Wheat Germ Agglutinin-conjugated Horseradish Peroxidase Research Articles

GABAergic neurons in the rat dentate gyrus are innervated by subcortical calretinin‐containing afferents

Fibers of supramammillary origin establish putatively excitatory asymmetric synaptic connections with dentate granule cells. The present study was designed to determine whether hippocampal γ-aminobutyric acid (GABA)-ergic nonprincipal cells are also targets of these calretinin (CR)-containing subcortical afferents. Light and electron microscopic double immunostaining for CR and parvalbumin (PA) or calbindin (CB) were performed in the rat dentate gyrus ipsilateral and contralateral to a unilateral fimbria-fornix transection. GABA-postembedding immunostaining was performed on ultrathin sections of this double-labeled material. Contralateral to the transection, CR-immunoreactive fibers formed multiple large boutons in the inner molecular layer. These fibers also impinged on PA-containing basket cells located adjacent to the granular layer and on CB-immunoreactive hilar neurons. Ipsilateral to the transection, CR-containing fibers in the inner molecular layer and boutons impinging on PA-containing or CB-immunoreactive neurons were absent. Parent cell bodies of extrinsic CR-containing afferents were traced using wheat germ agglutinin-conjugated horseradish peroxidase. Additional CR immunostaining of the subcortical region unveiled retrogradely labeled neurons that were also immunostained for CR only in the supramammillary area and the nucleus reuniens. The latter projection, however, terminates in CA1 and not in the dentate gyrus. Subcortical afferents impinging on dentate nonprincipal cells formed exclusively asymmetric synapses. Postembedding immunostaining demonstrated that CB-containing cells contain GABA, whereas CR-positive axon terminals forming asymmetric synapses are devoid of this labeling. These data indicate that dentate inhibitory neurons receive a putative excitatory input originating from the supramammillary nucleus. Thus, the supramamillo-hippocampal pathway may exert a powerful feed-forward inhibitory control of the signal flow in the rat dentate gyrus. © 1996 Wiley-Liss, Inc.

Innervation of propatagial musculature in a flying squirrel, Glaucomys volans (Rodentia, Sciuridae).

The propatagium of gliding and flying mammals is of both functional and phylogenetic interest. The innervation of the propatagial muscle, platysma II, was studied with the axonal tracer wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) in a flying squirrel, Glaucomys volans. Injections of WGA-HRP into the proximal third of platysma II labeled motoneurons in the lateral part of the medial subdivision of the ipsilateral facial nucleus and in the ipsilateral ventral horn of the brachial enlargement. Injections into distal regions of platysma II labeled motoneurons in the ipsilateral ventral horn of spinal segments C5-C8 but not in the facial nucleus. Injections along the whole length of the muscle labeled afferent axons in the ipsilateral dorsal horn of spinal segments C4-T1. These results demonstrate a mixed facial and spinal motor innervation of propatagial musculature in the flying squirrel and indicate that this pattern of mixed innervation is more widespread among flying and gliding mammals than previously reported. Mixed facial and cervical propatagial innervation, independently derived in different flying and gliding mammals, may represent a common solution in the design of the propatagium. These findings complicate the use of propatagial muscle innervation patterns for the establishment of phylogenetic relationships among flying and gliding mammals.

Loss of neurons in the dorsal root ganglia after transection of a peripheral sensory nerve. An anatomical study in monkeys.

Injury to a sensory nerve often results in a poor long term outcome, partly because of sensory motor mismatch of regenerating axons at the transection site. We studied nine macaque monkeys and found that 27% of nerve cells in the projecting dorsal root ganglia had been lost 21 months after transection and suturing of the radial sensory nerve. No specific cell sizes were lost and the reduction was evenly distributed in the affected ganglia in which neurons had been labelled with a mixture of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) and HRP alone.

The nucleus of the optic tract (NOT) and the dorsal terminal nucleus (DTN) of opossums (Didelphis marsupialis aurita).

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected unilaterally into the pretectocollicular region of opossums (Didelphis marsupialis aurita), primarily to investigate the existence of a commissural subcortical pathway but also to reveal afferents and efferents of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in this species. Labelled cells and terminals were observed in the contralateral NOT-DTN. Furthermore, HRP was injected bilaterally in the region of the inferior olive (IO) to verify if the distribution of labelled cells in the NOT-DTN overlapped the region of commissural labelled cells. The two subpopulations of retrogradely labelled cells coincided, being distributed within the retinal terminal field attributed to the NOT-DTN, as revealed by contralateral eye injections of HRP. The commissural cells were located slightly more ventral than the olivary cells in the optic tract. The pretectocollicular WGA-HRP injections also labelled cells and terminals bilaterally in the lateral terminal nucleus (LTN), interstitial nucleus of the superior fasciculus, posterior fibers (INSFp), ventral lateral geniculate nucleus (vLGN), and superior colliculus (SC) and ipsilaterally in the medial terminal nucleus (MTN). In addition, further caudally, labelled cells and terminals were observed bilaterally in the nuclei prepositus hypoglossi (PH) and in the medial (MVN) and lateral (LVN) vestibular nuclei. Labelled terminals were found in the ipsilateral nucleus reticularis tegmenti pontis (NRTP) and in the IO with ipsilateral predominance. This study allowed an anatomical delimitation of the NOT-DTN in this opossum species, as defined by the olivary and commissural subpopulations, as well as a hodological evaluation of this region. The existence of some common anatomical aspects with other mammalian species is discussed.

Early decline and late restoration of spinal cord binding and transganglionic transport of isolectin B4 from Griffonia simplicifolia I after peripheral nerve transection or crush

Isolectin B4 from Griffonia simplicifolia I (B4) has a high binding affinity to a large population of unmyelinated primary sensory neurons (Wang et al., Neuroscience 62 (1994) 539-551). Using immunohistochemical techniques, binding and transganglionic transport of B4 in the spinal cord was investigated, both at short and long survival times, after sciatic nerve transection and ligation or crush in the adult rat. Nerve transection and ligation resulted in nearly complete disappearance of B4 immunolabelling in the sciatic nerve territory of the superficial dorsal horn after B4 binding, as well as after transganglionic transport of B4 by 2 weeks postinjury. Partial recovery of both B4 binding and B4 transport was found by 8 months, and nearly complete recovery by 16 months, indicating that reappearance of B4 binding is not critically dependent on peripheral reinnervation. Crush injury made by jeweller's forceps resulted in partial depletion of binding and transport by 2 weeks and a nearly complete recovery by 10 weeks. The results show that binding and transganglionic transport of B4 can be used to label dorsal horn connections of unmyelinated primary afferents during the process of regeneration after crush injury. Furthermore, as B4 binding and transport recover at long survival times in the absence of reestablished peripheral connections, the same techniques can be used to study central primary afferent connections at long survival times after nerve transection. Binding and transganglionic transport of B4 offer alternatives to the use of previous techniques such as transganglionic transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) to study central connections of fine primary afferents after injury.

Connections between the reticular nucleus of the thalamus and pulvinar-lateralis posterior complex: a WGA-HRP study.

The present study utilises the capacity of wheat germ agglutinin-conjugated horseradish peroxidase to label both afferent and efferent projections from selected regions of the thalamic reticular nucleus (TRN) to the pulvinar lateralis-posterior complex (Pul-LP) of the cat. Fourteen injections into the TRN located between anterior-posterior levels 8.5 and 4.5 were analysed. The projection of the TRN to the Pul-LP complex is roughly organised in a topographic manner and is not widespread within the thalamus. Anterograde labelling in the Pul-LP extended rostrocaudally with a slight oblique dorsoventral orientation. Projections to the medial LP were predominantly but not exclusively from rostral areas of TRN, while projections to the lateral LP were largely from caudal areas of the TRN. Projections to other areas of the Pul-LP were sparse. The connections between TRN and Pul-LP were reciprocal, although the distribution of labelled cells and anterograde labelling was not completely overlapping. Reciprocal connections with the dorsal lateral geniculate nucleus were largely with the C-laminae and the medial interlaminar nucleus. The results are discussed with reference to the corticothalamic projections and the visuotopy of the Pul-LP.

Motor and somatosensory corticostriatal projection magnifications in the squirrel monkey.

1. Motor and somatosensory cortex project massively to the primate striatal matrix, terminating in distributed sets of overlapping projection zones (matrisomes) within the putamen. To study this system quantitatively, we have developed a computer-assisted estimation of the changes in magnification that occur as motor and somatosensory cortical body representations are projected onto the putamen. 2. Cortical and striatal body maps were assessed in squirrel monkeys by injecting anterograde tract tracers into electrophysiologically identified body-part representations in cortical areas 4, 3a, 3b, and 1. Relative projection magnification was defined as the ratio of the cortical injection site volume to the striatal projection site volume. 3. Magnification comparisons indicate that the tracers wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) and 35S-methionine have similar sensitivities. 4. The relative proportions of body-part representations in the striatal maps were not significantly different from those in cortical maps. Both had large representations of hand, foot, and mouth, and smaller representations of trunk. 5. The relative magnification of the motor cortex projection to the striatum was roughly twice as large as those of projections from individual somatosensory areas. 6. These findings suggest that, in the sensorimotor striatum, motor and somatosensory inputs may undergo different proportions of local processing at the borders of their distribution zones (striosomes and matrisomes).

Extrinsic and intrinsic substance P innervation of the rat lateral septal area calbindin cells.

The electrophysiological observations that substance P administration to the lateral septal area elicits both excitatory and inhibitory responses, together with earlier reports on the multiple sources of substance P innervation of the septum, implies that these axons with distinct origins have different functions. This prompted us to examine the origin and neurochemical character of substance P afferents to the lateral septal area. Chronic surgical isolation of the septum from its ventral afferents and retrograde tracer experiments using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for substance P, were employed to elucidate the origin of these axon terminals. In order to assess the possible co-existence of substance P with other neurotransmitter substances in the parent cells of the septopetal projections, co-localization studies for substance P and choline acetyltransferase, as well as substance P and GABA, were performed. The comparative distribution of substance P fibers and septal calbindin-containing neurons was also investigated using correlated light and electron microscopic double immunostaining. The results are summarized as follows: (i) the substance P innervation of the lateral septal area derives from several hypothalamic nuclei (including the lateral and lateroanterior hypothalamic area, tuber cinereum and ventromedial hypothalamic nucleus) and tegmental nuclei (the majority of fibers from the laterodorsal and a few from the pedunculopontine tegmental nucleus), as well as intrinsic septal cells; (ii) the septopetal substance P fibers of tegmental origin are cholinergic; intraseptal substance P neurons located in the dorsolateral part of the lateral septum also contain GABA, while substance P neurons seen on the border between the medial and lateral septal area and septopetal hypothalamic substance P cells do not contain GABA or acetylcholine; (iii) substance P fibers from pericellular baskets around calbindin-containing lateral septal neurons with a high degree of selectivity; (iv) approximately 90% of the entire calbindin cell population are postsynaptic targets of substance P axons; (v) their terminals contact the soma and the dendrites of these cells, among them the somatospiny neurons; and (vi) the extrinsic substance P boutons establish asymmetric, while the intrinsic substance P axon terminals form symmetric membrane specializations. Because neurons in the lateral septal area receive hippocampal input and project massively to hypothalamic areas, the different types of substance P input on these neurons can modify the information flow arriving from the hippocampus to diencephalic brain structures at the level of the lateral septal area.

Patchy and laminar terminations of medial geniculate axons in monkey auditory cortex.

The object of this study was to identify the terminal distributions of thalamocortical axons arising in chemically characterized subdivisions of the medial geniculate complex. Large injections of wheat germ agglutinin-conjugated horseradish peroxidase or small injections of Phaseolus vulgaris leucoagglutinin were made in the medial geniculate complex of Macaca fuscata. The terminal distributions of labeled axons in the cortex were correlated with auditory cortical fields demonstrable by different intensities of immunoreactivity for parvalbumin. Fibers from the ventral nucleus terminated mainly in layer IV and deep portion of layer III (IIIB), with additional terminations in layers I-IIIA and in layer VI. In layers IIIB-IV, a major terminal plexus was formed by a small number of dense patches, 300-500 microns in diameter, surrounded by smaller satellite patches. The patches conformed to a similarly lobulated pattern of parvalbumin fiber immunoreactivity. Terminations of some individually labeled thalamocortical fibers were restricted to a single patch, whereas others innervated more than one patch by collateral branches. Fibers from the dorsal nuclei ending in areas of less dense parvalbumin immunoreactivity surrounding the primary auditory cortex formed much larger terminal patches centered largely in layer IIIB. Fibers from the magnocellular nucleus had relatively few terminal branches but innervated extremely wide areas by collaterals of single axons. Two types of axons arose from the magnocellular nucleus, one terminating preferentially in middle cortical layers and the other exclusively in layer I. These may arise respectively from parvalbumin- and calbindin-immunoreactive cell populations in the magnocellular nucleus.

Morphological diversity and glutamate immunoreactivity of retinal terminals in the suprachiasmatic nucleus of the cat.

Although the cat visual system has been the subject of intensive investigation, little attention has been given to the morphological features of ganglion cell projections to the suprachiasmatic nucleus. The present study has utilized anterograde transport of horseradish peroxidase and wheat germ agglutinin-conjugated horseradish peroxidase to label ganglion cell terminals in the cat suprachiasmatic nucleus. Visualization of the reaction product was facilitated through the use of gold-substituted silver intensification. Ganglion cell terminals were found to be morphologically diverse, making both asymmetric and symmetric contacts with postsynaptic processes. Synaptic vesicles were either scattered or densely packed, sometimes forming paracrystalline arrays. In contrast to other retinorecipient areas in which ganglion cell terminals have been characterized by the presence of lightly staining mitochondria, many of the retinal terminals in the suprachiasmatic nucleus were seen to contain darkly stained mitochondria. Postembedding antiglutamate immunocytochemistry was used to evaluate the level of endogenous glutamate in these ganglion cell terminals. Although morphologically diverse, all of the retinal terminals in the suprachiasmatic nucleus were glutamate positive, consistent with the postulated role of glutamate as the neurotransmitter of retinal ganglion cells.

Hypothalamic Leu-enkephalin-immunoreactive fibers terminate on calbindin-containing somatospiny cells in the lateral septal area of the rat.

Correlated light and electron microscopic double-immunostaining experiments for Leu-enkephalin and calbindin were employed to determine the postsynaptic targets in the septal complex of Leu-enkephalin fibers. Chronic surgical isolation of the septal complex from its hypothalamic afferents and retrograde tracer studies using wheat germ agglutinin-conjugated horseradish peroxidase, both followed by an immunostaining for Leu-enkephalin, were performed to elucidate the location of the origin of these axon terminals. Furthermore, a colocalization study for glutamic acid decarboxylase and Leu-enkephalin was carried out on hypothalamic sections to determine their possible coexistence in cells projecting to the lateral septum. These studies revealed that 1) Leu-enkephalin-immunoreactive axons form pericellular baskets around a population of lateral septal area neurons; 2) they establish exclusively asymmetric synaptic contacts on their soma and initial dendritic segments; 3) 10% of the lateral septal area calbindin-containing cells, which are all of the gamma-aminobutyric acid (GABA)-ergic somatospiny type, are innervated by Leu-enkephalin-immunoreactive baskets; 4) only 40% of the Leu-enkephalin target neurons are calbindin immunopositive; 5) the septopetal Leu-enkephalin fibers derive from neurons located in the ipsilateral perifornical area and anterior hypothalamus; and 6) none of their cells of origin cocontains the inhibitory transmitter GABA. These observations indicate that hypothalamic Leu-enkephalin-containing neurons are non-GABAergic excitatory cells. Hence, they can effectively stimulate a population of lateral septal area neurons, including the somatospiny cells, which are all GABAergic. Therefore, after stimulatory Leu-enkephalin action, these neurons can inhibit their postsynaptic targets, including other projective lateral septal neurons.

EMG analysis of harmaline-induced tremor in normal and three strains of mutant mice with Purkinje cell degeneration and the role of the inferior olive

1. The effects of intraperitoneal injections of 10 mg/kg harmaline were tested in normal mice and three strains of cerebellar mutant mice with Purkinje cell degeneration. Ten normal (wild-type) mice (+/+), as well as five lurcher (lc/+), six nervous (nr/nr), and eight Purkinje cell degeneration (pcd/pcd) mutants were implanted with chronic electromyogram (EMG) electrodes in the hamstring and quadriceps muscle groups of the right hindlimb. 2. EMGs were recorded in each of the mice during spontaneous activity before and after intraperitoneal injections of 0.3 ml harmaline (10 mg/kg). Spectral analysis was used to quantify the amplitude and frequency of tremor found in the EMGs after harmaline administration. Normal mice responded to harmaline with strong, continuous 11- to 14-Hz tremor. Mutants from the pcd/pcd strain also reacted with continuous tremor, but of lower amplitude and frequency. In contrast, nr/nr mutants exhibited intermittent paroxysmal tremor lasting for only a few seconds, and lc/+ mutants showed no evidence of tremor whatsoever. 3. In order to detect covert tremor that was possibly not revealed by focal intramuscular EMG recordings, several mutant and normal mice were also tested on a suspended platform to which an accelerometer was attached. The results confirmed the findings from EMG recordings. 4. An incidental observation made during the course of this study was that harmaline tremor disappeared from the normal mouse during swimming and reappeared when the animal was withdrawn from the water. 5. Although Purkinje cells appeared to increase both the depth of modulation and the frequency of tremor, the inhibitory action of the cerebellar cortex does not seem to be essential for the generation of tremor. 6. Parasagittal cerebellar sections of the normal, wild-type mice and the three strains of cerebellar mutant mice of various ages were stained with cresyl violet and examined for Purkinje cell degeneration. Purkinje cell degeneration was found to be complete in the pcd/pcd and lc/+ strains. Although an initial examination of parasagittal sections of the nr/nr strain failed to find any surviving Purkinje cells, further examination of sections cut in the coronal plane revealed small clusters of Purkinje cells in the vermal area of the posterior lobe. 7. The retrograde transport of wheat-germ-agglutinin-conjugated horseradish peroxidase (WGA-HRP) pressure-injected into the cerebellar cortex was used to study the olivocerebellar projections in the wild-type mice and the three strains of cerebellar mutant mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Neural connections of auditory association cortex with the posterior cingulate cortex in the monkey

Clinical studies have indicated that the posterior cingulate cortex is intimately involved in verbal and auditory memory. The present study was performed to obtain anatomical evidence for the above proposal. The connections of the auditory cortical areas with the posterior cingulate cortex in the macaque monkey were examined by retrograde and anterograde tracing methods using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). WGA-HRP was injected into either area TA, TB or TC in the superior temporal auditory cortex. Area TA was reciprocally connected with the posterior cingulate cortex, whereas areas TC and TB were not. The rostral two-thirds of area TA had major connections with the caudomedial lobule in the retrosplenial cortex (CML of Goldman-Rakic et al., 1984) and a minor one with area 23b. The caudal third of area TA was connected only with area 23b. However, neither labeled cells nor terminals were observed in areas 23a, 23c, 29, 30 or 31 in the posterior cingulate cortex following a WGA-HRP injection into the caudal, intermediate or rostral portion of area TA. The present finding suggests that verbal and auditory memory impairment in patients with damage to the posterior cingulate cortex is largely due to damage to the CML and area 23b and not to the other posterior cingulate areas.

Origin of the calcitonin gene-related peptide-immunoreactive nerve fibers in the rat shoulder joint.

The rat shoulder joint capsule is innervated by thin sympathetic and sensory nerve fibers, most of which contain calcitonin gene-related peptide (CGRP). In order to establish the origin and distribution of CGRP-immunoreactive (IR) fibers, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the shoulder joints of rats via a dorsal surgical approach. After WGA-HRP injection, the cervico-thoracic dorsal root ganglia (DRG) were removed and processed using both HRP histochemistry and CGRP immunohistochemistry. In the C4 to C7 DRG, small to medium-sized neurons (20-40 microns) were labeled by this combined method. The number and size of the labeled neurons were measured in the cervical 4th-7th DRG. The number of double-labeled neurons was one quarter of the total number of HRP-labeled neurons and 1/20 of the CGRP-IR neurons. Most of the double-labeled cells were located in the C6 ganglion, and the mean number of double-labeled neurons was 13 at this level. This distribution and function of the CGRP-IR fibers in the rat shoulder joint capsule are discussed.

Synaptic organization of the interstitial subdivision of the nucleus tractus solitarii and of its laryngeal afferents in the rat

The nucleus tractus solitarii, the first central relay for gustatory and a variety of visceral afferents, is also an integrative center for numerous functions. Its interstitial subdivision is involved in swallowing and respiratory reflexes. The ultrastructural characteristics of this subdivision and of its laryngeal afferents were investigated in adult rat by a serial-section study and by application of wheat germ agglutinin-horseradish peroxidase conjugate to the peripheral afferent fibers. The interstitial subnucleus contained scattered small neuronal cell bodies with such ultrastructural features as a large nucleus with deep indentations and an organelle-poor cytoplasm. On the basis of their size and vesicular content, the axon terminals were classified into three categories. Group I and group II terminals were small or large, respectively, and contained mainly small, round, and clear synaptic vesicles. Group III terminals were also small but contained small, pleomorphic, and clear vesicles. Axodendritic synapses were the most numerous. They were either asymmetrical, comprised of group I and II terminals, or symmetrical, comprised of group III terminals. More than 50% were part of complex synaptic arrangements in the form of rosettes or glomeruli. Axosomatic contacts involved both group I and group III terminals and were always symmetrical. A high frequency of axoaxonic synapses was found. They were symmetrical, comprised of group III terminals on group I or II terminals. Different types of symmetrical synaptic contacts made by dendrites were also found. This study indicates also that the ipsilateral interstitial subdivision constitutes the preferential site of termination for superior laryngeal afferents. The labeled axon terminals belonged exclusively to groups I and II and were involved in both axodendritic and axoaxonic synapses. Some of the axodendritic synapses were part of rosettes or glomeruli. All these synaptic arrangements may be considered a morphological substrate for important processing of afferent information in the nucleus tractus solitarii. They may account for some of the integrative functions of the interstitial subnucleus such as physiological processes triggered from the superior laryngeal nerve.

Trisynaptic inhibition from the contralateral vertical semicircular canal nerves to neck motoneurons mediated by spinal commissural neurons.

1. Neck motoneurons usually receive disynaptic excitation and inhibition from individual semicircular canal nerves. However, in motoneurons of some neck muscles, trisynaptic inhibition is evoked by stimulation of the contralateral vertical canal nerves. The present study was performed to analyze this pathway and the location and properties of the last-order interneurons responsible for mediating this trisynaptic inhibition from the contralateral vertical canal nerves to neck motoneurons in anesthetized cats. 2. Bipolar stimulating electrodes were implanted on the contralateral anterior (ACN), lateral (LCN), and posterior canal nerve (PCN), and postsynaptic potentials (PSPs) evoked by electrical stimulation of individual canal nerves were intracellularly recorded from motoneurons of the obliquus capitis inferior (OCI), longus capitis (LC), and rectus capitis posterior (RCP) muscles. Stimulation of the contralateral ACN evoked trisynaptic inhibitory PSPs (IPSPs) in OCI and LC motoneurons and disynaptic excitatory PSPs (EPSPs) in RCP motoneurons. Stimulation of the contralateral PCN evoked di- and trisynaptic IPSPs in OCI and RCP motoneurons and disynaptic EPSPs in LC motoneurons. Stimulation of the contralateral LCN evoked disynaptic EPSPs in all of the motoneurons examined. 3. To determine the pathway that mediates these trisynaptic IPSPs from the vertical canal nerves to neck motoneurons, a lesion was made in the lower medulla, and the patterns of PSPs evoked by stimulation of the three contralateral canal nerves were compared before and after the lesion. Interruption of the ipsilateral medial longitudinal fascicle (MLF) abolished all disynaptic EPSPs and IPSPs from the three contralateral canal nerves in OCI, LC, and RCP motoneurons. In contrast, trisynaptic IPSPs evoked by stimulation of the contralateral ACN or PCN remained unaffected by sectioning the MLFs bilaterally. Sectioning of the contralateral lateral vestibulospinal tract (LVST) eliminated the trisynaptic IPSPs in OCI and LC motoneurons evoked by contralateral ACN stimulation and trisynaptic IPSPs in OCI and RCP motoneurons evoked by contralateral PCN stimulation but did not affect disynaptic EPSPs and IPSPs. 4. Stimulation of the contralateral LVST in the lower medulla after sectioning the bilateral MLFs evoked disynaptic IPSPs in OCI, LC, and RCP motoneurons. Because the LVST only projects ipsilaterally, this finding indicates that the last-order interneurons that mediate the trisynaptic inhibition through the LVST are most likely commissural neurons located in the spinal cord. 5. To determine the locations of last-order commissural neurons terminating on OCI motoneurons, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected into the OCI muscle nerve at C1.(ABSTRACT TRUNCATED AT 400 WORDS)

Olfactory bulb transplants establish afferent and efferent connections with host forebrain in rat

We are using wheat germ agglutinin-conjugated horseradish peroxidase (WIiRP) to study reconnectivity in the transplanted (TX) olfactory bulb (OB) in Sprague-Dawley rats. Tritium-labeled OBs from fetal rat donors of Embryonic Days 14–15 were immediately grafted into neonatal rats in the site from which the host OB had been removed. Following survival times of 7 weeks and longer, WHRP solution was injected into the TX OB, and subjects were perfused after 24 h. The WHRP transport is seen in fibers from the TX OB into layer I of the host olfactory peduncle (OP) and olfactory cortex (OC) and in cell bodies in layers II and III of the OP and OC, the lateral hypothalamus, and the contralateral anterior olfactory nucleus (AON). These findings reaffirm that the axons from a TX OB make connections with some appropriate areas of the host brain and also indicate that axons from cells in the target areas of the host brain, including contralateral AON, reinnervate the TX OB.

Organization of thalamic projections to the ventral striatum in the primate.

Although thalamic projections to the dorsal striatum are well described in primates and other species, little is known about thalamic projections to the ventral or "limbic" striatum in the primate. This study explores the organization of the thalamic projections to the ventral striatum in the primate brain by means of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and Lucifer yellow (LY) retrograde tracer techniques. In addition, because functional and connective differences have been described for the core and shell components of the nucleus accumbens in the rat and are thought to be similar in the primate, this study also explores whether these regions of the nucleus accumbens can be distinguished by their thalamic input. Tracer injections are placed in different portions of the ventral striatum, including the medial and lateral regions of the ventral striatum; the central region of the ventral striatum, including the dorsal part of the core of the nucleus accumbens; and the shell region of the nucleus accumbens. Retrogradely labeled neurons are located mainly in the midline nuclear group (anterior and posterior paraventricular, paratenial, rhomboid, and reuniens thalamic nuclei) and in the parafascicular thalamic nucleus. Additional labeled cells are found in other portions of the intralaminar nuclear group as well as in other thalamic nuclei in the ventral, anterior, medial, lateral, and posterior thalamic nuclear groups. The distribution of labeled cells varies depending on the area of the ventral striatum injected. All regions of the ventral striatum receive strong projections from the midline thalamic nuclei and from the parafascicular nucleus. In addition, the medial region of the ventral striatum receives numerous projections from the central superior lateral nucleus, the magnocellular subdivision of the ventral anterior nucleus, and parts of the mediodorsal nucleus. After injection into the lateral region of the ventral striatum, few labeled neurons are seen scattered in nuclei of the intralaminar and ventral thalamic groups and occasional labeled cells in the mediodorsal nucleus. The central region of the ventral striatum, including the dorsal part of the core of the nucleus accumbens, receives a limited projection from the midline thalamic, predominantly from the rhomboid nucleus. It receives much smaller projections from the central medial nucleus and the ventral, anterior, and medial thalamic groups. The shell of the nucleus accumbens receives the most limited projection from the thalamus and is innervated almost exclusively by the midline thalamic nuclei and the central medial and parafascicular nuclei. The shell is distinguished from the rest of the ventral striatum in that it receives the fewest projections from the ventral, anterior, medial, and lateral thalamic nuclei.

Cervical primary afferent input to vestibulospinal neurons projecting to the cervical dorsal horn: An anterograde and retrograde tracing study in the cat

Vestibulospinal neurons in the caudal half of the medial and descending vestibular nuclei terminate in the cervical spinal cord, not only in the ventral horn and intermediate zone but also in the dorsal horn. The purpose of the present study was to examine whether the areas containing these vestibulospinal neurons are reached by cervical primary afferents. In one group of experiments, wheat germ agglutinin-horseradish peroxidase conjugate and horseradish peroxidase were pressure injected into spinal ganglia C2-C8 and revealed anterogradely labeled fibers and boutons in the caudal part (caudal to the dorsal cochlear nucleus) of the ipsilateral medial and descending vestibular nuclei. This projection was verified in experiments in which wheat germ agglutinin-horseradish peroxidase conjugate was microiontophoretically injected into the caudal half of either the medial or the descending vestibular nuclei and revealed retrogradely labeled cells only in ipsilateral spinal ganglia C2-C7, with a maximum of cells in C3. In another group of experiments, after microiontophoretic injections of Phaseolus vulgaris leucoagglutinin or Biocytin into either the medial or the descending vestibular nuclei, anterogradely labeled fibers and boutons were present in the cervical spinal cord, mainly bilaterally in the dorsal horn (laminae I-VI) but also, to a lesser extent, in the ventral horn and intermediate zone. The existence of a loop that relays cervical primary afferent information to vestibulospinal neurons projecting to the cervical spinal cord, in particular the dorsal horn, may have implications for vestibular control over local information processing in the cervical dorsal horn.

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.