Wheat Germ Agglutinin-horseradish Peroxidase Research Articles

Role of Cerebellohypothalamic GABAergic Projection in Mediating Cerebellar Immunomodulation

ABSTRACTOur previous studies have shown that the cerebellar interposed nucleus (IN) modulates lymphocyte functions. As the cerebellum does not have a direct contact with the immune system, it is required to explore the pathway mediating the cerebellar immunomodulation. In this study, both lymphocyte percentage in peripheral leukocytes and lymphocyte proliferation induced by concanavalin A were reduced by the bilateral IN lesions with kainic acid. Anterograde tracing of nerve tracts with biotinylated dextran amine (BDA) from the cerebellum to the hypothalamus revealed that the BDA-labeled fibers from the cerebellar IN neurons traveled through superior cerebellar peduncle (SCP), crossed in SCP decussation, and primarily terminated in lateral hypothalamic area (LHA). Retrograde tracing with wheat germ agglutinin-horseradish peroxidase from the LHA to the cerebellar IN combined with immunohistochemistry for gamma-aminobutyric acid (GABA) or glutamate in the cerebellar sections displayed that the neuronal projections from the cerebellar IN to the LHA mostly were GABAergic. Blockage of GABAA receptors in the LHA with hydrastine led to a reduction in the lymphocyte percentage and proliferation, similar to the IN lesions. These results show a direct GABAergic projection from cerebellar IN to LHA and suggest that the projection mediates cerebellar immunomodulation.

Meal parameters and vagal gastrointestinal afferents in mice that experienced early postnatal overnutrition

Early postnatal overnutrition results in a predisposition to develop obesity due in part to hypothalamic and sympathetic dysfunction. Potential involvement of another major regulatory system component — the vagus nerve — has not been examined. Moreover, feeding disturbances have rarely been investigated prior to development of obesity when confounds due to obesity are minimized. To examine these issues, litters were culled on the day of birth to create small litters (SL; overnutrition), or normal size litters (NL; normal nutrition). Body weight, fat pad weight, meal patterns, and vagal sensory duodenal innervation were compared between SL and NL adult mice prior to development of obesity. Meal patterns were studied 18 h/day for 3 weeks using a balanced diet. Then vagal mechanoreceptors were labeled using anterograde transport of wheatgerm agglutinin–horseradish peroxidase injected into the nodose ganglion and their density and morphology were examined. Between postnatal day 1 and weaning, body weight of SL mice was greater than for NL mice. By young adulthood it was similar in both groups, whereas SL fat pad weight was greater in males, suggesting postnatal overnutrition produced a predisposition to obesity. SL mice exhibited increased food intake, decreased satiety ratio, and increased first meal rate (following mild food deprivation) compared to NL mice, suggesting postnatal overnutrition disrupted satiety. The density and structure of intestinal IGLEs appeared similar in SL and NL mice. Thus, although a vagal role cannot be excluded, our meal parameter and anatomical findings provided no evidence for significant postnatal overnutrition effects on vagal gastrointestinal afferents.

Accessory olfactory bulb function is modulated by input from the main olfactory epithelium

Although it is now established that sensory neurons in both the main olfactory epithelium and the vomeronasal organ may be activated by both general and pheromonal odorants, it remains unclear what initiates sampling by the vomeronasal organ. Anterograde transport of wheat germ agglutinin-horseradish peroxidase was used to determine that adequate intranasal syringing with zinc sulfate interrupted all inputs to the main olfactory bulb but left intact those to the accessory olfactory bulb. Adult male treated mice were frankly anosmic when tested with pheromonal and non-pheromonal odors and failed to engage in aggressive behavior. Treated juvenile females failed to show puberty acceleration subsequent to exposure to bedding from adult males. Activation of the immediate early gene c-Fos and electrovomeronasogram recording confirmed the integrity of the vomeronasal system in zinc sulfate-treated mice. These results support the hypothesis that odor detection by the main olfactory epithelium is required to initiate sampling by the vomeronasal system.

Evidence of a Gustatory-Vestibular Pathway for Protein Transport

To demonstrate anatomically a pathway for protein transport from the palate to the vestibular system. The vestibulofacial anastomosis and associated ganglion cells were identified in a collection of 160 horizontally sectioned human temporal bones that had been stained with hematoxylin and eosin. Wheat germ agglutinin-horseradish peroxidase (HRP) was applied to the greater superficial petrosal nerve in 4 Sprague-Dawley rats. After 30 hours, the rats were killed by intracardiac perfusion, and the seventh and eighth nerves with adjacent brainstem removed. Frozen sections cut at 30 mum through this block were then reacted for HRP, counterstained with neutral red, and mounted on slides for examination in the light microscope. Thirty-two of the 160 human temporal bones contained sections through the vestibulofacial anastomosis and its ganglion. In all cases, the ganglion was incorporated into the vestibular ganglion (VG) adjacent to the nervus intermedius. In all 4 experimental rats, HRP reaction product labeled a small number of ganglion cells in the VG adjacent to the nervus intermedius and facial nerve. These observations support the presence of a pathway from receptors in the palate to the VG.

Expression of the TRPM8-immunoreactivity in dorsal root ganglion neurons innervating the rat urinary bladder

The neurochemical phenotypes of the transient receptor potential melastatin-8 (TRPM8)-immunoreactive afferent neurons innervating the rat urinary bladder were examined by using a highly sensitive tyramide signal amplification method, combined with wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing. TRPM8-immunoreactivity was detected in a small proportion of the WGA-HRP-labeled bladder afferent neurons in the dorsal root ganglia of the Th13-L1 (1.14%) and the L6-S1 (1.27%), and these neurons were small in size (<600 μm 2). The 82.6 ± 3.8% of the TRPM8-immunoreactive bladder afferent neurons and 80.9 ± 1.5% of the total population of the TRPM8-immunoreactive afferent neurons in the observed dorsal root ganglia expressed NF200. On the other hand, the proportions of the co-expression of TRPM8 and nociceptive markers such as calcitonin gene-related peptide (CGRP), transient receptor potential vanilloid-1 (TRPV1), and isolectin B4 (IB4) in the bladder afferent neurons (81.5 ± 5.2% for CGRP, 36.1 ± 4.0% for TRPV1, and 15.8 ± 5.5% for IB4) were higher in comparison to those in the total population of the TRPM8-immunoreactive afferent neurons (21.9 ± 2.4% for CGRP, 16.6 ± 1.7% for TRPV1, and 5.4 ± 0.5% for IB4), although no significant difference existed for IB4. Our results suggest that the TRPM8-expressing bladder afferents should be classified as Aδ-fibers and C-fibers, while some of these afferents may be involved in nociceptive sensations.

Columnar organization of estrogen receptor-α immunoreactive neurons in the periaqueductal gray projecting to the nucleus para-retroambiguus in the caudal brainstem of the female golden hamster

In the hamster brainstem estrogen receptor-α-immunoreactive neurons (ER-α-IR) are present in the nucleus para-retroambiguus (NPRA), located in the caudal ventrolateral medulla (CVLM) ventrolaterally to the nucleus retroambiguus (NRA). NPRA neurons project mainly to the thoracic and upper lumbar cord and are probably involved in the autonomic adaptations during the estrous cycle. The periaqueductal gray (PAG), projecting to the CVLM, also contains ER-α-IR neurons. This raises the questions: how are these projections organized and are ER-α-IR neurons in PAG and NPRA linked directly? Combined retro- and anterograde tracing techniques, using wheat germ agglutinin–horseradish peroxidase (WGA-HRP), were carried out to demonstrate neuronal relationships between PAG and NPRA and/or NRA. Finally, a double-label immunostaining was performed in ovariectomized hamsters combining anti-ER-α antibody immunocytochemistry with cholera toxin B injections into the CVLM, to differentiate between ER-α-IR projections from the PAG to either NRA or NPRA. The experiments showed that retrograde labeling from the NRA mainly occurred in the rostral and intermediate ipsilateral PAG, while injections involving both NRA and NPRA resulted in numerous labeled neurons in the ipsilateral rostral, intermediate and especially the caudal PAG. The anterograde tracing studies confirmed these projections: from the rostral PAG almost exclusively to the NRA and from the caudal PAG to the NPRA, while the intermediate lateral PAG projects to both NPRA and NRA. Our double-immunostudies revealed that ER-α-IR projections descend only towards the NPRA and mainly originate from the ipsilateral caudal PAG. Retrogradely labeled ER-α-IR neurons in the PAG were observed in two separate columns, laterally and ventrolaterally in the caudal half of the PAG. The results provide evidence for the existence of differentiated PAG-CVLM projections to NRA and NPRA, respectively, originating from discrete longitudinal “PAG-columns.” Only the projection to the NPRA is estrogen receptive, supporting the hypothesis that the NPRA is involved in the adaptive changes in autonomic control during successive phases of the estrous cycle.

Morphological Changes of the Sensory Neurons in the Peripheral Neuropathy of Rat Tibial Nerve Using WGA-HRP Tracing Method

Neuropathy is a general term referring to disorders of nerves, and produces when the nerves are damaged. It is characterized by spontaneous pain, allodynia and hyperalgesia. The purpose of present study is to observe the number of WGA-HRP (wheat germ agglutinin-horseradish peroxidase) labelded sensory neurons of DRG (dorsal root ganglia), and distributions according to cell size of sensory neuron in tibial nerve ligation model (NLM). The tibial nerve ligation was performed with 3-0 silk by the application of three tight ligatures at the mid-thigh level. In the neuropathy model of rat tibial nerve ligation, morphological changes of sensory neurons in DRG were observed using WGA-HRP. Rats of NLM showed the neuropathic behaviors. Rats were shown guarding affected limb and limping. Their toes and ankle joint of operated limb were hyperflexed. Under light microscopy, tibial nerve showed degeneration of axons in NLM. In control and NLM, labeled sensory neurons of tibial nerve distributed L4 and L5 DRG. In control group, the labeled sensory neurons were round or oval in shape. They were large and small cells, and mixed pattern. Total number of labeled sensory neurons in NLM decreased significantly from control group. The number of labeled sensory neurons in L4 and L5 DRG decreased significantly from control group. Labeled large and small cells decreased significantly from control group. Present study may serve as the basic information about the changes of DRG sensory neurons in NLM.

West Nile virus preferentially transports along motor neuron axons after sciatic nerve injection of hamsters

Prior findings led us to hypothesize that West Nile virus (WNV) preferentially transports along motor axons instead of sensory axons. WNV is known to undergo axonal transport in cell culture and in infected hamsters to infect motor neurons in the spinal cord. To investigate this hypothesis, WNV was injected directly into the left sciatic nerve of hamsters. WNV envelope-staining in these hamsters was only observed in motor neurons of the ipsilateral ventral horn of the spinal cord, but not in the dorsal root ganglion (DRG). To evaluate the consequence of motor neuron infection by WNV, the authors inoculated wheat germ agglutinin-horseradish peroxidase (WGA-HRP) 9 days after WNV sciatic nerve injection, and stained the spinal cord and the DRG for HRP activity 3 days later. The degree of HRP-staining in DRG was the same in WNV- and sham-infected animals, but the HRP-staining in the motor neuron in the ventral horn was considerably less for WNV-infected hamsters. To investigate the mechanism of WNV transport, hamsters were treated with colchicine, an inhibitor of membranous microtubule-mediated transport. The intensity of the WNV-stained area in the spinal cord of colchicine-treated hamsters at 6 days after WNV infection were significantly reduced (P<or=.05) compared to the placebo-treated hamsters. These data suggest that WNV is preferentially transported through the motor axons, but not the sensory axons, to subsequently infect motor neurons and cause motor weakness and paralysis.

Activated autologous macrophage implantation in a large-animal model of spinal cord injury

Axonal regeneration may be hindered following spinal cord injury (SCI) by a limited immune response and insufficient macrophage recruitment. This limitation has been partially surmounted in small-mammal models of SCI by implanting activated autologous macrophages (AAMs). The authors sought to replicate these results in a canine model of partial SCI. Six dogs underwent left T-13 spinal cord hemisection. The AAMs were implanted at both ends of the lesion in 4 dogs, and 2 other dogs received sham implantations of cell media. Cortical motor evoked potentials (MEPs) were used to assess electrophysiological recovery. Functional motor recovery was assessed with a modified Tarlov Scale. After 9 months, animals were injected with wheat germ agglutinin-horseradish peroxidase at L-2 and killed for histological assessment. Three of the 4 dogs that received AAM implants and 1 of the 2 negative control dogs showed clear recovery of MEP response. Behavioral assessment showed no difference in motor function between the AAM-treated and control groups. Histological investigation with an axonal retrograde tracer showed neither local fiber crossing nor significant uptake in the contralateral red nucleus in both implanted and negative control groups. In a large-animal model of partial SCI treated with implanted AAMs, the authors saw no morphological or histological evidence of axonal regeneration. Although they observed partial electrophysiological and functional motor recovery in all dogs, this recovery was not enhanced in animals treated with implanted AAMs. Furthermore, there was no morphological or histological evidence of axonal regeneration in animals with implants that accounted for the observed recovery. The explanation for this finding is probably multifactorial, but the authors believe that the AAM implantation does not produce axonal regeneration, and therefore is a technology that requires further investigation before it can be clinically relied on to ameliorate SCI.

Central projections of the sensory innervation of the rat middle meningeal artery

Headaches, especially migraine, involve not only pain but also aspects such as vasodilation of cranial vessels and sensitization of nerve endings, processes dependent on and connected to the central nervous system. To understand pathogenic mechanisms of headache, it is important to elucidate the central projections of sensory nerves that innervate cranial vessels, of which the middle meningeal artery (MMA) is the largest artery supplying the dura mater. In this study, cholera toxin subunit b (CTb) or wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) was applied on the adventitia of MMA. After perfusion fixation, the brainstem, the C1–C4 spinal segments and the trigeminal and C2 dorsal root ganglia were removed and sections from these tissues were processed to visualize transported tracers. Labeled cell bodies were seen ipsilaterally in the trigeminal and C2 dorsal root ganglia. Labeled nerve terminations were found ipsilaterally in the lateral part of the spinal dorsal horn of segments C1–C3 and in the caudal and interpolar parts of the spinal trigeminal nucleus. WGA-HRP labeled terminations were mainly located in laminae I and II, whereas CTb labeled terminations located in laminae III–V. These results indicate that sensory information from the MMA is transmitted through both trigeminal and cervical spinal nerve branches to a region in the central nervous system extending rostrally from the C3 dorsal horn to the interpolar part of the spinal trigeminal nucleus. Our data further substantiates that the sensory innervation of the MMA, in addition to putative nociceptive afferents, include a population of large caliber afferents with an as yet unclear but presumably non-nociceptive role.

Sympathetic nerve fibers sprout into rat odontoblast layer, but not into dentinal tubules, in response to cavity preparation

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the dentinal tubules in response to artificial cavity preparation in dentin. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG). They were then observed using light and electron microscopes. In normal dental pulp (control), scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in the odontoblast layer and subodontoblastic region. In injured pulp 3 weeks after cavity preparation, reaction products were about 1.8-times more plentiful in the above areas (versus control pulp). However, no labeled nerve fibers were observed in the dentinal tubules in either control or injured dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in rat dental pulp in response to cavity preparation, they do not penetrate into the dentinal tubules in which postganglionic nerve endings derived from the SCG were not originally present.

Organization of histamine-immunoreactive, tuberomammillary neurons projecting to the dorsal tier of the substantia nigra compacta in the rat

Following the injection of a retrograde tracer, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold), into the dorsal tier of substantia nigra compacta (SNCD), histamine immunostaining was performed for the tuberomammillary nucleus (TMN) in order to investigate the projection from the TMN to the SNCD. At the rostral pole of the TMN, the retrograde labeling in the dorsomedial subdivision following medial SNCD injections was predominantly ipsilateral (78%), whereas only a few cells were located bilaterally following lateral SNCD injections. Following tracer injections along the medio-lateral location along the SNCD, the labeling at the ventrolateral TMN was bilateral with slightly ipsilateral (58–61%) dominance. At rostral and caudal TMN levels, clusters of labeled neurons were localized within two discrete columns of the ventrolateral TMN. At rostral TMN level, a lateral column of cells was located at the lateral tip of the ventrolateral TMN just medial to the internal capsule, while the medial column was close to the protruded region along the ventral, pial border. At the caudal TMN level, two columns were located on either side of the lateral mammillary nucleus. Taken together, the present study suggests that ventrolateral as well as dorsomedial TMN might provide arousal-related information to medial, intermediate, and lateral regions of the SNCD, which in turn influence extrapyramidal, behavioral functions performed by the substantia nigra compacta.

Adenovirus‐mediated retrograde transfer of neurotrophin‐3 gene enhances survival of anterior horn neurons of twy/twy mice with chronic mechanical compression of the spinal cord

Chronic mechanical compression of the spinal cord causes neural tissue damage, including loss of anterior horn cells around the level of injury. Exogenous delivery of neurotrophins to neuronal cells could provide neuroprotection to a spinal cord subjected to mechanical injury. We investigated the efficacy of retrograde gene delivery of adenoviral vector (AdV) carrying neurotrophin-3 (NT-3) gene into twy (twy/twy) mouse spinal cord anterior horn neurons with chronic and progressive mechanical compression at C1-C2 level. AdV-NT-3 was used for retrograde delivery via the sternomastoid muscle to the cervical spinal accessory motoneurons in 16-week-old adult twy mice with relatively mild spinal cord compression. Four weeks after the AdV-NT-3 or AdV-beta-galactosidase cDNA (LacZ) as a marker gene injection, the compressed cervical spinal cord was examined histologically, immunohistologically, and by immunoblot analysis. Immunoreactivity to NT-3 was significantly enhanced in the AdV-NT-3-injected twy mice compared with the AdV-LacZ-injected mice. The numbers of anterior horn neurons of Nissl-, choline acetyltransferase (ChAT)-, and trkC-stained and wheat germ agglutinin-horseradish peroxidase (WGA-HRP)-labeled neurons at the spinal cord level with maximum compression were significantly higher in AdV-NT-3-transfected than in AdV-LacZ-transfected twy mice. Retrograde NT-3 gene transfer to twy mouse anterior horn neurons increased neurite axonal length and arborization of WGA-HRP-labeled neurons. Our results suggest that targeted retrograde NT-3 gene delivery is feasible in the intact animal and that it enhances neuronal survival even under chronic mechanical compression of the spinal cord.

Impairment of Retrograde Neuronal Transport in Cardiac Vagal Motoneurons in Streptozotocin-Induced Diabetic Rats: A Wheat Ger Agglutinin-Horseradish Peroxidase Neurohistochemical study

Central projections of vagal motoneurons to the heart were studied in diabetic rats using Wheat germ Agglutinin-Horseradish peroxidase (WGA-HRP). Experimental rats were rendered diabetic by intraperitoneal injection of streptozotocin in citrate buffer. The diabetic rats were maintained in a stable diabetic state by daily injection of insulin for 24 weeks. Age-matched control rats were injected intraperitoneally with citrate buffer not containing streptozotocin. Control rats were also kept alive for 24 weeks after citrate buffer injection. At the end of 24 weeks the two groups were prepared for injection with WGA-HRP. Following anesthesia with sodium pentobarbitone, thoracotomy was performed on the left aspect of the thorax to expose the heart. The atrial and ventricular walls were then injected with 5% WGA-HRP by multiple intramuscular penetrations. Experimental and control rats were sacrificed 72 h after tracer injection by transcardial perfusion first with normal saline followed by fixative and then buffered sucrose. Transverse serial frozen sections of the brainstem were then taken and processed for WGA-HRP neurohistochemistry and analyzed under light and dark-field microscopy. Analysis of the sections taken from diabetic rats revealed fewer WGA-HRP labeled neurons in the nucleus ambiguus (nA) than sections taken from control rats. Sporadic labeling of the dorsal motor nucleus of the vagus nerve was observed in control rat but not in the diabetic rats. It was concluded that the depletion of labeled neurons in the diabetic rats compared with the normoglycaemic rats is indicative of impairment of retrograde neuronal transport of WGAHRP in chronic diabetic state.

The nose may help the brain: intranasal drug delivery for treating neurological diseases

While enormous progress has been made regarding our understanding of the pathogenic mechanisms of neurological diseases, there are only a small number of effective drugs for treating these illnesses. A key obstacle for developing effective drugs for treating neurological diseases is the blockage of drug entrance into the CNS by the BBB [1]. Less than 2% of all small-molecule drugs, and virtually no large-molecule drugs, can cross the BBB. Therefore, it is of critical significance to search for drug-delivery strategies that can effectively deliver drugs into the CNS. An increasing number of studies on both animals and human subjects have suggested that intranasal drug delivery could be used to deliver both smalland largesized drugs into the CNS by bypassing the BBB. It appears increasingly reasonable to conduct clinical trials to determine if intranasal drug delivery may be used to treat neurological diseases. In the 1970s and 1980s there were multiple studies suggesting that intranasal administration may enable substances to directly enter into the brain by pathways involving the olfactory epithelium and olfactory bulb [2]. In 1995 Thorne et al. reported the first quantitative study indicating that intranasal administration could deliver large-sized molecules into the brain by bypassing the BBB [3]. Intranasal administration of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) led to a significant presence of WGA-HRP in the olfactory bulb of rats, while there was no detectable amount of WGA-HRP in the olfactory bulb after intravenous injection of the same concentration of WGA-HRP. Since 1997, many studies have indicated that intranasal administration can enable large-sized molecules, such as IGF-1, FGF-2, TGF-β1, erythropoietin, IFN-β, HIV-1 Tat, insulin and leptin, to be transported into the CNS at least partially through direct nose-to-brain routes [2,4–9]. A number of studies using animal models of neurological diseases have demonstrated that intranasal delivery of large-sized molecules can produce beneficial effects. For example, intranasal nerve growth factor (NGF) administration can attenuate memory deficits and neurodegeneration in transgenic models of Alzheimer’s disease (AD) [10]; administration of erythropoietin [8] or IGF-I [11] by the intranasal approach can significantly decrease ischemic brain damage; and intranasal delivery of growth factors can also increase neurogenesis in rat brains [9]. We have conducted studies to directly compare the efficacy of intranasal drug delivery with that of intravenous drug delivery in treating brain ischemia. Based on the cell-culture studies showing the protective effects of gallotannin (GT) and nobotanin B – two inhibitors of poly(ADP-ribose) glycohydrolase – against oxidative cell injury [12], we found that intranasal delivery of GT is much more effective than intravenous injection of GT in decreasing ischemic brain injury [13]. On the basis of the in vitro findings that nicotinamide adenine dinucleotide (NAD+) treatment can prevent oxidative stress-induced cell death [14], we also found that intranasal NAD+ administration can produce up to 90% decreases in infarct formation when given 2 h after ischemia, which is the most profound protection ever reported from drugs that have been administered hours after ischemic onset [15,16].

Manganese-Enhanced MRI of Brain Plasticity in Relation to Functional Recovery after Experimental Stroke

Restoration of function after stroke may be associated with structural remodeling of neuronal connections outside the infarcted area. However, the spatiotemporal profile of poststroke alterations in neuroanatomical connectivity in relation to functional recovery is still largely unknown. We performed in vivo magnetic resonance imaging (MRI)-based neuronal tract tracing with manganese in combination with immunohistochemical detection of the neuronal tracer wheat-germ agglutinin horseradish peroxidase (WGA-HRP), to assess changes in intra- and interhemispheric sensorimotor network connections from 2 to 10 weeks after unilateral stroke in rats. In addition, functional recovery was measured by repetitive behavioral testing. Four days after tracer injection in perilesional sensorimotor cortex, manganese enhancement and WGA-HRP staining were decreased in subcortical areas of the ipsilateral sensorimotor network at 2 weeks after stroke, which was restored at later time points. At 4 to 10 weeks after stroke, we detected significantly increased manganese enhancement in the contralateral hemisphere. Behaviorally, sensorimotor functions were initially disturbed but subsequently recovered and plateaued 17 days after stroke. This study shows that manganese-enhanced MRI can provide unique in vivo information on the spatiotemporal pattern of neuroanatomical plasticity after stroke. Our data suggest that the plateau stage of functional recovery is associated with restoration of ipsilateral sensorimotor pathways and enhanced interhemispheric connectivity.

Evidence for corticocortical connections between areas 7 and 17 in cerebral cortex of the cat

This study provides the first evidence of direct corticocortical connections between areas 7 and 17 of the cat. Wheat germ agglutinin horseradish peroxidase (WGA-HRP) was administrated by micro-electrophoresis and micro-injection, respectively, into area 17 and area 7 in different hemispheres in eight cats. WGA-HRP labeled pyramidal neurons were observed primarily in layer 5 of areas 7 and 17 indicating that there are reciprocal connections between these areas. Optical imaging was used to guide WGA-HRP injections to single orientation columns in area 17. After such restricted injections labeled pyramidal cells were observed in layer 5 of area 7. These pyramidal cells were arranged as discontinuous patches extending across a broad region of area 7. These results suggest that feedback from area 7 to area 17 may arise from specific functional columns in area 7.

Commissural Mirror-Symmetric Excitation and Reciprocal Inhibition Between the Two Superior Colliculi and Their Roles in Vertical and Horizontal Eye Movements

The functional roles of commissural excitation and inhibition between the two superior colliculi (SCs) are not yet well understood. We previously showed the existence of strong excitatory commissural connections between the rostral SCs, although commissural connections had been considered to be mainly inhibitory. In this study, by recording intracellular potentials, we examined the topographical distribution of commissural monosynaptic excitation and inhibition from the contralateral medial and lateral SC to tectoreticular neurons (TRNs) in the medial or lateral SC of anesthetized cats. About 85% of TRNs examined projected to both the ipsilateral Forel's field H and the contralateral inhibitory burst neuron region where the respective premotor neurons for vertical and horizontal saccades reside. Medial TRNs received strong commissural excitation from the medial part of the opposite SC, whereas lateral TRNs received excitation mainly from its lateral part. Injection of wheat germ agglutinin-horseradish peroxidase into the lateral or medial SC retrogradely labeled many larger neurons in the lateral or medial part of the contralateral SC, respectively. These results indicated that excitatory commissural connections exist between the medial and medial parts and between the lateral and lateral parts of the rostral SCs. These may play an important role in reinforcing the conjugacy of upward and downward saccades, respectively. In contrast, medial SC projections to lateral SC TRNs and lateral SC projections to medial TRNs mainly produce strong inhibition. This shows that regions representing upward saccades inhibit contralateral regions representing downward saccades and vice versa.

Projection patterns from the amygdaloid nuclear complex to subdivisions of the dorsal raphe nucleus in the rat

The goal of the present study was to identify the projection from the subdivisions of the amygdaloid nuclear complex to specified subregions of the dorsal raphe (DR) nucleus and to attempt to compare the density of amygdaloid input to the DR with that of inputs from other limbic structures. Use of a retrograde tracer, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold), demonstrated that amygdaloid input to midline DR subdivision originates mainly from the medial portion of the medial amygdaloid nucleus, whereas that to lateral wing subdivision derives from the region extending from the lateral portion of the medial amygdaloid nucleus to the commissural stria terminalis. Use of the retrograde tracer Fluorogold (FG) produced relatively large but circumscribed injection sites comprising midline DR as well as portions of lateral wing subdivision and confirmed that the medial amygdaloid nucleus provides the major input to the DR. We also demonstrated that although amygdaloid input was not as extensive as inputs from other limbic structures such as the medial prefrontal cortex or the lateral habenular nucleus, it was comparable to input from the lateral septal nucleus. Based on these observations, we suggest that the medial amygdaloid nucleus provides substantial input to the DR and may contribute an emotional influence on sleep–wakefulness cycle or pain–stress modulation. Furthermore, it seems that the medial amygdaloid–DR projection might be anatomically and functionally distinct from the well-characterized central amygdaloid–periaqeductal gray (PAG) circuit which is essential for conditioned fear.

Topographical Representation of Motoneurons Innervating the Transverse Mandibular Muscle in the Trigeminal Motor Nucleus, with Special Reference to Rats

Localization of motoneurons of the transverse mandibular muscle (TM) was examined by a retrograde tracing method (horseradish peroxidase (HRP) and wheat germ agglutinin-HRP (WGA-HRP) ) in the rat (rodent), house musk shrew (Suncus murinus, insectivore) and miniature pig (even-toed, ungulate). In the injection of HRP solution into the TM of the rat, labeled neurons with HRP appeared at the lateral and ventral margins of the dorsolateral division in the rostral two-thirds level of the trigeminal motor nucleus. This distribution area was close to or overlapped that of medial pterygoid and lateral pterygoid motoneurons. TM motoneurons of the shrew and pig were located in the ventromedial part of the dorsolateral division at the rostral half level of the nucleus. The distribution patterns of each masticatory muscle motoneuron in the rat were also confirmed for comparison with those of TM motoneurons. Electromyography during feeding in the rat showed that TM activity almost synchronized with the activities of both jaw-closer and jaw-opener muscles.