Wheat Germ Agglutinin-horseradish Peroxidase Research Articles

1615 Laminar differences in functional properties of cells in inferotemporal cortex of macaque

Blind individuals often demonstrate enhanced non-visual perceptual abilities. Neuroimaging and transcranial magnetic stimulation experiments have suggested that computations carried out in the occipital cortex may underlie these enhanced somatosensory or auditory performances. Thus, cortical areas that are dedicated to the analysis of the visual scene may, in the blind, acquire the capacity to participate in other sensory processing. However, the neural substrate that underlies this transfer of function is not fully characterized. Here we studied the synaptic and anatomical basis of this phenomenon in cats that were visually deprived by dark rearing, either early visually deprived after birth (EVD), or late visually deprived after the end of the critical period (LVD); data were compared with those obtained in normally reared cats (controls). The presence of synaptic and spike responses to auditory stimulation was examined by means of intracellular recordings in area 17 and the border between areas 17 and 18. While none of the cells recorded in control and LVD cats showed responses to sound, 14% of the cells recorded in EVD cats showed both subthreshold synaptic responses and suprathreshold spike responses to auditory stimuli. Synaptic responses were of small amplitude, but well time-locked to the stimuli and had an average latency of 30±12 ms. In an attempt to identify the origin of the inputs carrying auditory information to the visual cortex, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was injected in the visual cortex and retrograde labeling examined in the cortex and thalamus. No significant retrograde labeling was found in auditory cortical areas. However, the proportion of neurons projecting from supragranular layers of the posteromedial and posterolateral parts of the lateral suprasylvian region to V1 was higher than that in control cats. Retrograde labeling in the lateral geniculate nucleus showed no difference in the total number of neurons between control and visually deprived cats, but there was a higher proportion of labeling in C-laminae in deprived cats. Labeled cells were not found in the medial geniculate nucleus, a thalamic relay for auditory information, in either control or visually deprived cats. Finally, immunohistochemistry of the visual cortex of deprived cats revealed a striking decrease in pavalbumin- and calretinin-positive neurons, the functional implications of which we discuss.

Distinct cell groups in the lumbosacral cord of the cat project to different areas in the periaqueductal gray.

The periaqueductal gray (PAG) is involved in aggressive and defensive behavior, micturition, and lordosis. Especially for the latter two functions, PAG afferents from the lumbosacral cord are of vital importance because, in addition to information regarding homeostasis and thermoregulation, they convey information from the pelvic viscera and sex organs. In the present retro- and antero-grade tracing study, the projection patterns of different lumbosacral cell groups in the PAG were determined. In the retrograde study, wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) injections were made in the PAG and/or adjacent tegmentum, and in the anterograde study, WGA-HRP was injected in different lumbosacral segments. The results revealed that lumbosacral-PAG neurons could be divided into three groups. The first and largest group was present in lumbar 7-sacral 3 segments (L7-S3) and consisted of small, oval, and fusiform neurons. It extended from the dorsolateral part of lamina I in L7, along the lateral part of the dorsal horn in S1, and into lamina V of S2. In the lateral part of S2, some of its neurons formed clusters with intervals of +/- 230 microns. The location of the first group overlapped extensively with the termination area of pelvic and pudendal afferents. The main midbrain target of the first group was the medial part of the lateral PAG. The second group consisted of small to large multipolar neurons in laminae VIII and medial VII of caudal L6, L7, and rostral S1. This group projected strongly to a distinct region in the lateral part of the lateral PAG and the laterally adjacent tegmentum. About 10% of the labeled neurons did not fit in the two groups. They were evenly distributed throughout lumbar 4-coccygeal 3 segments (L4-Co3) and consisted of large multipolar lamina V neurons and small lamina I neurons that projected diffusely to the lateral and dorsal PAG. The large lamina V neurons also targeted the laterally adjacent tegmentum. The possible involvement of the lumbosacral-PAG projections in micturition, lordosis, and defensive and aggressive behavior is discussed.

An ultrastructural study of the relationship between sensory trigeminal nerves and odontoblasts in rat dentin/pulp as demonstrated by the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP).

Because the ultrastructure of the trigeminal sensory nerves in dentin, especially in relation to odontoblasts, remains to be clarified, we investigated the relationship between the trigeminal sensory nerves and the odontoblast processes using the anterograde axonal transport technique by injecting wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the rat trigeminal ganglion. Light microscopically, the nerves labeled with WGA-HRP were mainly concentrated at the pulpal horn, forming a nerve plexus at the subodontoblastic region and penetrating the predentin/dentin about 50 to 70 microns. Ultrastructurally, HRP reaction products were observed intra-axonally in the myelinated (A delta) and unmyelinated (C) axons in the subodontoblastic region. Most nerves lost the Schwann sheath and were naked in the predentin/dentin. The labeled varicosities were close to the odontoblast processes in the dentinal tubules. No synaptic structures could be detected between the varicosities and the odontoblasts, but a gap about 20 nm wide was found between them. One type of varicosity was a rich mitochondria-containing varicosity, while the other was a rich vesicle-containing (large dense core vesicles and small clear vesicles) one. The reaction products were also found in the extracellular spaces surrounding the axons. Sometimes the reaction products were seen in the coated pits or the endocytotic vesicles of the odontoblast processes. The present study demonstrated that nerve endings (varicosities) derived from the trigeminal ganglion were present in the dentinal tubules, and that WGA-HRP extracellularly extruded from the sensory nerves in the odontoblastic layer or predentin/dentin. These findings thus suggest that sensory nerves may have some (e.g., trophic) effect on either odontoblasts or the environment around the sensory nerves in the dentin/pulp.

Spinal Cord Terminations of the Medial Wall Motor Areas in Macaque Monkeys

We used anterograde transport of wheat germ agglutinin-horseradish peroxidase to examine the pattern of spinal termination of efferents from the supplementary motor area (SMA) and the two caudal cingulate motor areas (CMAd and CMAv). Our analysis was limited to cervical segments of the macaque. For comparison, we also examined the pattern of termination of efferents from the primary motor cortex (M1). The SMA, CMAd, CMAv, and M1 all terminate in the ventral horn (lamina IX). Thus, all of these motor areas appear to have direct connections with spinal motoneurons, particularly those innervating muscles of the fingers and wrist. All of the motor areas also terminate in the intermediate zone of the spinal cord (laminae V-VIII). Terminations from the SMA and M1 were densest in three regions: (1) dorsolaterally within laminae V-VII; (2) dorsomedially within lamina VI; and (3) ventromedially within lamina VII and adjacent lamina VIII. In contrast, efferents from the CMAd terminate most densely in the dorsolateral portion of the intermediate zone, whereas those from the CMAv were concentrated in the dorsomedial region. Thus, the CMAd and CMAv may innervate distinct sets of interneurons that project directly to motoneurons, and thereby influence specific aspects of segmental motor control. These results suggest that corticospinal projections from the SMA, CMAd, and CMAv are in many respects similar to those of efferents from M1. Consequently, each of the motor areas on the medial wall has the potential to generate and control movement at the level of the spinal cord and may provide an anatomical substrate for the recovery of motor function that follows damage to M1.

Projections and fiber characteristics of sensory afferents of the anterior cruciate ligament in an animal experiment

The sensory innervation of the rabbit anterior cruciate ligament was studied by retrograde tracing technique using wheat-germ-agglutinin-horseradish-peroxidase (WGA-HRP) and Fast Blue as neuronal tracers. Injection of the tracer into the ligament was followed by histo- and immunohistochemical investigation of labelled nerve cell bodies located in the dorsal root ganglia. In 4 animals we injected the tracer into the joint cavity to label general joint afferents. The segmental distribution of retrogradely labelled neurons following injection into the anterior cruciate ligament (L6, L7, S1) is significantly different from the distribution pattern after injection into the knee joint (L4-S2). Retrogradely labelled nerve cells innervating the anterior cruciate ligament were further investigated using immunohistochemical and morphometric analysis. The sensory innervation of the anterior cruciate ligament is therefore comprised of at least 2 different qualities of sensory afferent nerves: 1. Small neurones immunoreactive to the inflammatory peptide substance P most likely transmitting nociceptive information centrally (44%). 2. Large, presumably fast conducting A-fibre-afferents characterized by neurofilament proteins transmitting proprioceptive information from corpuscular mechanoreceptors (43%). The results of this study put further weight to the importance of the sensory role of the anterior cruciate ligament using neuroanatomical and immunohistochemical techniques.

Pontine and medullary projections to the nucleus retroambiguus: a wheat germ agglutinin-horseradish peroxidase and autoradiographic tracing study in the cat.

The nucleus retroambiguus (NRA) in the caudal medulla oblongata plays a role in expiration, vocalization, vomiting, and possibly lordosis. The present study tried to determine which structures, in turn, control the NRA. One cell group is the periaqueductal gray (PAG), which is considered to be the final integrator of defensive and aggressive behaviors, micturition, vocalization, and lordosis. Structures rostral to the PAG seem to bypass the NRA. With respect to the existence of cell groups caudal to the PAG projecting to the NRA, the situation is less clear. Therefore, in five adult female cats, injections of wheat germ agglutinin-horseradish peroxidase were centered on the NRA, and the resulting retrogradely labeled neurons were plotted. In the areas containing retrogradely labeled cells, the anterograde autoradiographic tracer [3H]-leucine was injected in 66 cats. The combined results demonstrated that NRA afferents not only originate from the PAG but also from specific cell groups in the pontine and medullary lateral tegmental field, i.e., the ventrolateral parabrachial nucleus, the nucleus Kölliker-Fuse, the retrotrapezoid nucleus, and the ventrolateral part of the medulla caudal to the facial nucleus including the Bötzinger and pre-Bötzinger complex and the periambigual region. Afferents also originate from the solitary nucleus and two cell groups in the ventral part of the medullary medial tegmental field, one at the level of facial nucleus and one just rostral to the hypoglossal nucleus. It can be concluded that many respiratory-related cell groups have direct access to the NRA. The cell groups in the medial tegmental field, which have not yet been found to play an important role in respiration, might serve as relay for certain limbic system cell groups to reach the NRA in the context of specific emotional behavior.

Anatomical Demonstration of Ocular Dominance Columns in Striate Cortex of the Squirrel Monkey

The squirrel monkey is the only primate reported to lack ocular dominance columns. Nothing anomalous about the visual capacity of squirrel monkeys has been found to explain their missing columns, leading to the suggestion that ocular dominance columns might be "an epiphenomenon, not serving any purpose" (Livingstone et al., 1995). Puzzled by the apparent lack of ocular dominance columns in squirrel monkeys, we made eye injections with transneuronal tracers in four normal squirrel monkeys. An irregular mosaic of columns, averaging 225 microns in width, was found throughout striate cortex. They were double-labeled by placing wheat germ agglutinin-horseradish peroxidase into the left eye and [3H]proline into the right eye. The tracers labeled opposite sets of interdigitating columns, proving they represent ocular dominance columns. The columns were much clearer in layer IVc alpha (magno-receiving) than IVc beta (parvo-receiving). In the lateral geniculate body, the parvo laminae showed extensive mixing of ocular inputs, suggesting that increased label spillover contributes to the blurred columns in layer IVc beta. The cytochrome oxidase (CO) patches were organized into distinct rows, but they bore no consistent relationship to the ocular dominance columns. These experiments indicate that ocular dominance columns are less well segregated in squirrel monkeys than macaques, but they are present. This fact is pertinent to a recent study reporting that ocular dominance columns are absent in normal squirrel monkeys, but induced to form by strabismus (Livingstone, 1996).

Topographic patterns of V2 cortical connections in macaque monkeys

Patterns of connections of dorsal and ventral portions of the second visual area (V2) were used to evaluate and extend current theories of cortical organization and processing streams in macaque monkeys. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and up to four different fluorochromes in V2 labeled neurons and terminations in V2 and in 1) caudal (DLc) and rostral (DLr) subdivisions of dorsolateral cortex between V2 and the middle temporal area (MT); 2) regions we define as dorsomedial (DM) and dorsointermediate (DI) areas; 3) MT, medial superior temporal area (MST), and fundal superior temporal area (FST); 4) the dorsal part of inferior temporal (TEO) cortex; and 5) two locations in posterior parietal cortex. The largest extrastriate connection zone was DLc, which occupied the caudal one-third to one-half of the fourth visual area (V4) region of other proposals. Based on the connection pattern, foveal vision in DLc is represented adjacent to foveal vision in V2, with the lower quadrant represented dorsally and the upper quadrant ventrally, as in V2, but within a much less extensive region of cortex. The sparser connections of DLr formed a more compressed but parallel visuotopic pattern. A third visuotopic pattern of connections was located in a moderately myelinated region of cortex just rostral to dorsomedial V2. Whereas the region would include parts of dorsal visual area 3 (V3), V3a, and possibly other areas of other proposals, we interpret the connection pattern as reflecting a dorsomedial visual area, DM, with foveal vision represented caudolaterally and other parts of the lower and upper quadrants represented more medially and rostrally. A fourth pattern of label in dorsointermediate cortex suggested the location and organization of another visual area (DI). Most of a fifth connection pattern with MT was congruent with the known visuotopic organization of MT area, but visuotopically mismatched foci of connections were observed as well. Sparser foci of label in MST suggested a rostrodorsal representation of foveal vision, with paracentral vision represented more caudally. Separate dorsal and ventral foci of label in FST were consistent with previous evidence for dorsal (FSTd) and ventral (FSTv) visual areas. Finally, connections with TEO and posterior parietal cortex were sparse. Our results suggest that much of visual cortex organization is similar in New and Old World monkeys. © 1996 Wiley-Liss, Inc.

Topographic patterns of V2 cortical connections in macaque monkeys.

Patterns of connections of dorsal and ventral portions of the second visual area (V2) were used to evaluate and extend current theories of cortical organization and processing streams in macaque monkeys. Injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and up to four different fluorochromes in V2 labeled neurons and terminations in V2 and in 1) caudal (DLc) and rostral (DLr) subdivisions of dorsolateral cortex between V2 and the middle temporal area (MT); 2) regions we define as dorsomedial (DM) and dorsointermediate (DI) areas; 3) MT, medial superior temporal area (MST), and fundal superior temporal area (FST); 4) the dorsal part of inferior temporal (TEO) cortex; and 5) two locations in posterior parietal cortex. The largest extrastriate connection zone was DLc, which occupied the caudal one-third to one-half of the fourth visual area (V4) region of other proposals. Based on the connection pattern, foveal vision in DLc is represented adjacent to foveal vision in V2, with the lower quadrant represented dorsally and the upper quadrant ventrally, as in V2, but within a much less extensive region of cortex. The sparser connections of DLr formed a more compressed but parallel visuotopic pattern. A third visuotopic pattern of connections was located in a moderately myelinated region of cortex just rostral to dorsomedial V2. Whereas the region would include parts of dorsal visual area 3 (V3), V3a, and possibly other areas of other proposals, we interpret the connection pattern as reflecting a dorsomedial visual area, DM, with foveal vision represented caudolaterally and other parts of the lower and upper quadrants represented more medially and rostrally. A fourth pattern of label in dorsointermediate cortex suggested the location and organization of another visual area (DI). Most of a fifth connection pattern with MT was congruent with the known visuotopic organization of MT area, but visuotopically mismatched foci of connections were observed as well. Sparser foci of label in MST suggested a rostrodorsal representation of foveal vision, with paracentral vision represented more caudally. Separate dorsal and ventral foci of label in FST were consistent with previous evidence for dorsal (FSTd) and ventral (FSTv) visual areas. Finally, connections with TEO and posterior parietal cortex were sparse. Our results suggest that much of visual cortex organization is similar in New and Old World monkeys.

NOS-positive preganglionic neurons innervate a subpopulation of postganglionic neurons in superior cervical ganglion in rats

To determine the postganglionic targets of NOS-containing preganglionic neurons, we studied the association of NADPH-diaphorase positive preganglionic fibers and retrogradely labeled postganglionic neurons in the superior cervical ganglion (SCG) in rats. Wheat germ agglutinin-horseradish peroxidase solution was applied to the anterior chamber of the eye, middle cerebral artery, subcutaneous layer of the facial skin, or submucosal layer of the inside of the lip. Two days after tracer application, the rats were perfused with fixative solution. Serial sections of the SCG were stained histochemically for NADPH-diaphorase followed by diaminobenzidine reaction. More than 80% of the labeled postganglionic neurons innervating the structures in the subcutaneous or submucosal layer showed close association with NADPH-diaphorase positive preganglionic nerve terminals; approximately one-third of these labeled neurons were encircled by dense baskets of pericellular terminals. On the other hand, most of the postganglionic neurons innervating the iris (69%) or the cerebral artery (90%) did not show a distinct association with NADPH-diaphorase positive terminals. These results suggest that one of the principal roles of the NOS-containing preganglionic neurons may be in controlling the postganglionic neurons which innervate the structures in the subcutaneous or submucosal layer.

Hippocampal afferents to the rat prefrontal cortex: Synaptic targets and relation to dopamine terminals

Afferents to the prefrontal cortex (PFC) from the hippocampal formation and from midbrain dopamine (DA) neurons have been implicated in the cognitive and adaptive functions of this cortical region. In the present study, we investigated the ultrastructure and synaptic targets of hippocampal terminals, as well as their relation to DA terminals within the PFC of adult rats. Hippocampal afferents were labeled either by anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from the ventral hippocampal formation or by anterograde degeneration following fimbria lesion. Hippocampal terminals in the PFC, identified by either method, formed primarily asymmetric axospinous synapses, with a small percentage forming asymmetric axodendritic synapses. Dopamine terminals in the PFC were identified by peroxidase immunocytochemistry for either tyrosine hydroxylase or DA and formed primarily symmetric synapses onto dendritic spines and small caliber dendritic shafts. Spines that received symmetric synaptic contact from DA terminals invariably also received an asymmetric synapse from an unlabeled terminal, forming a triadic complex. Hippocampal and DA terminals in the PFC were not often observed in the same area of the neuropil, and no examples of convergence of hippocampal and DA terminals onto common postsynaptic targets were observed. Further analysis revealed that spines receiving synaptic contact from hippocampal terminals did not receive additional synaptic contact from any other source. However, when localized to the same area of the neuropil, hippocampal and DA terminals were often in direct apposition to one another, without forming axo-axonic synapses. These results suggest that 1) hippocampal terminals primarily form excitatory synapses onto spiny pyramidal neurons, 2) hippocampal afferents are unlikely to be synaptically modulated by DA or non-DA terminals at the level of the dendritic spine, and 3) appositions between hippocampal and DA terminals may facilitate presynaptic interactions between these afferents to the PFC.

Reevaluation of synaptic relationships of cerebellar terminals in the ventral lateral nucleus of the rhesus monkey thalamus based on serial section analysis and three-dimensional reconstruction.

Terminals of cerebellar afferents (CB) to different regions of the ventral lateral nucleus (VL) of the rhesus monkey thalamus were labeled with wheat germ agglutinin-horseradish peroxidase following injections into the dentate nucleus. Synaptic relationships of 17 CB with projection neuron dendrites (PNd) and local circuit neuron dendrites (LCNd) were analyzed in serial ultrathin sections from dorsal and ventral VL regions, which are known to differ cytoarchitecturally and functionally. Three terminals were reconstructed using three-dimensional (3D) computer image analysis techniques to obtain volumetric and planar measurements. CB in the ventral VL were often flat and elongated with synaptic vesicles arranged in clusters. Each CB was engaged with one PNd and one to four LCNd. A single bouton formed 8-50 synaptic contacts, with those on PNd outnumbering the ones on LCNd 4.1:1. Only some CB in the ventral VL were engaged in complex synaptic arrangements such as triads and serial synapses. Most CB in the dorsal VL displayed a roundish shape and numerous uniformly distributed synaptic vesicles. They formed 5-25 synaptic contacts with a 3:1 ratio of contacts on PNd compared with those on LCNd. CB in the dorsal VL participated in a variety of complex synaptic arrangements. Two types of triads were found: classic with CB, PNd and LCNd, and unconventional with CB and two LCNd. CB were also involved in serial synapses with two LCNd or LCNd and another PNd, and serial sequential synapses with two LCNd and a PNd. Three glomerulus-like structures were encountered in the dorsal VL. 3D reconstruction and volumetric measurements revealed that synaptic contacts formed by CB on PNd had varying shapes and sizes (0.022-0.274 microns2). Synapses formed on LCNd were larger (0.09-0.407 microns2). The total area of all active zones of a single CB on LCNd was either equal to or about 40% smaller than that of synapses on PNd. The entire active zone area comprised 1-1.6% of the total CB surface area and did not seem to correlate with the volume. Synaptic contacts formed by associated LCNd on PNd in complex arrangements were usually small (0.021-0.044 micron2). The results suggest that: synapses formed by CB on PNd and LCNd, and synapses formed by LCNd on PNd may differ in strength; a variety of different circuits participate in the processing of cerebellar afferent information in the primate VL; and these circuits differ in functionally different VL subdivisions.

A nigro-rubro-bulbar pathway to the parvicellular reticular formation in the rat.

A possible pathway from the substantia nigra pars reticulata (SNr) to the parvicellular reticular formation (RFp) via the red nucleus (RN) was examined light and electron microscopically by combining anterograde and retrograde tracing techniques. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the SNr and cholera toxin B subunit (CTb) into the RFp, many CTb-labelled neurones were distributed contralaterally in the dorsal part of the RN, where numerous BDA-labelled axon terminals originating from the ipsilateral SNr were found. After contralateral injections of BDA into the dorsolateral part of the SNr and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the RFp, ipsilateral axons labelled with BDA were found to make synaptic contacts with the somata and dendrites of contralateral neurones labelled with WGA-HRP in the dorsal part of the RN.

Contrasting locations of pallidal-receiving neurons and microexcitable zones in primate thalamus.

1. In two awake, juvenile male Macaca fascicularis monkeys, microstimulation was applied in ventralis anterior (VA), ventralis lateralis (VL), or ventralis posterior lateralis (VPL) of the thalamus. Thalamic recording was used to identify the region that contained pallidal-receiving (PR) thalamic neurons, cells that responded orthodromically to stimulation in the internal pallidal segment (GPi). Thalamic stimulation was used to elicit motor responses. Penetrations in the thalamus and the pallidum focused on areas with activity related to contralateral arm movement. Fifty-one PR cells were identified electrophysiologically in VL oralis (VLo), VL caudalis (VLc) and VA pars parvocellularis (VApc). 2. With a subject at rest, trains of stimuli were applied through the thalamic microelectrode. Palpable or visible muscle twitches or joint movements were evoked by short trains (12 pulses) of stimuli applied in VLc and VPL oralis (VPLo); thresholds there ranged from 5 to 75 microA. In VA and VLo, areas where PR neurons were located, even longer trains (24 pulses) of stimuli with currents up to 200 microA usually failed to evoke movement. In the caudal portions of VLo, near VPLo, there were some microexcitable sites found near PR cells where stimuli at approximately 50 microA elicited movement. 3. From microstimulation studies combined with histological reconstruction, Ashe and co-workers hypothesized that microexcitable zones were cerebellar receiving areas (CR) and nonexcitable zones were PR areas. Our data support theirs and add electrophysiological identification of PR areas. Further, we injected wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the thalamus at one of the more rostral microexcitable sites, just caudal and lateral to identified PR cells. The tetra-methyl benzidine-reacted HRP label was found in the contralateral deep cerebellar nuclei (DCN) but not in ipsilateral GPi, showing that even this rostral microexcitable zone was a CR area. 4. Together with evidence from the literature, the data are consistent with the hypothesis that PR cells have relatively weak access to spinal-destined motor outputs, whereas thalamocortical neurons from VPLo and VLc have more secure access. In addition to characteristics of cell discharge and responses to somatosensory stimulation, microexcitability may be a further aid in tentative electro-physiological identification of PR versus CR areas of the motor thalamus without necessarily recording thalamic neuronal responses to stimulation in GPi or the DCN.

Crossed thalamo-cortical and cortico-thalamic projections in adult mice

The crossed thalamo-cortical and cortico-thalamic connections of the mouse are drawn using the tracer wheat germ agglutinin-horseradish peroxidase. After injections in the frontal cortex of the right hemisphere cells labeled retrogradely and axons labeled anterogradely are observed in the thalamus ipsilateral and contralateral to the cortical injections. The retrograde and anterograde labeling in the contralateral thalamus is less intense than ipsilaterally and involves the mediodorsal, ventral medial, central medial, and paracentral nuclei. Crossed fronto-thalamic axons reach more lateral regions than those containing contralateral thalamo-frontal neurons. Our results demonstrate that the thalamo-cortical system of mice has a bilateral component. The functional significance of this pathway and analogies with crossed thalamo-cortical connections in other species are discussed.

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.

Functional Demarcation of a Border Between Areas V6 and V6A in the Superior Parietal Gyrus of the Macaque Monkey

We have compared physiological data recorded from three alert macaque monkeys with separate observations of local connectivity, to locate and characterize the functional border between two related but distinct visual areas on the caudal face of the superior parietal gyrus. We refer to these areas as V6 and V6A. The occupy almost the entire extent of the anterior bank of the parieto-occipital sulcus, V6A being the more dorsal. These two areas are strongly interconnected. Anatomically, we have defined the border as the point at which labelled axon terminals first adopt a recognizably 'descending' pattern in their laminar characteristics, after injections of wheatgerm agglutinin-horseradish peroxidase into the dorsal half of the gyrus (in presumptive V6A). A similar principle was used to recognize the same border by the pattern of input from area V5, except that in this case the relevant transition in laminar characteristics is that between an 'intermediate' pattern (in V6) and an 'ascending' pattern (in V6A). V6A was found to be distinct from V6 in a number of its physiological properties. Unlike V6, it contains visually unresponsive cells as well as units with craniotopic receptive fields ('real-position' cells), units tuned to very slow stimulus speeds, units with complex visual selectivities and units with activity related to attention. V6A was also found to have a larger mean receptive field size and scatter than V6. By contrast, response properties related to the basic orientation and direction of moving bar stimuli were indistinguishable between V6 and V6A, as was the influence of gaze direction on cell activity in the two areas. Two-dimensional maps of the recording sites allowed reconstruction of the V6/V6A border. For comparison, the anatomical results were rendered on two-dimensional maps of identical format to those used to summarize the physiological data. After normalizing for relative size, the physiological and connectional estimates of the border between V6 and V6A were found to coincide, at least within the range of individual variation between hemispheres. An architectonic map in the same format was also made from a hemisphere stained for myelin and Nissl substance. Area PO, defined by its general density of myelination was not distinct in this material, but several architectural features were traceable and one of these was also found to approximate the V6/V6A border. The particular criteria that distinguish V6 from V6A differ from a recent description of areas PO and POd in the Cebus monkey; we believe it most likely that PO and POd together may correspond to V6.

Concanavalin A- and Wheat Germ Agglutinin-Conjugated Lectins as a Tool for the Identification of Multiple N-Glycosylation Sites in Heterologous Protein Expressed in Yeast

We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A–horseradish peroxidase. Wheat germ agglutinin–horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein. Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry. This methodology was applied to a model glycoprotein, α-amylase fromBacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeastPichia pastoris.

Patterns of transmitter labelling and connectivity of the cat's nucleus of Darkschewitsch: A wheat germ agglutinin‐horseradish peroxidase and immunocytochemical study at light and electron microscopical levels

Immunocytochemical studies using antibodies raised against a number of probable synaptic transmitters of the mesodiencephalic area, and fibre-tracing studies using wheat germ agglutinin-horseradish peroxidase (WGA-HRP), have been performed in adult cats. Glutamate and aspartate immunoreactivity produced a strong labelling of many cell bodies and terminals in the nucleus of Darkschewitsch (ND). gamma-Aminobutyrate (GABA) immunoreactivity in the ND appeared as a moderate label in some small neurones, and as a strong label in a few glial-like cells, in addition to being present in high levels to produce strong labelling in many GABA-immunopositive terminals that possessed pleomorphic vesicles. Some choline acetyltransferase-positive terminals and dendrites and a few substance P-positive fine fibres possessing varicosities also were observed in the ND. Following WGA-HRP injection in the ND, dense terminal labelling was seen ipsilaterally in the rostral half of the medial accessory olive, suggesting that there may be a certain degree of mediolateral and dorsoventral topographic correspondance within the ND-olive projection. In the same cases, many cell bodies containing HRP reaction product also were found 1) ipsilaterally in the motor cortex, anterior pretectal nucleus, and a restricted area of the caudal part of the substantia nigra pars reticulata; 2) contralaterally in the anterior and posterior interposed cerebellar nuclei as well as in a portion of the lateral cerebellar nucleus; and 3) bilaterally in the zona incerta, the posterior pretectal nucleus, the pedunculopontine tegmental nuclei, the spinal trigeminal nucleus, the dorsal column nuclei, and the spinal cord. Details of the interrelationships and functional considerations amongst the ND, adjacent nuclei, and longitudinal zones of the cerebellum are discussed.

Ultrastructure and synaptology of the nucleus ambiguus in the rat: the compact formation.

The fine structure of the esophagomotor compact formation of the nucleus ambiguus was studied. Esophageal motoneurons are atypical in that they have extensive direct somato-somatic and somato-dendritic appositions without intervening glial processes. A unique feature is the presence of finger- and leaf-like somatic protrusions which partially wrap longitudinally oriented dendrites and, occasionally, small groups of dendrites and axons. The neuropil contains many longitudinally oriented, small-diameter dendrites of relatively uniform size (1.1 +/- 0.4 S.D. micrograms in diameter). Motoneuronal somatic profiles have 0-5 synapses per profile which represents a synaptic density of 10.6 synapses per soma. Axodendritic synapses measure 0.5 x 0.7 microgram in the transverse plane and are up to 3.0 micrograms long in the sagittal plane. Many axon terminals contact both a soma and dendrite in close apposition. Most axon terminals (> 90%) contain round vesicles and form asymmetric junctions with somata and dendrites. Axon terminal degeneration after electrolytic lesions and labelling after injection of wheat germ agglutinin-horseradish peroxidase in the nucleus of the tractus solitarius show that afferent connections to the compact formation form axodendritic synapses. The ultrastructure and synaptology of esophageal motoneurons is characterized by the close apposition of somata and dendrites (somatic-dendritic bundling), and the longitudinal orientation of dendrites (dendritic bundling), axons and axon terminals in the neuropil. These features may be important morphological substrates for synchronization and coordination of esophageal motoneuronal activity and esophageal peristalsis.