Injections Of Wheat Germ Agglutinin Research Articles

Polyethylene glycol fusion repair of severed rat sciatic nerves reestablishes axonal continuity and reorganizes sensory terminal fields in the spinal cord.

JOURNAL/nrgr/04.03/01300535-202507000-00030/figure1/v/2024-09-09T124005Z/r/image-tiff Peripheral nerve injuries result in the rapid degeneration of distal nerve segments and immediate loss of motor and sensory functions; behavioral recovery is typically poor. We used a plasmalemmal fusogen, polyethylene glycol (PEG), to immediately fuse closely apposed open ends of severed proximal and distal axons in rat sciatic nerves. We have previously reported that sciatic nerve axons repaired by PEG-fusion do not undergo Wallerian degeneration, and PEG-fused animals exhibit rapid (within 2-6 weeks) and extensive locomotor recovery. Furthermore, our previous report showed that PEG-fusion of severed sciatic motor axons was non-specific, i.e., spinal motoneurons in PEG-fused animals were found to project to appropriate as well as inappropriate target muscles. In this study, we examined the consequences of PEG-fusion for sensory axons of the sciatic nerve. Young adult male and female rats (Sprague-Dawley) received either a unilateral single cut or ablation injury to the sciatic nerve and subsequent repair with or without (Negative Control) the application of PEG. Compound action potentials recorded immediately after PEG-fusion repair confirmed conduction across the injury site. The success of PEG-fusion was confirmed through Sciatic Functional Index testing with PEG-fused animals showing improvement in locomotor function beginning at 35 days postoperatively. At 2-42 days postoperatively, we anterogradely labeled sensory afferents from the dorsal aspect of the hindpaw following bilateral intradermal injection of wheat germ agglutinin conjugated horseradish peroxidase. PEG-fusion repair reestablished axonal continuity. Compared to unoperated animals, labeled sensory afferents ipsilateral to the injury in PEG-fused animals were found in the appropriate area of the dorsal horn, as well as inappropriate mediolateral and rostrocaudal areas. Unexpectedly, despite having intact peripheral nerves, similar reorganizations of labeled sensory afferents were also observed contralateral to the injury and repair. This central reorganization may contribute to the improved behavioral recovery seen after PEG-fusion repair, supporting the use of this novel repair methodology over currently available treatments.

Renal sensory nerves increase sympathetic nerve activity and blood pressure in 2-kidney 1-clip hypertensive mice.

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to define the function, anatomy, and contribution of mouse renal sensory neurons to a renal nerve-dependent model of hypertension. First, electrical stimulation of mouse renal afferent nerves produced frequency-dependent increases in ABP that were eliminated by ganglionic blockade. Stimulus-triggered averaging revealed renal afferent stimulation significantly increased splanchnic, renal, and lumbar sympathetic nerve activity (SNA). Second, kidney injection of wheat germ agglutinin into male C57Bl6 mice (12-14 wk; Jackson Laboratories) produced ipsilateral labeling in the T11-L2 dorsal root ganglia. Next, 2-kidney 1-clip (2K1C) hypertension was produced in male C57Bl6 mice (12-14 wk; Jackson Laboratories) by placement of a 0.5-mm length of polytetrafluoroethylene tubing around the left renal artery. 2K1C mice displayed an elevated ABP measured via telemetry and a greater fall in mean ABP to ganglionic blockade at day 14 or 21 vs. day 0. Renal afferent discharge was significantly higher in 2K1C-clipped vs. 2K1C-unclipped or sham kidneys. In addition, 2K1C-clipped vs. 2K1C-unclipped or sham kidneys had lower renal mass and higher mRNA levels of several proinflammatory cytokines. Finally, both ipsilateral renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33 mM capsaicin) at time of clipping resulted in lower ABP of 2K1C mice at day 14 or 21. These findings suggest mouse renal sensory neurons are activated to increase SNA and ABP in 2K1C hypertension. NEW & NOTEWORTHY This study documents the function, anatomy, and contribution of mouse renal sensory nerves to neurogenic hypertension produced by renal stenosis. Activation of renal afferents increased sympathetic nerve activity and blood pressure. Renal afferent activity was elevated in hypertensive mice, and renal afferent denervation lowered blood pressure. Clinically, patients with renal stenosis have been excluded from clinical trials for renal denervation, but this study highlights the potential therapeutic efficacy to target renal nerves in these patients.

Central Terminals of Trigeminal Nerves that Innervate the Nose and Nasal Passages Colocalize with Medullary Dorsal Horn Neurons Activated during Voluntary Diving in Rats

Underwater submersion in mammals initiates the diving response, an autonomic cardiorespiratory reflex that includes apnea, a parasympathetically‐mediated bradycardia, and a sympathetically‐mediated increase in total peripheral resistance. The initial synapse of this reflex occurs within the ventral medullary dorsal horn (MDH) of the spinal trigeminal nucleus caudalis. The anterior ethmoidal and nasopalatine nerves, trigeminal nerves that innervate the internal nasal passages, also send their central terminations to the ventral MDH. The objective of this research was to detect colocalization of secondary MDH neurons activated by voluntary diving with the central terminations of the trigeminal nerves innervating the internal nasal passages. MDH activated neurons were identified by Fos protein production, while central terminals of the trigeminal nerves were located by the transganglionic tracer Wheat Germ Agglutinin (WGA). Sprague‐Dawley rats (N=12) were trained to voluntarily dive through a 5 m underwater Plexiglas maze. After training, 6 rats then received WGA injections into the anterior ethmoidal nerve, while 6 rats received WGA injections into the nasopalatine nerve. Four days post‐WGA injection, 3 rats from each nerve injection group performed 12 dive trials over 1 hour to activate brainstem neurons and initiate Fos production; 3 rats from each nerve injection group were non‐diving controls. Brainstem tissue from all rats was immunohistochemically processed to visualize both Fos and WGA, and was inspected using a Nikon C1 confocal microscope and Nikon Eclipse Ti2 inverted fluorescent microscope. Results indicate repetitive voluntary diving increases the number of Fos‐positive neurons within the ventral tip of the superficial MDH and adjacent paratrigeminal nucleus. WGA results show central termination sites from both the anterior ethmoidal and nasopalatine nerves within this same MDH location. Additionally, MDH Fos‐positive neurons from diving animals show extensive colocalization with WGA injected into both the anterior ethmoidal nerve and nasopalatine nerve compared to non‐diving control rats. We conclude both the anterior ethmoidal and nasopalatine nerves can provide afferent information necessary to activate MDH neurons during initiation of the mammalian diving response.Support or Funding InformationSupported by MWU Intramural funding (PFM) and CGS MBS program (MM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wheat Germ Agglutinin as a Potential Therapeutic Agent for Leukemia.

Dietary lectins are carbohydrate-binding proteins found in food sources. We used a panel of seven dietary lectins to analyze cytotoxicity against hematological cancers. Wheat germ agglutinin (WGA), even at low doses, demonstrated maximum toxicity toward acute myeloid leukemia (AML) cells. Using AML cell lines, we show time- and dose-dependent killing by WGA. We also show that low doses of WGA kills primary patient AML cells, irrespective of subtype, with no significant toxicity to normal cells. WGA caused AML cell agglutination, but failed to agglutinate RBC's at this dose. WGA, primarily, binds to N-acetyl-D-glucosamine (GlcNAc) and is also reported to interact with sialic-acid-containing glycoconjugates and oligosaccharides. After neuraminidase pre-treatment, which catalyzes the hydrolysis of terminal sialic acid residues, AML cells were less sensitive to WGA-induced cell death. AML cells were also not sensitive to succinyl-WGA, which does not react with sialic acid. Incubation with LEL lectin, which recognizes GlcNAc or SNA, which binds preferentially to sialic acid attached to terminal galactose in α-2,6 and to a lesser degree α-2,3 linkage, did not alter AML cell viability. These data indicate that WGA-induced AML cell death is dependent on both GlcNAc binding and interaction with sialic acids. We did not observe any in vitro or in vivo toxicity of WGA toward normal cells at the concentrations tested. Finally, low doses of WGA injection demonstrated significant in vivo toxicity toward AML cells, using xenograft mouse model. Thus, WGA is a potential candidate for leukemia therapy.

Morphological analysis for neuronal pathway from the hindbrain ependymocytes to the hypothalamic kisspeptin neurons.

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine β-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1–A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

Disruption of the Centrifugal Visual System Inhibits Early Eye Growth in Chicks

Emmetropization, the process by which neonatal refractive errors are reduced toward zero, is partially dependent on brain-retina connectivity. Here, we investigated the role of the centrifugal visual system, a visually driven retinal feedback projection, as one potential influence on this complex mechanism. Lesions of the isthmo-optic nucleus/tract or sham surgeries were performed in fifty-four 4- to 5-day-old chicks to disrupt centrifugal efferents to the contralateral retina. Prior to surgery, baseline refractive error measurements were made using streak retinoscopy. Postoperative ocular phenotyping, which (in addition to retinoscopy) comprised A-scan ultrasonography and infrared keratometry, was performed 7 days and 21 days postsurgery. A pathway-tracing paradigm was used to determine lesion success, whereby an injection of wheat-germ agglutinin was made into the vitreous chamber contralateral to the lesion. Postmortem, tissue processing, immunohistochemistry, and stereological analysis of intact centrifugal neurons were performed. Subsequently, chicks were divided into quartile groups based on percentage lesion success. Seven days postsurgery, chicks in the quartile of highest percentage lesion success exhibited significant axial hyperopia in the "treated eye" (contralateral to the lesion) relative to the "control eye" (ipsilateral to the lesion) eye, when compared with subjects within quartile groups of lower percentage lesion success (P = 0.004). However, by 21 days postsurgery, the induced hyperopia was no longer evident. Unilateral disruption of centrifugal efferents to the retina of the contralateral eye induces an initial axial hyperopia, which is subsequently reversed through increased vitreous elongation in the affected eyes.

Secretion-related structures of hypothalamo-hypophysial terminals in the rat posterior pituitary

Hypothalamic terminals were investigated in the rat posterior pituitary (PP). Injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and co-injection of WGA-HRP with Rab3A-siRNA were made into the hypothalamus, respectively. Additional injection of WGA-HRP was made into the hypothalamus in the animals exposed to ethanol. These injections resulted in heavy labeling of fibers exclusively confined to the PP. Ultrastructural observations showed terminals, fibers, pituicytes, capillaries and vascular spaces in the PP. Although the majority of terminals were observed to contain large dense core vesicles (LDCVs) and HRP-reaction products (HRP-RPs), exocytosis of LDCVs in close proximity to cell membrane was not found. Interestingly, a few terminals showed alteration of cell membrane called "apocrine-like structure" containing LDCV and RP. The narrow neck portion of the structure gave the appearance that it may have been in some stage of separating from terminals. Other remarkable feature was that terminals occasionally reveal the structure of "leakage" of RP discharged into vascular spaces crossing cell membrane. Such hormone-releasing mechanism might be involved in one of "diacrine-like secretion". In the present study secretion-related structures of hypothalamic terminals in the PP are quite different from normal vesicular exocytosis.

A Neurobehavioral Analysis of the Prevention of Visual Impairment in the DBA/2J Mouse Model of Glaucoma

Timoptic-XE treatment was used to examine the relationship between age-related changes in intraocular pressure (IOP), retinal cell loss, visual ability, and neuronal labeling in the superior colliculus in the DBA/2J mouse model of pigmentary glaucoma. Mice were administered Timoptic-XE (0.0%, 0.25%, or 0.50%) daily from 9 weeks to 12 months of age. Visual ability and IOP were evaluated at 3, 6, 9, and 12 months of age. Mice from each group were then given intraocular injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), and estimates of the number of cells in the ganglion cell layer of the retina, WGA-HRP transneural labeling of cells, cell count, and cross-sectional area of Nissl-stained cells in the superior colliculus were obtained. Mice treated with 0.50% and 0.25% Timoptic-XE maintained a high level of performance in behavioral vision tasks, while 12-month-old untreated mice (0.0% Timoptic-XE) exhibited impaired visual performance. Timoptic-XE therapy reduced IOP and cell loss in the ganglion cell layer of the retina and prevented somal shrinkage and the decrease in WGA-HRP transneural labeling in the superior colliculus that occurred in untreated mice at 12 months of age. This study provides a comprehensive assessment of the efficacy of Timoptic-XE in DBA/2J mice by correlating age-related visual system changes in the retina and brain with changes in IOP and visual ability. These results showed that reducing IOP not only rescued retinal ganglion cell atrophy but also restored visual function and altered patterns of neurodegeneration that occur with blindness.

Morphological Evidence of an Altered Process of Synaptic Transcytosis in Adult Rats Exposed to Ethanol

The effects of ethanol exposure on synaptic structure were investigated in the nucleus of solitary tract (NST) in rats, using the horse-radish peroxidase (HRP) method. Eight-week-old experimental rats were allowed free access to a liquid diet containing ethanol for 3 weeks, while controls were given an isocaloric diet. Some of the control and experimental animals were given an injection of wheat germ agglutinin conjugated with HRP (WGA-HRP) into the vagus nerve toward the end of the treatment period. After the treatment, the neuropil region of the NST was examined under an electron microscope. We observed that a few terminals were characterized by deep indentation of axodendritic membranes into the post-synaptic neurons. This appeared to be similar to that commonly seen in exocrine glands. Interestingly, the indented portion often contained various sizes of vacuoles and flattened cisternae. HRP-reaction product (RP) transported to terminals was recognized easily as an electron-dense lysosomal substance when lead citrate staining was omitted. Terminals containing HRP-RP also revealed quite a similar structure with indentation of axodendritic membranes as described earlier. The results are considered to confirm that terminals forming 'apocrine-like structures' observed in the ethanol-fed animals with no injection of WGA-HRP originate from afferent fibers of the vagus nerve. The present study suggests the possibility that the alteration of the synaptic structure induced by ethanol exposure can lead to the neuronal transcytosis of materials including proteins which is different from the normal vesicular exocytosis involved in chemical synaptic transmission.

Direct synaptic connections between superior colliculus afferents and thalamo-insular projection neurons in the feline suprageniculate nucleus: A double-labeling study with WGA-HRP and kainic acid

The suprageniculate nucleus (Sg) of the feline thalamus, which subserves largely unimodal sensory and orientation behavior, receives input from the deep layers of the superior colliculus (SC), and projects to the suprasylvian cortical areas, such as the anterior ectosylvian visual area and the insular visual area (IVA), which contain visually responsive neurons. Through a double tract-tracing procedure involving the injection of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) into the IVA and the injection of kainic acid into the SC, this study sought to determine the nature of the synaptic relationship between the SC afferents and the thalamo-cortical projection neurons. WGA-HRP injections labeled numerous neurons in the Sg, while kainic acid injections destroyed many tectothalamic terminals in the Sg. The distributions of the WGA-HRP-labeled neurons and the degenerated axon terminals overlapped in the dorsal part of the Sg. Electron microscopic observations demonstrated that the degenerated axon terminals made synaptic contacts with the dendrites of the WGA-HRP-labeled neurons in this overlapping region of the Sg. These results provide the first anatomical evidence that the Sg may play a role in the key relay of visual information from the SC to the IVA, within an identified extrageniculo-cortical pathway.

Reticulospinal neurons in the pontomedullary reticular formation of the monkey (Macaca fascicularis)

Recent neurophysiological studies indicate a role for reticulospinal neurons of the pontomedullary reticular formation (PMRF) in motor preparation and goal-directed reaching in the monkey. Although the macaque monkey is an important model for such investigations, little is known regarding the organization of the PMRF in the monkey. In the present study, we investigated the distribution of reticulospinal neurons in the macaque. Bilateral injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) were made into the cervical spinal cord. A wide band of retrogradely labeled cells was found in the gigantocellular reticular nucleus (Gi) and labeled cells continued rostrally into the caudal pontine reticular nucleus (PnC) and into the oral pontine reticular nucleus (PnO). Additional retrograde tracing studies following unilateral cervical spinal cord injections of cholera toxin subunit B revealed that there were more ipsilateral (60%) than contralateral (40%) projecting cells in Gi, while an approximately 50:50 ratio contralateral to ipsilateral split was found in PnC and more contralateral projections arose from PnO. Reticulospinal neurons in PMRF ranged widely in size from over 50 μm to under 25 μm across the major somatic axis. Labeled giant cells (soma diameters greater than 50 μm) comprised a small percentage of the neurons and were found in Gi, PnC and PnO. The present results define the origins of the reticulospinal system in the monkey and provide an important foundation for future investigations of the anatomy and physiology of this system in primates.

Identification of hypophysiotropic luteinizing hormone-releasing hormone (LHRH) neurons by combined retrograde labeling and immunocytochemistry.

The decapeptide luteinizing hormone-releasing hormone (LHRH) is produced by telencephalic and diencephalic neurons and transported to the median eminence (ME). After having been released from nerve terminals, it is carried by the hypophysial portal vessels to the anterior pituitary, where it stimulates the production and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Those LHRH neurons which project to the ME represent the final common pathway for the regulation of the pituitary/gonadal axis. We identified these neurons by injecting a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Eight to 24 hours later colchicine was given into the lateral ventricle and 24-48 hours after the WGA injection the animals were sacrificed. Vibratome sections of the brains were stained simultaneously for WGA and LHRH with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus were double-labeled, indicating that they projected to the ME. Double labeled LHRH cells were either smooth, fusiform or "spiny". WGA-accumulating LHRH perikarya were intermixed with single-labeled LHRH cells. The remaining 30% of the LHRH neurons which were not labeled with WGA appeared to project to different hypothalamic and extrahypothalamic areas of the brain. Our results suggest that there are at least two populations of LHRH neurons, one with access to the portal capillaries of the ME and functionally related to the regulation of the pituitary, and one without access to capillaries of the ME, functionally probably related to intracerebral neurotransmission or modulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Overlap of nigrothalamic terminals and thalamostriatal neurons in the feline lateralis medialis-suprageniculate nucleus

The lateralis medialis-suprageniculate nucleus (LM-Sg) of the feline posterior thalamus is a relay nucleus with a clear visuomotor function. In this study, we examined the distribution of axon terminals of the nigral afferent to the LM-Sg following injection of an anterograde tracer, biocytin, into the substantia nigra pars reticulata, and the distribution of the thalamostriatal projection neurons in the LM-Sg following the injection of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) as a retrograde tracer into the caudate nucleus. The biocytin-labeled terminal-like puncta were located in the ventromedial portion of this nucleus in such a way that most of the labeled elements took the form of swellings having boutons in places, while a minority appeared in clusters of 3-5 large terminal-like puncta. The retrograde WGA-HRP-labeled neurons were also found in the ventromedial part of the LM-Sg, and the distributions of labeled nigrothalamic axon terminals and labeled thalamostriatal projection neurons therefore overlapped in this region. The present results indicate that the nigral afferent may make synaptic contacts directly with the thalamostriatal projection neurons within the LM-Sg.

Somatosensory areas S2 and PV project to the superior colliculus of a prosimian primate, Galago garnetti

As part of an effort to describe the connections of the somatosensory system in Galago garnetti, a small prosimian primate, injections of tracers into cortex revealed that two somatosensory areas, the second somatosensory area (S2) and the parietal ventral somatosensory area (PV), project densely to the ipsilateral superior colliculus, while the primary somatosensory area (S1 or area 3b) does not. The three cortical areas were defined in microelectrode mapping experiments and recordings were used to identify appropriate injection sites in the same cases. Injections of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) were placed in S1 in different mediolateral locations representing body regions from toes to face in five galagos, and none of these injections labeled projections to the superior colliculus. In contrast, each of the two injections in the face representation of S2 in two galagos and three injections in face and forelimb representations of PV in three galagos produced dense patches of labeled terminations and axons in the intermediate gray (layer IV) over the full extent of the superior colliculus. The results suggest that the higher-order somatosensory areas, PV and S2, are directly involved in the visuomotor functions of the superior colliculus in prosimian primates, while S1 is not. The somatosensory inputs appear to be too widespread to contribute to a detailed somatotopic representation in the superior colliculus, but they may be a source of somatosensory modulation of retinotopically guided oculomotor instructions.

The pupillary and ciliary components of the cat Edinger-Westphal nucleus: a transsynaptic transport investigation.

The distribution of preganglionic motoneurons supplying the ciliary ganglion in the cat was defined both qualitatively and quantitatively. These cells were retrogradely labeled directly, following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the ciliary ganglion, or were transsynaptically labeled following injections of WGA into the vitreous chamber. Almost half of the cells are distributed rostral to the oculomotor nucleus, both in and lateral to the anteromedian nucleus. Of the remaining preganglionic motoneurons, roughly 20% of the total are located dorsal to the oculomotor nucleus. Strikingly few of these neurons are actually found within the Edinger-Westphal nucleus proper. Instead, the majority are found in the adjacent supraoculomotor area or along the midline between the two somatic nuclei. An additional population, roughly 30% of the total, is located ventral to the oculomotor nucleus. This study also provides evidence for a functional subdivision of this preganglionic population. Pupil-related preganglionic motoneurons were transsynaptically labeled by injecting WGA into the anterior chamber, while lens-related preganglionic motoneurons were transsynaptically labeled by injecting WGA into the ciliary muscle. The results suggest that the pupil-related preganglionic motoneurons, that is, those controlling the iris sphincter pupillae muscle, are located rostrally, in and lateral to the anteromedian nucleus. In contrast, lens-related preganglionic motoneurons, that is, those controlling the ciliary muscle are particularly prevalent caudally, both dorsal and ventral to the oculomotor nucleus. Thus, the cat intraocular muscle preganglionic innervation is spatially organized with respect to function, despite the dispersed nature of its distribution.

Identification of tuberculo-ventral neurons in the polymorphic layer of the rat dorsal cochlear nucleus.

The tuberculo-ventral tract represents a short nervous circuit within the auditory cochlear nuclei. Tuberculo-ventral neurons of the dorsal cochlear nucleus send isofrequency inhibitory inputs to bushy cells of the ventral cochlear nucleus. Injection of wheat germ agglutinin conjugated to horseradish peroxidase into the rat ventral cochlear nucleus, labelled tuberculo-ventral neurons retrogradely in the deep polymorphic layer of the ipsilateral dorsal cochlear nucleus. Five to 20% of the perimeter of these cells was covered by synaptic boutons, most of which contained flat and pleomorphic vesicles. These boutons contained glycine and sometimes GABA. Occasional small axo-somatic boutons contained round vesicles and were immunonegative for both glycine and GABA. This study shows that the synaptic profile of tuberculo-ventral neurons is different from that of other medium-size glycinergic neurons within the polymorphic layer or more superficial regions of the dorsal cochlear nucleus like cartwheel neurons. In fact the latter mostly receive boutons that contain pleomorphic vesicles.

Substance P-immunoreactive neurons in the rostromedian area of the midbrain send axons to the lower spinal cord in the chicken

Substance P-immunoreactivity in neurons projecting to the spinal cord was examined using retrograde tract-tracing method combined with immunohistochemical techniques in chickens. Many small substance P-immunoreactive neurons were densely clustered in the midline area in the rostral midbrain, the rostromedian area (80% of the neurons in the rostromedian area). Some of these substance P-immunoreactive neurons in the rostromedian area (about 20% of substance P-immunoreactive neurons) were retrogradely labeled by small injections of wheat germ agglutinin conjugated horseradish peroxidase into the central part of the lumber segments including the intermediomedial nucleus, suggesting the projections from the rostromedian area to the lower spinal preganglionic regions. From the present data and mammalian previous studies, it was suggested that the midline area in the midbrain has fiber connections with the regions related autonomic functions, and all of which exhibit substance P-immunoreactivity.

Pallidal and cerebellar afferents to pre-supplementary motor area thalamocortical neurons in the owl monkey: A multiple labeling study

In the present study, we determined where thalamic neurons projecting to the pre-supplementary motor area (pre-SMA) are located relative to pallidothalamic and cerebellothalamic inputs and nuclear boundaries. We employed a triple-labeling technique in the same owl monkey (Aotus trivirgatus). The cerebellothalamic projections were labeled with injections of wheat germ agglutinin conjugated to horseradish peroxidase, and the pallidothalamic projections were labeled with biotinylated dextran amine. The pre-SMA was identified by location and movement patterns evoked by intracortical microstimulation and injected with the retrograde tracer cholera toxin subunit B. Brain sections were processed sequentially using different chromogens to visualize all three tracers in the same section. Alternate sections were processed for Nissl cytoarchitecture or acetylcholinesterase chemoarchitecture for nuclear boundaries. The cerebellar nuclei primarily projected to posterior (VLp), medial (VLx), and dorsal (VLd) divisions of the ventral lateral nucleus; the pallidum largely projected to the anterior division (VLa) of the ventral lateral nucleus and the parvocellular part of the ventral anterior nucleus (VApc). However, we also found zones of overlapping projections, as well as interdigitating foci of pallidal and cerebellar label, particularly in border regions of the VLa and VApc. Thalamic neurons labeled by pre-SMA injections occupied a wide band and were especially concentrated in the VLx and VApc, cerebellar and pallidal territories, respectively. Labeled thalamocortical neurons overlapped cerebellar inputs in the VLd and VApc and overlapped pallidal inputs in the VLa and the ventral medial nucleus. The results demonstrate that inputs from both the cerebellum and globus pallidus are relayed to the pre-SMA.

Third group of neostriatofugal neurons: neurokinin B-producing neurons that send axons predominantly to the substantia innominata.

Neostriatal neurons that produce neurokinin B were investigated immunocytochemically in the rat brain with an antibody against the C-terminal portion of the precursor prepropeptide of neurokinin B, preprotachykinin B (PPTB). PPTB-immunoreactive neurons were scattered throughout the neostriatum and constituted 5.1% of neostriatal neurons. They were immunopositive for projection neuron markers, such as precursor peptides of substance P, enkephalins, and dynorphins, but negative for intrinsic neuron markers, suggesting that PPTB was expressed in neostriatal projection neurons. However, PPTB-immunoreactive neurons were immunonegative for dopamine- and cyclic AMP-regulated phosphoprotein, which is known to be produced by striatopallidal and striatonigral neurons. Furthermore, almost no PPTB-immunoreactive axon terminals were observed in the substantia nigra or globus pallidus. The authors then made large kainic acid lesions in the neostriatum to reveal the target areas of PPTB-producing neurons and observed a decrease in PPTB-immunoreactive fibers in the sublenticular portion of the substantia innominata and, to much lesser extent, in the bed nucleus of the stria terminalis and central nucleus of the amygdala. After injection of wheat germ agglutinin into the substantia innominata, PPTB immunoreactivity was detected in many retrogradely labeled neostriatal neurons. In contrast, no PPTB immunoreactivity was observed in striatonigral or striatopallidal neurons after injection of retrograde tracers into the substantia nigra or globus pallidus. Thus, neurokinin B-producing neostriatal neurons were considered to send projection fibers predominantly to the substantia innominata. Furthermore, PPTB-immunoreactive axonal swellings were closely apposed to neurokinin B receptor-immunoreactive dendrites in the substantia innominata. Overall, the present results indicate that the rat brain possesses a chemically and hodologically unique neostriatofugal pathway in addition to the direct and indirect pathways.

Neurotransmitter characteristics of neurons projecting to the supramammillary nucleus of the rat.

Retrograde labelling was combined with immunohistochemistry to localize neurons containing choline acetyltransferase, gamma-aminobutyric acid (GABA), glutamate, serotonin, somatostatin, Leu-enkephalin, neurotensin, and substance P-immunoreactivity in neurons projecting to the supramammillary nucleus in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the supramammillary nucleus resulted in retrogradely labelled neurons in the medial septal nucleus, the nuclei of the diagonal band of Broca, the infralimbic cortex, the medial and lateral preoptic nucleus, the subiculum, the laterodorsal tegmental nucleus, the compact subnucleus of the central superior nucleus and the dorsal raphe nucleus. In the medial septal nucleus and in the nuclei of the diagonal band of Broca, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for choline acetyltransferase and small numbers of WGA-HRP-labelled neurons were immunoreactive for GABA, glutamate, neurotensin or substance P. In the medial preoptic nucleus, 85-90% of retrogradely labelled neurons (25-30 per section) were immunoreactive for somatostatin and a few WGA-HRP-labelled neurons displayed neurotensin-immunoreactivity. In the rostroventral part of the subiculum, small numbers of retrogradely labelled neurons were also immunoreactive for neurotensin or for glutamate. In the laterodorsal tegmental nucleus, 90% of WGA-HRP-labelled neurons (20-25 per section) were immunoreactive for choline acetyltransferase and small numbers of retrogradely labelled neurons also displayed substance P immunoreactivity. In the compact subnucleus of the central superior nucleus, 50-60% of retrogradely labelled neurons (15-20 per section) were also immunolabelled for GABA and approximately 30-40% of WGA-HRP-labelled neurons (10-12 per section) were immunoreactive for Leu-enkephalin. The compact subnucleus of the central superior nucleus also contained small numbers of retrogradely labelled neurons that displayed neurotensin immunoreactivity. In the dorsal raphe nucleus, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for serotonin and small numbers of retrogradely labelled neurons displayed neurotensin or glutamate immunoreactivity. These results suggest that the multiple neurochemicals contained in ascending and descending projections to the SuM participate in complex interactions in the transmission process of SuM neurons.