Henry J. Ralston

The distribution of dorsal root axons in laminae I, II and III of the macaque spinal cord: a quantitative electron microscope study.

This study examines the projection of dorsal root fibers to the upper dorsal horn of the monkey lumbar spinal cord utilizing degeneration and autoradiographic methods. The animals survived dorsal rhizotomy for periods varying from 18 hours to 28 days. Electron microscopy reveals the earliest degeneration to be neurofilamentous alteration of large synaptic profiles in lamina III and the inner zone of the substantia gelatinosa (IIi). This degeneration begins 18 hours after rhizotomy, reaches a peak at three days postoperatively and disappears by the end of the first week. Degenerating myelinated axons in the spinal gray matter, dorsal column white matter and Lissauers tract first appear three days postoperatively.

Localization of Postsynaptic Density-93 to Dendritic Microtubules and Interaction with Microtubule-Associated Protein 1A

Postsynaptic density-93 (PSD-93)/Chapsyn-110 is a member of the membrane-associated guanylate kinase (MAGUK) family of PDZ domain-containing proteins. MAGUKs are widely expressed in the brain and are critical elements of the cytoskeleton and of certain synapses. In the ultrastructural studies that are described here, PSD-93 localizes to both postsynaptic densities and dendritic microtubules of cerebellar Purkinje neurons. The microtubule localization is paralleled by a high-affinity in vivo interaction of PSD-93 via its guanylate kinase (GK) domain with microtubule-associated protein 1A (MAP1A). GK domain truncations that mimic genetically identified mutations of a Drosophila MAGUK,discs-large, disrupt the GK/MAP-1A interaction. Additional biochemical experiments demonstrate that intact MAGUKs do not bind to MAP1A as effectively as do isolated GK domains. This appears to be attributable to an intramolecular inhibition of the GK domain by the PDZs, because GK binding activity of full-length MAGUKs is partially restored by a variety of PDZ ligands, including the C termini of NMDA receptor 2B, adenomatous polyposis coli (APC), and CRIPT. Beyond demonstrating a novel cytoskeletal link for PSD-93, these experiments support a model in which intramolecular interactions between the multiple domains of MAGUKs regulate intermolecular associations and thereby may play a role in the proper targeting and function of MAGUK proteins.

Reticularis thalami afferents to the ventrobasal complex of the rat thalamus: an electron microscope study

Afferents from the nucleus reticularis thalami (RT) to the thalamic ventrobasal complex were studied in the rat by looking for degenerating terminals after selective neurotoxic lesion of RT using injections of kainic acid. Several lines of evidence are presented indicating that RT afferents terminate in the VB by F type (Gray type II) terminals and that F type terminals in the VB all depend of RT neurons.

The primate dorsal spinothalamic tract: evidence for a specific termination in the posterior nuclei (Po/SG) of the thalamus.

&NA; The spinothalamic tract in primates and other mammals arises primarily from cells in lamina 1 of the dorsal horn, from lamina V cells and to a lesser extent from other laminae. Most of the neurons of lamina I respond only to noxious mechanical or thermal stimuli. Spinothalamic tract (STT) cells of lamina V tend to respond to both innocuous and noxious stimuli. Recent studies have suggested that the classical STT in the anterolateral quadrant (ALQ) contains primarily the axons of lamina V cells and that the axons of lamina I cells travel more dorsally in the dorsolateral quadrant (DLQ) to constitute the dorsal spinothalamic tract (DSTT). Using the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA‐HRP) injected into the spinal cord in conjunction with a contralateral anterolateral cordotomy, we have found there is a substantial projection of the DSTT to the posterior nuclei of the caudal‐ventral thalamus, designated Po/SG. This projection is almost entirely abolished when the lesion includes the area of spinal cord white matter at the level of the denticulate ligament. Larger lesions that destroy the ALQ and much of the lateral column white matter, but that spare the dorsolateral column white matter in the region of the corticospinal tract, abolish all transport of WGA‐HRP to the thalamus. We conclude that the spinothalamic pathway in the non‐human primate encompasses a continuous fiber bundle that extends dorsally to include the region of lateral column white matter opposite the denticulate ligament and that the more dorsal aspect of this pathway projects primarily to Po/SG of thalamus.

Monosynaptic projections from the lateral periaqueductal gray to the nucleus retroambiguus in the rhesus monkey: Implications for vocalization and reproductive behavior

The periaqueductal gray (PAG) is known to be essential for vocalization and reproductive behavior. The PAG controls components of these behaviors by means of projections to the nucleus retroambiguus (NRA), a group of premotor neurons in the caudal medulla oblongata. In the accompanying study (VanderHorst et al., 2000 [accompanying study]), the NRA and its lumbosacral projections have been identified in the rhesus monkey. The present light and electron microscopical tracing study describes the PAG‐NRA pathway in primates. To locate midbrain neurons projecting to the NRA, wheat germ agglutinin horseradish peroxidase (WGA‐HRP) was injected into the NRA in six monkeys. To determine the distribution pattern of PAG axons in the medulla oblongata, WGA‐HRP was injected into the PAG and adjacent tegmentum in three additional monkeys. In one of these three monkeys, biotinylated dextran amine and cholera toxin subunit b were injected into the lumbosacral cord to retrogradely identify NRA neurons. The results show that a compact group of neurons in the medial part of the lateral PAG at the intercollicular level sends a dense projection to the NRA. The projection is bilateral with a clear ipsilateral predominance. At the ultrastructural level, there are monosynaptic contacts between PAG fibers and NRA neurons, including NRA neurons that project to the lumbosacral cord. The synaptic contacts were primarily asymmetrical and the labeled terminal profiles contained spherical and dense core vesicles. It is concluded that there exists a strong and direct PAG‐NRA pathway in the rhesus monkey. Because NRA neurons projecting to the lower lumbar cord are included, the PAG‐NRA projection is likely to be involved not only in vocalization but also in other behaviors, such as receptive posture. J. Comp. Neurol. 424:251–268, 2000.

Monosynaptic projections from the nucleus retroambiguus to motoneurons supplying the abdominal wall, axial, hindlimb, and pelvic floor muscles in the female rhesus monkey

The nucleus retroambiguus (NRA) consists of premotor neurons in the caudal medulla. It is involved in expiration, vomiting, vocalization, and probably reproductive behavior by means of projections to distinct motoneuronal cell groups. Because no information is available about the NRA and its efferent pathways in primates, the present study examines NRA projections to the lumbosacral spinal cord in female rhesus monkeys. To identify the NRA, wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP) was injected into the lumbosacral cord in three monkeys. To study the distribution of NRA axons in the lumbosacral cord, WGA‐HRP injections were made into the NRA in seven monkeys. To identify motoneuronal cell groups receiving input from the NRA, the same seven monkeys also received cholera toxin subunit b (CTb) injections into different hindlimb, axial, and pelvic floor muscles. The results show that NRA neurons projecting to the lumbosacral cord are mainly located between 1 to 4 mm caudal to the obex. They send numerous axons to external oblique and pelvic floor motoneurons, whereas projections to iliopsoas and axial motoneurons are less numerous. The projections are bilateral, but show a clear contralateral predominance in the iliopsoas, axial, and pelvic floor motoneuronal cell groups. At the ultrastructural level, NRA‐terminal profiles make asymmetrical contacts with labeled and unlabeled dendrites in these motoneuronal cell groups and contain large amounts of spherical and a few dense core vesicles. It is concluded that the NRA is well developed in the monkey and that there exists a direct pathway from the NRA to lumbosacral motoneurons in this species. The finding that the NRA projects to a somewhat different set of motoneuronal cell groups compared with other species fits the concept that it is not only involved in expiration‐related activities but also in species specific receptive and submissive behavior. J. Comp. Neurol. 424:233–250, 2000.

The differential synaptic organization of the spinal and lemniscal projections to the ventrobasal complex of the rat thalamus. Evidence for convergence of the two systems upon single thalamic neurons

The synaptic organization of terminals originating either from the spinal cord (spinothalamic) or from the dorsal column nuclei (lemniscal) was investigated in the ventrobasal complex of the rat thalamus. Wheatgerm agglutinin conjugated to horseradish peroxidase was used as an anterogradely transported axonal tracer, using benzidine dihydrochloride as a chromogen for the identification by electron microscopy of spinal and lemniscal projections to the ventrobasal thalamus. A double anterograde tract tracing strategy, based labeling by wheatgerm agglutinin conjugated to horseradish peroxidase of spinal terminals and simultaneous visualization of lemniscal terminals identified by Wallerian degeneration induced by lesion of the neurons of origin in the dorsal column nuclei, was used to compare the postsynaptic elements contacted by the two pathways and to look for a possible convergence of the two pathways onto single thalamic neurons. Spinal and lemniscal terminals are large (2-2.5 microns mean average diameter) terminals containing several mitochondria and numerous rounded vesicles. A quantitative analysis of the mean average diameters of the terminals revealed that one could not differentiate between synapses formed by the two pathways on a morphological basis. Terminals of the two pathways make asymmetrical contacts (Gray type I) with dendrites of varying diameter, dendritic protrusions, and cell somata. A quantitative analysis of the least diameter of the postsynaptic elements demonstrates projections of the two systems to different, partially overlapping regions of thalamic neurons. Lemniscal terminals originating from the dorsal column nuclei frequently contact cell somata; axosomatic spinothalamic contacts are uncommon. In addition, lemniscal projections tend to contact more proximal dendrites than do spinal projections, and this differential synaptic organization is statistically significant. From a functional point of view, this differential synaptic organization might indicate that lemniscal inputs have greater influence than spinal inputs in affecting the activity of thalamic neurons. Labeled spinothalamic terminals contact the same dendritic profile as do degenerating lemniscal terminals in about 10% of single sections. Because the present study did not include a complete reconstruction of ventrobasal complex neurons of the thalamus or even regions of dendritic arbors, the degree of convergence is likely to be significantly underestimated. These findings indicate that the anatomical basis exists for an interaction between nociceptive and non-nociceptive somesthetic systems at the level of single ventrobasal neurons of the thalamus of the rat.

Transneuronal changes of the inhibitory circuitry in the macaque somatosensory thalamus following lesions of the dorsal column nuclei.

The inhibitory circuitry of the ventroposterolateral nucleus (VPL) of the macaque somatosensory thalamus was analyzed in normal animals and in those surviving for a few days or several weeks following a unilateral lesion of the cuneate nucleus, the source of medial lemniscal (ML) axons carrying information from the contralateral upper extremity. Inhibitory synaptic terminals in the VPL were defined as those that contain flattened or pleomorphic synaptic vesicles and that can be shown to be immunoreactive for γ‐aminobutyric acid (GABA). There are two types of these profiles: F axon terminals that arise from neurons of the thalamic reticular nucleus, and perhaps from VPL local circuit neurons (LCNs); and the dendritic appendages of LCNs that form presynaptic dendrites (PSDs). ML terminals normally have extensive synaptic interactions with PSDs but not with F axon terminals. Electron microscopic analyses revealed that cuneatus lesions resulted in a rapid loss of ML terminals and a statistically significant reduction in both F and PSD synaptic profiles. Confocal scanning microscopy also demonstrated a profound loss of GABA immunoreactivity in the deafferented VPL. These changes persisted for more than 20 weeks, without any evidence of reactive synaptogenesis of surviving sensory afferents or of inhibitory synapses. The changes in GABA circuitry are transneuronal, and the possible mechanisms that may underlie them are discussed. It is suggested that the altered GABAergic circuitry of the VPL in the monkey may serve as a model for understanding changes in somatic sensation in the human following peripheral or central deafferentation.

Ultrastructural Analysis of the Terminals of Various Somatosensory Pathways in the Ventrobasal Complex of the Raft Thalamus: An Electron-Microscopic Study Using Wheatgerm Agglutinin Conjugated to Horseradish Peroxidase as an Axonal Tracer

We used wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) as an anterograde tracer to label the terminals of the lemniscal, spinothalamic, and trigeminothalamic pathways in the ventrobasal complex of the rat thalamus (VB). The use of benzidine dihydrochloride (BDHC) as the chromogen allowed us to view the labeled profiles with the electron microscope and permitted us to compare the morphology of the terminals from the various pathways. We found that all the labeled somatosensory pathways terminate in the VB in the form of large terminals that contain round synaptic vesicles and make numerous asymmetrical synaptic contacts, usually with dendritic protrusions and proximal dendrites. The present results demonstrate that pathways conveying noxious and non-noxious somatosensory information terminate upon thalamic neurons with synaptic terminals having similar morphological features.

Monosynaptic projections from the nucleus retroambiguus region to laryngeal motoneurons in the rhesus monkey

Vocalization and straining-related activities require the activation of laryngeal muscles. The control of laryngeal muscles during these activities is thought to be mediated by a pathway from the periaqueductal gray via premotor neurons in the nucleus retroambiguus to laryngeal motoneurons in the nucleus ambiguus. However, direct contacts between the nucleus retroambiguus and laryngeal motoneurons have never been demonstrated anatomically. Moreover, data in primates about the nucleus retroambiguus-nucleus ambiguus pathway are lacking. Therefore, the present study examines the projection from the nucleus retroambiguus region to laryngeal motoneurons in the rhesus monkey at the light and electron microscopic levels. Injections with wheat germ agglutinin-horseradish peroxidase were made into the nucleus retroambiguus in five rhesus monkeys to anterogradely label fibers in the nucleus ambiguus. In two of these animals, the cricothyroid muscle was injected with cholera toxin subunit b to identify the motoneurons that supply it. The results show that the nucleus retroambiguus region most densely projects to the compact formation of the nucleus ambiguus, whereas cricothyroid motoneurons, which surround the compact formation, receive a moderate projection. The projections are bilateral, with a contralateral predominance. Ultrastructurally, anterogradely labeled terminal profiles from the nucleus retroambiguus contact cholera toxin subunit b-labeled dendrites of cricothyroid motoneurons. The terminal profiles contain primarily spherical vesicles and form asymmetrical contacts with cricothyroid motoneurons. This study demonstrates that the nucleus retroambiguus region projects to the nucleus ambiguus in the primate. Some of these projections include monosynaptic connections to laryngeal motoneurons. This pathway is important for the control of the vocal folds during vocalization and straining-related activities.