Fluoro-ruby retrograde tracing and three-dimensional visualization of the corticospinal tract in the guinea pig

Fluoro-ruby was injected into the posterior funiculus of the spinal cord in the cervical （C5-T2） and lumbar （L3-6） segments of adult guinea pigs. The spinal cord was cut into serial frozen sections. The Fluoro-ruby labeling was clearly delineated from the surrounding structure. The labeling traversed the cervical, thoracic and lumbar segments, and was located on the ventral portion of the posterior funiculus on the injected side, proximal to the intermediate zone of the dorsal gray matter. The fluorescence area narrowed rostro-caudally. The spinal cord, spinal cord gray matter and corticospinal tract were reconstructed using 3D-DOCTOR 4.0 software, resulting in a robust three-dimensional profile. Using functionality provided by the reconstruction software, free multi-angle observation and sectioning could be conducted on the spinal cord and corticospinal tract. Our experimental findings indicate that the Fluoro-ruby retrograde fluorescent tracing technique can accurately display the anatomical location of corticospinal tract in the guinea pig and that three-dimensional reconstruction software can be used to provide a three-dimensional image of the corticospinal tract.

Sensory innervation of the anterior eye segment in rats: aretrograde tracing study

This study aimed to investigate the sensory innervation of the anterior eye segment in rats by retrograde tracing with 1,1-dioleyl-3,3,3,3-tetrameth-ylindocarbocyanine,4-chlorobenesulfonate(FAST Dil)injected into the anterior chamber.The sensory innervation of distinct elements of the anterior segment of the rat’s eye,i.e.the cornea,ciliary body,iris,and trabecular meshwork,were studied by retrograde tracing using FAST Dil as a tracer.FAST Dil was injected into the anterior chambers of the rat’s eyes.The animals were sacrificed at different time points,i.e.,2,3,4,and 5 days after the injection.FAST Dil localization in trigeminal ganglions was studied with afluorescent microscope.Two days after FAST Dil injection into the anterior chambers,the cornea,the ciliary body,the iris,and the trabecular meshwork were heavily labeled.Neurons in the ipsilateral trigeminal ganglion were also consistently labeled.The number of labeled cells increased over time until 4 days after FAST Dil injection.FAST Dil-labeled neurons could be divided into two parts.Most of the Dil-labeled neurons were concentrated in a sharp,longitudinal,spindle-like stripe,located in the dorso-medial side of the trigeminal ganglion,approximately two thirds of the dorsal portion.The other part of Dil-labeled neurons scattered laterally to the stripe,but just in one third of the dorsal region.Thus,with our preliminary results,we conclude that the primary afferent sensory neurons innervating the rat’s anterior eye segments aggregate in the dorso-medial part of the ipsilateral trigeminal ganglion.It is feasible to identify them using retrograde tracing with FAST Dil anterior chamber injection.

The Double Projecting Neuron of the Cat Spinal Dorsal Horn——A Study with Double Retrograde Fluorescent Tracing Technique

The new double projecting neurons were found in the cat spinal dorsal horn by the double retrograde fluorescent tracing technique.Fast Blue(FB)was injected into unilateral dorsal column nucleus(DCN)of adult cats anesthetized with pentobarbital.Nuclear Yellow(NY)was injected ipsilaterally into the lateral cervical nucleus(LCN)8-9 days later.After an additional 18-30 hrs.

A"messenger zone hypothesis"based on the visual three-dimensional spatial distribution of motoneurons innervating deep limb muscles

Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneuro ns.Howeve r,current research on the spatial location of the spinal motoneurons innervating differe nt muscles is limited.In this study,we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs,which were innervated by the obturator nerve,femoral nerve,inferior gluteal nerve,deep pe roneal nerve,and tibial nerve.Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared o rgans(3DISCO)and 3-D imaging technology based on lightsheet fluorescence microscopy(LSFM).Additionally,we propose the hypothesis that"messenger zones"exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups.We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons.Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse.Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles,but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.

Comparison of commonly used retrograde tracers in rat spinal motor neurons

The purpose of this study was to investigate the effect of four fluorescent dyes, True Blue（TB）, Fluoro-Gold（FG）, Fluoro-Ruby（FR）, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate（Di I）, in retrograde tracing of rat spinal motor neurons. We transected the muscle branch of the rat femoral nerve and applied each tracer to the proximal stump in single labeling experiments, or combinations of tracers（FG-Di I and TB-Di I） in double labeling experiments. In the single labeling experiments, significantly fewer labeled motor neurons were observed after FR labeling than after TB, FG, or Di I, 3 days after tracer application. By 1 week, there were no significant differences in the number of labeled neurons between the four groups. In the double-labeling experiment, the number of double-labeled neurons in the FG-Di I group was not significantly different from that in the TB-Di I group 1 week after tracer application. Our findings indicate that TB, FG, and Di I have similar labeling efficacies in the retrograde labeling of spinal motor neurons in the rat femoral nerve when used alone. Furthermore, combinations of Di I and TB or FG are similarly effective. Therefore, of the dyes studied, TB, FG and Di I, and combinations of Di I with TB or FG, are the most suitable for retrograde labeling studies of motor neurons in the rat femoral nerve.

Distribution of serotonin 5-HT_(2A) and 5-HT_7 receptors in the Onuf’s nucleus of the rat spinal cord

BACKGROUND：Motoneurons from the Onuf’s nucleus of the spinal cord, which innervate the striated muscle of the pelvic floor, play an important role in erection, ejaculation, and urine control. Serotonin （5-hydroxytryptamine, 5-HT） regulates motoneuron activity from the Onuf’s nucleus of the spinal cord. However, few studies exist that describe 5-HT receptor distribution in the Onuf’s nucleus. In addition, the nature of the effects of 5-HT receptor on the innervating striated muscle of the pelvic floor is controversial. OBJECTIVE： To investigate the distribution of serotonin 5-HT2A and 5-HT7 receptors in motoneurons of Onuf’s nucleus in the spinal cord of male rats, and to analyze the relationship of 5-HT2A and 5-HT7 receptor to central modulation of urogenital function. DESIGN, TIME AND SETTING： The neural morphology experiment was performed at the Ultramicro-structure Laboratory of Reproductive Medicine, Basic Medical College, Chongqing Medical University, China from April to December 2007. MATERIALS： Ten adult, Sprague Dawley rats （eight males and two females） were randomly divided into gender control group （n = 4, 50% male and 50% female） and a retrograde tracing group （n = 6, 100% male） Recombinant pseudorabies virus （PRV-152） was provided by Professor LW Enquist from Princeton University, USA. Rabbit anti-5-HT2A and 5-HT7 receptor antibodies were purchased from Diasorin, France. METHODS： In the gender control group, the spinal L5-6 segments were harvested, sliced, and then incubate antibodies specific against 5-HT2A or 5-HT7 receptors for immunohistochemical staining. In the retrograde tracing group, PRV-152 was separately injected into the right ischiocavernosus （ischiocavernosus subgroup, n = 3） and the right external urethral sphincter （external urethral sphincter subgroup, n = 3）. Four days after injection, L5-6 segments were harvested, sliced, and incubated with antibodies specific against 5-HT2A or 5-HT7 receptors for double-labeling immunofluorescence staining. MAIN OUTCOME MEASURES： Distribution analysis of 5-HT2A and 5-HT7 receptors in Onuf’s nucleus utilizing optical or laser confocal microscopy. RESULTS： 5-HT2A receptor immunoreactivity was revealed primarily in the medial region of the dorsolateral nucleus of Onuf’s nucleus. 5-HT7 receptor expression was observed in the lateral part of the dorso-lateral nucleus. 5-HT2A and 5-HT7 receptor expressions in the Onuf’s nucleus were significantly greater in male rats, compared to female rats. Double-labeling immunofluorescence demonstrated that 5-HT2A recepto were distributed primarily in the surrounding motoneurons innervating the ischiocavernosus, and 5-HT7 receptors were primarily expressed in motoneurons innervating the external urethral sphincter. CONCLUSION： Motoneurons innervating the ischiocavernosus and external urethral sphincter are located primarily in the medial and lateral region of the dorsolateral nucleus of L5-6 segments. The 5-HT2A receptor-innervating ischiocavernosus may be preferentially involved in the regulation of sexual reflex, and the 5-HT7 receptor-innervating external urethral sphincter may mainly join in regulating micturition reflex.

Is Marcus Gunn jaw winking a primitive reflex？ Rat neuroanatomy

AIM： To investigate a possible trigeminal proprioceptive- oculomotor neural pathway and explore possible synaptic connections between neurons in this pathway. Attempt to bring a new insight to mechanism of Marcus Gunn syndrome （MGS）. METHODS： Anterograde and retrograde tract tracing was applied and combined with immunofluorescent stain in rats. After electrophysiological identifying mesencephalic trigeminal nucleus （Vine） neurons, intracellular injection of tracer was performed to trace axon trajectory. RESULTS： Following injections of anterograde tracers into the Vine, labeled terminals were observed ipsilateral in oculomotor and trochlear nuclei （Ill/IV）, as well as in their premotor neurons in interstitial nucleus of Cajal and Darkschewitsch nucleus （INC/DN）. Combining with choline acetyltransferase （CHAT） immunofluorescent stain, it showed that Vme projecting terminals contact upon ChAT positive Ill/IV motoneurons under confocal microscope. By retrograde labeling premotor neurons of the III, it showed that Vme neuronal terminals contact with retrogradely labeled pre-oculomotor neurons in the INCIDN. Axons of intraceiiularly labeled Vme neurons that respond to electric stimuli of the masseter nerve traveled into the ipsilateral III. CONCLUSION： There may exist a trigeminal propdoceptive- oculomotor system neural circuit in the rat, which is probably related to vertical-torsional eye movements. Possible association of this pathway with MGS etiology was discussed.

End-to-side neurorrhaphy repairs peripheral nerve injury：sensory nerve induces motor nerve regeneration

End-to-side neurorrhaphy is an option in the treatment of the long segment defects of a nerve.It involves suturing the distal stump of the disconnected nerve（recipient nerve） to the side of the intimate adjacent nerve（donor nerve）.However,the motor-sensory specificity after end-to-side neurorrhaphy remains unclear.This study sought to evaluate whether cutaneous sensory nerve regeneration induces motor nerves after end-to-side neurorrhaphy.Thirty rats were randomized into three groups：（1） end-to-side neurorrhaphy using the ulnar nerve（mixed sensory and motor） as the donor nerve and the cutaneous antebrachii medialis nerve as the recipient nerve;（2） the sham group：ulnar nerve and cutaneous antebrachii medialis nerve were just exposed;and（3） the transected nerve group：cutaneous antebrachii medialis nerve was transected and the stumps were turned over and tied.At 5 months,acetylcholinesterase staining results showed that 34% ± 16% of the myelinated axons were stained in the end-to-side group,and none of the myelinated axons were stained in either the sham or transected nerve groups.Retrograde fluorescent tracing of spinal motor neurons and dorsal root ganglion showed the proportion of motor neurons from the cutaneous antebrachii medialis nerve of the end-to-side group was 21% ± 5%.In contrast,no motor neurons from the cutaneous antebrachii medialis nerve of the sham group and transected nerve group were found in the spinal cord segment.These results confirmed that motor neuron regeneration occurred after cutaneous nerve end-to-side neurorrhaphy.

Whole-Brain Map of Long-Range Monosynaptic Inputs to Different Cell Types in the Amygdala of the Mouse

The amygdala,which is involved in various behaviors and emotions,is reported to connect with the whole brain.However,the long-range inputs of distinct cell types have not yet been defined.Here,we used a retrograde trans-synaptic rabies virus to generate a whole-brain map of inputs to the main cell types in the mouse amygdala.We identified 37 individual regions that projected to neurons expressing vesicular glutamate transporter 2,78 regions to parvalbumin-expressing neurons,104 regions to neurons expressing protein kinase C-δ,and 89 regions to somatostatin-expressing neurons.The amygdala received massive projections from the isocortex and striatum.Several nuclei,such as the caudate-putamen and the CA1 field of the hippocampus,exhibited input preferences to different cell types in the amygdala.Notably,we identified several novel input areas,including the substantia innominata and zona incerta.These findings provide anatomical evidence to help understand the precise connections and diverse functions of the amygdala.

Sexual Dimorphism of Inputs to the Lateral Habenula in Mice

The lateral habenula(LHb),which is a critical neuroanatomical hub and a regulator of midbrain monoaminergic centers,is activated by events resulting in negative valence and contributes to the expression of both appetitive and aversive behaviors.However,whole-brain cell-type-specific monosynaptic inputs to the LHb in both sexes remain incompletely elucidated.In this study,we used viral tracing combined with in situ hybridization targeting vesicular glutamate transporter 2(vGlut2)and glutamic acid decarboxylase 2(Gad2)to generate a comprehensive whole-brain atlas of inputs to glutamatergic andγ-aminobutyric acid(GABA)ergic neurons in the LHb.We found>30 ipsilateral and contralateral brain regions that projected to the LHb.Of these,there were significantly more monosynaptic LHb-projecting neurons from the lateral septum,anterior hypothalamus,dorsomedial hypothalamus,and ventromedial hypothalamus in females than in males.More interestingly,we found a stronger GABAergic projection from the medial septum to the LHb in males than in females.Our results reveal a comprehensive connectivity atlas of glutamatergic and GABAergic inputs to the LHb in both sexes,which may facilitate a better understanding of sexual dimorphism in physiological and pathological brain functions.