this gland. Nerve backfilling was used in an attempt to identify the neurons involved in this innervation. In M. abbreviatus, the DB has a uniform appearance throughout their supraesophageal and subesophageal portions. Dorsal body cells have several features in common with steroid-secreting gland cells, such as the presence of many lipid droplets, numerous mitochondria with tubular cristae and a developed smooth endoplasmic reticulum cisternae. Throughout the DB in M. abbreviatus numerous axonal endings were seen to be in contact with the DB cells exhibiting a synaptic-like structure. The axon terminals contained numerous electron-dense and scanty electron-lucid vesicles. In addition, the DB nerve endings exhibited FMRFamide immunoreactive vesicles. Injection of neural tracer into the DB yielded retrograde labelling of neurons in the metacerebrum lobe of the cerebral ganglia and in the parietal ganglia of the subesophageal ganglia complex. The possibility that some of these retrograde-labelled neurons might be FMRFamide-like neurons that may represent a neural control to the DB in M. abbreviatus is discussed.

1002 ACUTE INFLAMMATION IN THE PELVIS MODULATES THE EXCITABILITY OF BLADDER EXTRINSIC SENSORY NEURONS VIA ACTIVATION OF VOLTAGE GATED SODIUM CHANNELS AND NON-SELECTIVE CATION CHANNELS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Neurogenic Voiding Dysfunction1 Apr 20101002 ACUTE INFLAMMATION IN THE PELVIS MODULATES THE EXCITABILITY OF BLADDER EXTRINSIC SENSORY NEURONS VIA ACTIVATION OF VOLTAGE GATED SODIUM CHANNELS AND NON-SELECTIVE CATION CHANNELS Qi Lei, Xiao-Qing Pan, Alan Wein, and Anna Malykhina Qi LeiQi Lei More articles by this author , Xiao-Qing PanXiao-Qing Pan More articles by this author , Alan WeinAlan Wein More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.2016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chronic pelvic pain is a symptom of many functional pelvic disorders and develops as a result of cross-sensitization in the pelvis. Our recent studies demonstrated increased excitability of convergent dorsal root ganglion neurons receiving input from the distal colon and urinary bladder. The objective of this study was to clarify the mechanisms modulating the expression of voltage gated sodium (Na+) channels in bladder sensory neurons after acute inflammation in the pelvis. METHODS Sprague-Dawley male rats underwent survival surgeries for retrograde labeling of sensory neurons using Fast Blue (1.5 % w/v in water, 20-22 μl). Na+ channels were recorded in lumbosacral (L6-S2) dorsal root ganglia (DRG) neurons isolated from 4 groups of animals: control, resiniferatoxin (RTX) instillation in the colon, trinitrobenzene sulfonic acid (TNBS) induced experimental colitis and double treatment (RTX+TNBS). Animals were sacrificed 3 days after the last treatment. RESULTS Acute colonic inflammation increased the peak amplitude of total Na+ current in bladder DRG neurons by 2-fold at -30 mV (-102.7±41.0 pA/pF in control group vs -229.3±18.1 pA/pF in experimental group, p≤0.05). Activation of TRPV1 receptors in the colon had similar effects on the amplitude of total Na+ current in bladder neurons as in the colitis group. However, colonic application of RTX followed by TNBS-induced colitis significantly enhanced Na+ current in bladder DRG neurons from -102.7±41.0 pA/pF to – 364.6±60.1 pA/pF (n=12, p≤0.05). Acute colitis did not change voltage dependence of steady-state activation nor inactivation in bladder sensory neurons. However, these parameters were affected by RTX treatment. Thus, V1/2 of activation curve of total Na+ current after RTX application had a leftward shift from -27.80±4.78 mV in the control group to -42.20±1.75 mV in RTX group (n=10, p≤0.05) whereas inactivation curve was shifted from -37.75±3.64 mV to -42.94±5.80 mV. Pretreatment with RTX followed by experimental colitis did not modulate the activation kinetics of Na+ current in bladder DRG neurons but caused a leftward shift in inactivation curve from -37.75±3.64 mV to -47.62±1.5 mV (n=11, p≤0.05). CONCLUSIONS Our data provide evidence that acute experimental colitis can modify the expression of Na+ channels in bladder extrinsic sensory neurons via TRPV1 signaling pathways. This data can provide a basis for the development of new therapeutic protocols to treat chronic pelvic pain of genitourinary origin. Glenolden, PA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e389-e390 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qi Lei More articles by this author Xiao-Qing Pan More articles by this author Alan Wein More articles by this author Anna Malykhina More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Application of Gold Nanoprobes Using an In Vitro Tissue Slice Preparation for the Phenotypical Classification, Connectivity Assessment, and Electrophysiological Recording of Hypothalamic Thermoregulatory Neurons

Thermoregulatory neurons in the anterior hypothalamus form synaptic networks, which affect responses that regulate body temperature. In order to characterize these pathways of activation, connections to efferent control areas, like the dorsomedial hypothalamus or the raphe pallidus, require identification. Traditional retrograde labeling using fluorescent dyes may label neurons with axon terminals near an injection site, but also label passing axon fibers. Here, we describe a novel methodology for retrograde neuronal labeling in live tissue slices, by using nanometer‐sized, gold nanoprobes, which allow for specific neuronal entry, via axon terminals in close proximity to their injection site. Upon entry, these nanoprobe complexes diffuse to the soma, where they are readily visualized and quantified. Depending on nanoprobe specifications, this method is highly specific for particular neuronal populations, thereby enabling targeted electrophysiological recording.

Invitro application of gold nanoprobes in live neurons for phenotypical classification, connectivity assessment, and electrophysiological recording

Thermoregulatory neurons in the preoptic area of the anterior hypothalamus (POA) form synaptic networks, which affect responses that regulate body temperature. To characterize these pathways of activation, projections to effector control areas, like the dorsomedial hypothalamus (DMH), require labeling in live tissue slices. Traditional fluorescent dyes label axon terminals near an injection site, but unfortunately, also that of nearby fibers of passage. Here, we describe a novel methodology for retrograde labeling of neurons in vitro, which will allow for further electrophysiological recording. To determine if POA neurons project to the DMH, we have used nanometer-sized, gold nanoprobes, which provide for specific neuronal entry, via synapses in close proximity to the injection site. Upon neuronal entry, these nanoprobe complexes diffuse to the soma, where they are readily visualized and quantified. We found that conjugation of these gold nanoprobes with VGLUT-2 antibodies and polyethyleneimine (PEI) facilitates neuronal entry and a high level of labeling efficacy. This novel method, adapted from emerging cancer therapy technologies, is highly specific for determining axon terminal projections within particular neuronal populations, while maintaining neuronal viability for targeted live cell electrophysiological recording.

Alterations in the vascular architecture of the dorsal root ganglia in a rat neuropathic pain model

An alteration in the structural arrangement of blood vessels identified by RECA immunohistochemistry was studied in a rat L4 dorsal root ganglia (L4-DRG) neuropathic pain model. We compared a three-dimensional (3-D) reconstruction of the vascular architecture surrounding bodies of the primary sensory neurons in the L4-DRG of naïve rats with that of rats that had surgically undergone unilateral sciatic nerve ligature. Rhodamine-conjugated dextran (Fluoro-Ruby) was used for retrograde labelling of neurons, the axons of which had been injured by nerve ligature. In contrast to DRG from naïve rats and contralateral DRG from operated rats, an increased proportion of RECA+ vascular area and the appearance of nest-like arrangements of blood vessels around neuronal bodies with injured axons were observed in L4-DRG ipsilateral to the sciatic nerve ligature. Fractal analysis confirmed a higher degree of vascular branching, irregularity, and tortuosity in L4-DRG related with sciatic nerve injury. The results suggest that nerve injury induces changes in vascular architecture in associated DRG.

Tecto-reticulospinal pathway (TRS)와 dorsal lateral geniculate nucleus (dLGN)에서 역행성이동추적물질 이용 햄스터 상구에서 GluR1-, GluR4- 면역반응 신경세포 연구

본 연구자들의 앞선 연구에서는 햄스터 상구에서 AMPA 수용체의 아형인 GluR1과 GluR4의 분포와 눈 적출 이후 이들 수용체의 분포를 면역세포화학적 방법으로 연구하였다. 또한, 표지한 GluR1과 GluR4를 칼슘결합단백질인 calbindin D28K, calretinin, parvalbumin과 GABA로 표지하여 비교하였다. 본 연구에서 역행성이동추적물질(retrograde tracer)인 호스래디시퍼옥시다아제(horseradish peroxidase, HRP)를 상구의 각 주요 상행로와 하행로에 주입함으로써 GluR1- 면역반응 신경세포들과 GluR4- 면역반응 신경세포들이 투사신경세포(projection neurons)임을 밝혀내었다. Tecto-reticulospinal pathway (TRS)와 dorsal lateral geniculate nucleus (dLGN)으로 HRP 를 주입한후, 햄스터들은 회복을 위해 48시간 동안 살려둔 뒤 관류(perfusion)하였다. GluR- 면역반응 처리된 절편들은 역행성 표지된 신경세포를 지님을 확인하였다. HRP를 주입하였더니 단지 적은 수의 GluR1- 면역반응 신경세포들이 TRS (1.4%)와 dLGN (2.6%)으로 투사되었고, 반면에 많은 수의 GluR4- 면역반응 신경세포들이 TRS (32.7%)로 투사되었다. 사이/투사 신경세포(inter/projection neurons)들은 GluR 아단위들의 분류된 분포와 차별화된 양상을 보였고 이들의 이러한 분포는 칼슘결합단백질들과 GABA와는 겹쳐지지 않았으며, 일전에 발표했던 시각적 행동 반응에서 안구 적출 후 수용체 아단위들의 기능적 다양화와는 차별화된 양상을 보였다. We recently reported the distributions of AMPA (<TEX>$\alpha$</TEX>-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptor subtypes glutamate receptors (GluR) 1 and GluR4 in the superior colliculi (SC) of hamsters with antibody immunocytochemistry and the effect of enucleation on these distributions. We also compared these labelings to those of calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. In the present study, we investigated whether the GluR1- and GluR4-immunoreactive (IR) neurons are interneurons or projection neurons by injection of the retrograde tracer horseradish peroxidase (HRP) into one of each major ascending and descending pathways of the SC. HRP injections were made into a tecto-reticulospinal pathway (TRS) and dorsal lateral geniculate nucleus (dLGN). Animals were then allowed to recover and to survive for 48 hr before perfusion. Sections containing retrograde-labeled neurons were then treated for GluR-immunoreactivity. HRP injections proved that only a small population of the GluR1-IR cells project into TRS (1.4%) and dLGN (2.6%). However, a large subpopulation of GluR4-IR cells project into TRS (32.7%). The differential compositions of inter/projection neurons, along with our previous studies on the separate distribution of the GluR subunits, its differential co-localization with calcium-binding proteins and GABA, and differential reactions to enucleations, strongly imply the functional variety of the receptor subunits in visual behavior responses.

Distinct startle responses are associated with neuroanatomical differences in pufferfishes

Despite the key function of the Mauthner cells (M-cells) in initiating escape responses and thereby promoting survival, there are multiple examples of M-cell loss across the teleost phylogeny. Only a few studies have directly considered the behavioral consequences of naturally occurring M-cell variation across species. We chose to examine this issue in pufferfishes, as previous research suggested that there might be variability in M-cell anatomy in this group of fish. We characterized the M-cell anatomy and fast-start responses of two pufferfish species, Tetraodon nigroviridis and Diodon holocanthus. T. nigroviridis showed robust fast-starts to both tactile and acoustic startling stimuli. These fast-starts occurred with a latency typical of M-cell initiation in other fish, and retrograde labeling of spinal-projection neurons revealed that T. nigroviridis does have M-cells. By contrast, D. holocanthus only rarely exhibited fast-start-like behavior, and these responses were at a substantially longer latency and were much less extensive than those of T. nigroviridis. Using three complementary anatomical techniques we were unable to identify obvious M-cell candidates in D. holocanthus. These results provide a clear correlation between M-cell presence or absence and dramatic differences in fast-start behavior. The rich diversity within the pufferfish clade should allow future studies investigating the factors that contribute to this correlated anatomical and behavioral variation.

Neuroanatomical tracing combined with in situ hybridization: Analysis of gene expression patterns within brain circuits of interest

Most of our current understanding of brain circuits is based on hodological studies carried out using neuroanatomical tract-tracing. Our aim is to advance one step further by visualizing the functional correlate in a given circuit. In this regard, we believe it is feasible to combine retrograde tracing with fluorescence, non-radioactive in situ hybridization (ISH) protocols. The subsequent detection at the single-cell level of the expression of a given mRNA within retrograde-labeled neurons provides information regarding cellular function. This may be of particular interest when trying to elucidate the performance of brain circuits of interest in animal models of brain diseases. Several combinations of retrograde tracing with either single- and double-ISH are presented here, together with some criteria that influence the selection of the tracer to be used in conjunction with the strong demands of the ISH.

Chronic morphine treatment induces functional delta-opioid receptors in amygdala neurons that project to periaqueductal grey

Chronic morphine treatment and persistent pain stimuli trigger translocation of delta-opioid receptors (DORs) from cytosolic pools to the surface membrane. Previously, we reported that chronic treatment with morphine induces functional DORs on GABAergic nerve terminals impinging on some neurons in the midbrain periaqueductal grey. In the present investigation, we used chronic administration of morphine in adult rats to study delta and mu-opioid receptors in the central nucleus of amygdala (CeA), a brain region with a substantial (presumed) GABAergic projection to the periaqueductal grey. Chronic morphine treatment increased the proportion of neurons displaying an increased potassium conductance in response to a selective DOR-agonist. There was a corresponding reduction in responsiveness of CeA neurons to a selective mu-opioid agonist. By combining retrograde labelling and live cell recording of CeA-periaqueductal grey projection neurons, we found nearly all (6/7 or 86%) projection neurons responded to delta agonist after chronic treatment with morphine versus only 2/7 neurons (29%) from vehicle-treated animals. Other physiological properties of amygdala neurons did not differ between neurons from vehicle and morphine-treated animals. Taken together, these results indicate that chronic treatment with morphine upregulates functional DORs in neurons projecting from the CeA to periaqueductal grey. CeA-periaqueductal grey projections form part of the descending antinociceptive and autonomic control systems suggesting an upregulation of functional DOR in antinociception, emotion and anxiety following chronic morphine treatment.

Mosaic hoxb4a Neuronal Pleiotropism in Zebrafish Caudal Hindbrain

To better understand how individual genes and experience influence behavior, the role of a single homeotic unit, hoxb4a, was comprehensively analyzed in vivo by clonal and retrograde fluorescent labeling of caudal hindbrain neurons in a zebrafish enhancer-trap YFP line. A quantitative spatiotemporal neuronal atlas showed hoxb4a activity to be highly variable and mosaic in rhombomere 7–8 reticular, motoneuronal and precerebellar nuclei with expression decreasing differentially in all subgroups through juvenile stages. The extensive Hox mosaicism and widespread pleiotropism demonstrate that the same transcriptional protein plays a role in the development of circuits that drive behaviors from autonomic through motor function including cerebellar regulation. We propose that the continuous presence of hoxb4a positive neurons may provide a developmental plasticity for behavior-specific circuits to accommodate experience- and growth-related changes. Hence, the ubiquitous hoxb4a pleitropism and modularity likely offer an adaptable transcriptional element for circuit modification during both growth and evolution.

Characterization of sheep ( Ovis aries) palatine tonsil innervation

Palatine tonsils (PTs), together with ileal Peyer's patches, rank among the first colonization sites for infectious prions. After replicating in these lymphoid tissues, prions undertake the process of “neuroinvasion,” which is likely mediated by the peripheral nerves connecting lymphoid tissues to the central nervous system (CNS). To study the connections between the tonsils and the CNS, we injected fluorescent tracers into the PTs of lambs; the highest number of Fast Blue (FB)–labeled neurons was found in cranial cervical ganglia (CCG), whereas a progressively decreasing number of cells were detected in proximal glossopharyngeal, proximal vagal, trigeminal, pterygopalatine, and cervicothoracic ganglia. Immunohistochemistry was carried out on tonsil and ganglia cryosections. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and calcium-binding protein S100 (S100), was observed in the fibers around and within PT lymphoid nodules. In the trigeminal, proximal glossopharyngeal and vagal ganglia the retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR. In all ganglia some retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR co-localization. It is worth noting that only 66±19% and 75±13% of retrogradely-labeled neurons in CCG showed TH- and DBH-IR, respectively. The present results allow us to attribute PT innervation mainly to the sympathetic component and to the glossopharyngeal, vagal and trigeminal cranial nerves. Furthermore, these data also provide a plausible anatomic route through which infectious agents, such as prions, may access the CNS, i.e. by traveling along several cranial and sympathetic nerves, as well as by migration via glial cells.

Paraventricular thalamus mediates context‐induced reinstatement (renewal) of extinguished reward seeking

Paraventricular thalamus (PvTh) is uniquely placed to contribute to reinstatement of drug and reward seeking. It projects extensively to regions implicated in reinstatement including accumbens shell (AcbSh), prefrontal cortex (PFC) and basolateral amygdala (BLA), and receives afferents from other regions important for reinstatement such as lateral hypothalamus. We used complementary neuroanatomical and functional approaches to study the role of PvTh in context-induced reinstatement (renewal) of extinguished reward-seeking. Rats were trained to respond for a reward in context A, extinguished in context B and tested in context A or B. We applied the neuronal tracer cholera toxin B subunit (CTb) to AcbSh and examined retrograde-labelled neurons, c-Fos immunoreactivity (IR) and dual c-Fos/CTb labelled neurons in PvTh and other AcbSh afferents. In PvTh there was c-Fos IR in CTb-positive neurons associated with renewal showing activation of a PvTh-AcbSh pathway during renewal. In PFC there was little c-Fos IR in CTb-positive or negative neurons associated with renewal. In BLA, two distinct patterns of activation and retrograde labelling were observed. In rostral BLA there was significant c-Fos IR in CTb-negative neurons associated with renewal. In caudal BLA there was significant c-Fos IR in CTb-positive neurons associated with being tested in either the extinction (ABB) or training (ABA) context. We then studied the functional role of PvTh in renewal. Excitotoxic lesions of PvTh prevented renewal. These lesions had no effect on the acquisition of reward seeking. These results show that PvTh mediates context-induced reinstatement and that this renewal is associated with recruitment of a PvTh-AcbSh pathway.