Behavioral and neural toxicity of the artemisinin antimalarial, arteether, but not artesunate and artelinate, in rats.

Three artemisinin antimalarials, arteether (AE), artesunate (AS), and artelinate (AL) were evaluated in rats using an auditory discrimination task (ADT) and neurohistology. After rats were trained on the ADT, equimolar doses of AE (25 mg/kg, in sesame oil, n=6), AS (31 mg/kg, in sodium carbonate, n=6), and AL (36 mg/kg, in saline, n=6), or vehicle (sodium carbonate, n=6) were administered (IM) for 7 consecutive days. Behavioral performance was evaluated, during daily sessions, before, during, and after administration. Histological evaluation of the brains was performed using thionine staining, and damaged cells were counted in specific brainstem nuclei of all rats. Behavioral performance was not significantly affected in any rats treated with AS, AL, or vehicle. Furthermore, histological examination of the brains of rats treated with AS, AL, and vehicle did not show damage. In stark contrast, all rats treated with AE showed a progressive and severe decline in performance on the ADT. The deficit was characterized by decreases in accuracy, increases in response time and, eventually, response suppression. When performance on the ADT was suppressed, rats also showed gross behavioral signs of toxicity that included tremor, gait disturbances, and lethargy. Subsequent histological assessment of AE-treated rats revealed marked damage in the brainstem nuclei, ruber, superior olive, trapezoideus, and inferior vestibular. The damage included chromatolysis, necrosis, and gliosis. These results demonstrate distinct differences in the ability of artemisinins to produce neurotoxicity. Further research is needed to uncover pharmacokinetic and metabolic differences in artemisinins that may predict neurotoxic potential.

Acute high dose arteether toxicity in rats.

Acute high dose administration of the artemisinin antimalarial, beta-arteether (AE), was evaluated in rats using an auditory discrimination task (ADT) and histology. After rats were trained on the ADT, AE (25, 75, 125 mg/kg, i.m.) or vehicle (sesame oil) was administered and behavioral performance was evaluated for 11 consecutive days. Histological evaluation of the brains was performed using thionine and cupric-silver staining. Damaged cells were counted in specific brainstem nuclei of all rats and a qualitative analysis of the rostral-caudal extent of selected brains was performed. Behavioral performance was not significantly affected by any treatment although some evidence of disruption was observed, particularly after the largest dose. At 125 mg/kg, AE produced statistically significant neuropathology, including chromatolysis, in the nucleus trapezoideus and nucleus superior olive. AE at 75 mg/kg, produced significant neuropathology in the nucleus trapezoideus. Neither AE at 25 mg/kg, nor vehicle produced damage. Qualitative analysis revealed a pattern of neuropathology focused in the brainstem. The results show that, in rats, a single dose of AE can produce a pattern of brainstem neuropathology and that specific brainstem nuclei, including auditory nuclei, are particularly vulnerable. These results are consistent with, and extend, previous studies demonstrating brainstem neurotoxicity from repeated AE administration. Moreover, early detection of AE-induced neuropathology is problematic and may require selective examination of brainstem functions.

Behavioral and neural toxicity of arteether in rats.

Repeated administration of the artemisinin antimalarial compound, 3-arteether (AE) (25 mg/kg, i.m.) was evaluated in rats using a two-choice, discrete trial, auditory discrimination task and subsequent neurohistology. Rats were trained to choose one of two response levers following presentation of white noise or a tone + white noise. Increasing and decreasing the intensity of the tone increased and decreased discriminability, respectively, and differential reinforcement density produced systematic changes in response bias. AE (n = 5) or vehicle (n = 5) was injected daily (9-12 days). Initial injections of AE did not affect behavioral performance. Continuing daily injections produced significant decreases in choice accuracy and significant increases in choice reaction time. When overt signs of severe toxicity were observed, rats were sacrificed and significant neural pathology was observed in the nucleus trapezoideus of AE-treated rats. In a subsequent experiment, AE was injected for 3 (n = 5), 5 (n = 5), or 7 (n = 5), consecutive days and performance was examined for an additional 7 days. Behavioral disruption was only observed in rats receiving AE for 7 days and the greatest degree of disruption occurred after AE injections were completed. Histopathological examination showed significant neural pathology in the nuclei trapezoideus, superior olive, and ruber of rats receiving 7- and 5-day AE regimens, and in the nucleus trapezoideus of rats receiving the 3-day regimen. Thus, behavioral disruption reflected, but did not predict, neuropathology. These results confirm and extend earlier results demonstrating neurotoxicity of AE in rats. Further, these results demonstrate that the auditory discrimination task provides an objective behavioral measure of AE neurotoxicity, and thus, can serve as a valuable tool for the safety development of AE and other artemisinin antimalarial compounds.

Dose-dependent brainstem neuropathology following repeated arteether administration in rats.

Histopathological effects of the artemisinin antimalarial, beta-arteether, were evaluated in rats. Arteether (3.125-12.5 mg/kg/day, IM, in sesame oil) was administered for 7 consecutive days. Seven days following the last injection, histological evaluation of the brainstem was performed. Rats treated with 12.5 mg/kg showed significant neuropathology, including chromatolysis, in the nucleus trapezoideus and nucleus superior olive. To a lesser extent, neuropathology was present in the nucleus ruber. Mild neuropathology was also detected in other brainstem regions examined. Although no statistically significant neuropathology was found for the groups treated with 6.25 mg/kg/day and 3.125 mg/kg/day, substantial neuropathology was observed in a single rat in each of these treatment conditions. These results confirm and extend previous studies demonstrating brainstem neurotoxicity from artemisinin antimalarials. Furthermore, these results suggest that, in rats, brainstem auditory pathways may be particularly vulnerable. Early detection of arteether neuropathology may, therefore, require examination of auditory functions.

Factors relating to neurotoxicity of artemisinin antimalarial drugs "listening to arteether".

The discovery of the occult brainstem neurotoxicity subsequent to widespread deployment of artemisinin derivatives has created particular problems. That is, the clinical setting for artemisinin use is problematic for accomplishing what ordinarily would be addressed in phase I-II clinical trials. Nevertheless, it is clear that an urgent and vital need exists for the deployment and widespread availability of artemisinins. The work done to date has already yielded a substantial body of evidence that, while incomplete, provides guidelines for artemisinin use to minimize the risk of these drugs while preserving their much-needed efficacy. The evidence thus far shows that route of administration, oil/water solubility and concentration-duration of drug level, are critical determinants of toxicity and can be given appropriate consideration in the clinical decisions regarding route, choice of drug used, and drug regimens. In this regard, an oral, water-soluble drug with moderately rapid clearance may be the most attractive choice in the absence of significant differences in efficacy. The same body of evidence clearly shows that toxicity can, and does, develop with no obvious or useful clinical marker. Therefore, the development and validation of a test that can reliably detect the onset of injury, at a reversible stage, is a critical path task for any future development in this class. More complete understanding of mechanisms, kinetics, and molecular targets of neurotoxicity, will certainly be forthcoming. A continuing, more generalized use of these drugs, however, cannot be fully endorsed without a useful, practical clinical test of toxicity. The requirement is especially critical in light of the reality that those patients receiving artemisinin derivatives live in high risk environments and are likely to receive repeated courses of therapy with little likelihood of close, post marketing surveillance.

Brainstem reticular nuclei that project to the thalamus in rats: a retrograde tracer study.

The precise nuclear origins of projections from the brainstem reticular formation to the thalamus were identified in rats using two retrograde tracing substances: wheat germ agglutinin-horseradish peroxidase conjugate, and Fluoro-Gold. Injections of these tracers were made into a variety of thalamic nuclei, including the intralaminar nuclei (most of these also involved the lateral part of the mediodorsal nucleus), the central part of the mediodorsal nucleus, the ventrolateral/ventromedial nuclei, and the ventral posterolateral/ventral posteromedial nuclei. Counts of retrogradely labeled cells were done on a large sample of select cases. The data generated by these cell counts indicate that brainstem reticular projections to the intralaminar/lateral mediodorsal complex are fairly strong, as are those to the ventrolateral/ventromedial nuclear complex. Ascending reticular projections to the mediodorsal nucleus per se are somewhat weaker, while those to the ventrobasal complex (or at least the ventral posterolateral nucleus) are weaker still. As a whole, reticular neurons projecting to the thalamus are by far most numerous in the midbrain, and then decline gradually at successively caudal levels through the pons and medulla. Midbrain reticular groups evincing very strong ascending projections include nucleus reticularis (n.r.) pedunculopontinus (particularly its pars compactus), n.r. cuneiformis and n.r. subcuneiformis (together known as the deep mesencephalic nucleus). Strong thalamic projections arise from the medial part of n.r. pontis oralis, the medial (beta) part of n.r. pontis caudalis, and the mid-pontine dorsomedial tegmental area. Within the medullary reticular formation, a 'trans-nuclear field' of neurons encompassing n.r. paragigantocellularis dorsalis and dorsal parts of n.r. gigantocellularis and n.r. parvocellularis was consistently labeled contralateral to the injection site. In general, ascending reticulothalamic projections are largely ipsilateral from midbrain reticular groups, bilateral from pontine reticular groups, and contralateral from medullary reticular groups. Within individual reticular nuclei, the morphology of labeled neurons is identical to that reported previously by this laboratory subsequent to spinal, cortical, or cerebellar tracer injections, thus strengthening our hypothesis that the various brainstem reticular nuclei can be distinguished on the basis of neuronal morphology. As a whole, thalamic-projecting reticular neurons are mostly small or medium-sized cells.

Brainstem reticular nuclei that project to the cerebellum in rats: a retrograde tracer study.

The nuclear origins of projections from the brainstem reticular formation to the cerebellum were examined using four retrograde tracer substances: horseradish peroxidase, wheat germ agglutinin-horseradish peroxidase conjugate, Fluoro-Gold, and rhodamine beads. Tracer injections were made into each of the three major longitudinal zones of the cerebellar cortex (vermis, paravermal hemisphere, and lateral hemisphere) as well as into the various deep cerebellar nuclei. Counts of retrogradely labeled cells were done on a large sample of select cases. The data generated by these cell counts indicate that the strongest reticulocerebellar projections arise from the three specialized pre-cerebellar reticular nuclei: the lateral reticular nucleus, the medullary paramedian reticular nucleus, and the reticulotegmental nucleus. The presumed noradrenergic locus coeruleus (A6 cell group) was also densely packed with retrogradely labeled neurons. However, strong reticulocerebellar projections also arose from other presumed catecholamine cell groups such as those in the ventrolateral medulla (the A1/C1 complex) and the caudal pons (A5). Substantial cerebellar projections originated from most of the various presumed serotonergic brainstem raphe cell groups (particularly raphe obscurus in the medulla), as well as from the presumed cholinergic Ch5 cell group (the pedunculopontine pars compactus nucleus). Labeled cells were also seen in several nonaminergic isodendritic reticular nuclei thought to be involved in visuomotor activity (e.g. paragigantocellularis dorsalis, raphe interpositus, and the pontine dorsomedial tegmental area), as well as in the lateral reticular zone of the medulla and lower pons (reticularis dorsalis and parvocellularis). Tracer injections into the deep nuclei produced relatively greater numbers of labeled neurons in large-celled medial reticular nuclei associated with skeletomotor activity, such as gigantocellularis, magnocellularis, and pontis caudalis. Reticular nuclei conspicuous in their lack of projections to the cerebellum included reticularis ventralis in the medulla, pontis oralis, and both subdivisions of the midbrain reticular formation (cuneiformis and subcuneiformis). As a whole, the various isodendritic reticular nuclei project most strongly to midline cerebellar structures (vermal cortex or fastigial nuclei), less strongly to the paravermal cortex or interposed nuclei, and least strongly to the lateral cortex or dentate nucleus. Within individual reticular nuclei, the morphology of labeled neurons is identical to that reported previously by this laboratory subsequent to spinal or cortical HRP injections, thus strengthening this laboratory's hypothesis that the various brainstem reticular nuclei can be distinguished on the basis of neuronal morphology.

Projections from the medial agranular cortex to brain stem visuomotor centers in rats.

Projections from medial agranular cortex to brain stem in rat were determined by use of the anterograde tracers Phaseolus vulgaris leucoagglutinin, or wheat germ agglutinin conjugated horseradish peroxidase. Axonal trajectories were also followed by means of the Wiitanen modification of the Fink-Heimer degeneration technique. AGm was identified on the basis of its cytoarchitectonics. AGm projected to the anterior pretectal nucleus, the rostral interstitial nucleus of the medial longitudinal fasciculus, the medial accessory oculomotor nucleus of Bechterew, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch, the nucleus cuneiformis and subcuneiformis, intermediate and deep superior collicular layers, the paramedian pontine reticular formation (reticularis pontis oralis and caudalis, and reticularis gigantocellularis), and raphe centralis superior. Differences in connections between rostral and caudal injections were observed: pontine and medullary projections were lighter from the rostral portion of AGm than from the more caudal portions of AGm. The heaviest projections to the anterior pretectal nucleus were from the caudal portion of AGm. The subcortical projections were very similar to those described for the frontal eye field in monkeys, and the majority of them targeted areas thought to be involved in coordination of gaze with head and neck movements. Thus AGm in rats may contain the homologue of the primate frontal eye fields.

Nuclear terminations of corticoreticular fiber systems in rats.

Corticoreticular fiber systems were examined in adult albino and hooded rats using anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and anterograde degeneration. WGA-HRP injections were made stereotactically into the medial prefrontal cortex, the medial agranular cortex, the anterior cingulate cortex, the face motor cortex, the forelimb motor cortex, the trunk-hindlimb motor cortex, the face somatosensory cortex, the primary auditory cortex, the secondary visual cortex and the primary visual cortex. With exception of the cingulate cortex (which is relatively inaccessible to lesioning methods) and the primary visual cortex, electrocautery lesions were made into these same cortical areas. The precise locations of cortical injection/lesion sites were corroborated on the basis of cortical cytoarchitectonic criteria, patterns of retrograde and anterograde thalamic labeling, and patterns of anterograde labeling in non-reticular brainstem nuclei such as the red nucleus, trigeminal nuclei and dorsal column nuclei. The heaviest corticoreticular projections arise from the medial agranular cortex. The medial prefrontal cortex also gives rise to consistently strong corticoreticular projections. The anterior cingulate cortex sends robust corticoreticular projections to the upper brainstem but relatively weak projections to the lower brainstem. With respect to the primary motor cortex, the face area gives rise to the densest corticoreticular projections, rivaling those emanating from the medial agranular cortex. The trunk-hindlimb area gives rise to substantial corticoreticular projections, but those originating from the forelimb area are modest and directed chiefly to midbrain and medullary levels. The face area of the somatosensory cortex gives rise to rather weak corticoreticular projections, while those arising from the primary auditory cortex are fewer still. Descending projections from the secondary visual cortex are sparse, with labeled terminals occurring in a few pontine and medullary reticular nuclei. Only one brainstem reticular nucleus (nucleus cuneiformis) was found to receive projections from the primary visual cortex, and this input was extremely sparse. Corticoreticular projections to the upper brainstem terminate predominantly ipsilateral to the cortical injection site, whereas medullary corticoreticular projections distribute bilaterally. Corticoreticular fibers from the medial agranular, face motor and trunk-hindlimb motor cortex terminate heavily in somatomotor brainstem reticular nuclei such as the pontis oralis, the pontis caudalis and the gigantocellularis.(ABSTRACT TRUNCATED AT 400 WORDS)

Reticulo- and trigemino-hypoglossal connections: a quantitative comparison of ultrastructural substrates.

Axon terminals were identified and characterized by electron microscopy after injections of horseradish peroxidase (HRP) into the spinal V nucleus (SPVN) or the medullary reticular formation adjacent to the XIIth nucleus. The synaptic organization and topology of these two different populations of hypoglossal afferents (T-XII and R-XII respectively) were determined by quantitative comparisons. Significant differences were obtained in the ratios of morphological types of terminals, sizes of axonal endings and their location on postsynaptic structures. Axon terminals containing spherical vesicles (S-terminals) and those with flattened/pleomorphic vesicles (F-terminals) were anterogradely labeled with HRP from both injection sites. However, the S/F ratio for R-XII terminals was 1.2:1 compared to 2.6:1 for T-XII afferents. Asymmetrical membrane densities (Gray Type I) were the predominant form of junctional specialization for S-terminal synapses. Asymmetrical densities with subjunctional dense bodies/bars (S-Taxi) were associated with a higher proportion of T-XII synapses than R-XII synapses. Almost all of the F-terminals from both sources had symmetrical densities (Gray Type II). The average diameter of R-XII terminals was greater than that of T-XII terminals. R-XII-F terminals were the largest terminals. The majority of axon terminals from both sources formed axodendritic synapses. However, R-XII terminals had a higher incidence (10% vs. 3%) of axosomatic contacts. The proportion of R-XII-F-terminals decreased from the central toward the distal dendrites, whereas the opposite was found for T-XII-F and T-XII-S-terminals. In contrast to these findings, R-XII-S-terminals were more uniformly distributed on dendrites of all sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolution of the reticular formation.

The reticular formation of mammals contains numerous nuclei which can be recognized by their projection patterns, cytoarchitectonics, and neuropeptide/neurotransmitter content. We have identified reticular nuclei in representatives from numerous reptilian groups and ascertained presence or absence of these reticular nuclei in an attempt to use neuronal occurrence as a tool to determine phylogenetic relationships. Recently these studies have been extended to two elasmobranchs, a galeomorph shark and a ray. In this report, we concentrate on three medullary spinal projecting reticular nuclei, reticularis gigantocellularis, reticularis magnocellularis, and reticularis paragigantocellularis. We found that all three nuclei were present in rats, lizards, and elasmobranchs, but one nucleus was absent in crocodilians, and two nuclei were absent in turtles. Thus brain organization may give us clues to phylogenetic relationships. Moreover, these three reticular nuclei exhibited remarkably similar cellular morphology in mammals, reptiles, and elasmobranchs.

Nuclear origins of brainstem reticulocortical systems in the rat.

Stereotaxic injections of 5% Fast Blue or 1% horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) were made into various cytoarchitectonic or functional regions of the cerebral cortex of anesthetized adult albino or hooded rats. Sections through the brainstems of these animals were then scrutinized for the presence of retrogradely labeled neurons. The data generated by this study indicate that at least 33 distinct nuclei or subnuclei within the brainstem reticular formation of the rat project directly to the cerebral cortex. More than half of these ascending reticulocortical systems are probably aminergic. The strongest reticulocortical projections emanate from presumed aminergic reticular-cell groups located at isthmic levels: specifically, the rostral serotonin-containing cell groups, as well as the noradrenergic locus coeruleus. However, relatively strong direct reticulocortical projections also originate from lower medullary cell groups which are probably catecholaminergic. Moderately strong reticulocortical projections emanate from cholinergic cell groups located at isthmic levels (the pars compacta of the pedunculopontine nucleus and the X area of Sakai). The most surprising finding in this study was that the classic isodendritic, nonaminergic central core of the brainstem gives rise to direct reticulocortical projections. The ventromedial areas of the medullary brainstem reticular formation give rise to the strongest nonaminergic ascending reticular projections, but all levels of the classic isodendritic reticular core give rise to direct reticulocortical projections. As a whole, cortically projecting reticular neurons are mostly small (10-25 microns in greatest diameter) or medium sized (26-35 microns in greatest diameter) neurons. Previous studies have shown that many of the cortically projecting reticular nuclei also project to the spinal cord, and within these nuclei, reticulocortical neurons often strongly resemble their reticulospinal counterparts with respect to details of neuronal morphology. This in turn suggests that some reticulocortical neurons may also project to spinal levels.

Distinguishing rat brainstem reticulospinal nuclei by their neuronal morphology. I. Medullary nuclei.

While cytoarchitectonic and hodological investigations suggest that the brainstem reticulospinal nuclei (BRN) are complexly organized, previous Golgi studies claimed that BRN comprise a homogeneous population with respect to neuronal morphology. To determine whether this is indeed the case, neurons of the various BRN of adult albino or hooded rats were either backfilled with horseradish peroxidase (HRP) from spinal injections, stained with a Nissl method or impregnated with a Golgi-Kopsch variant. The results suggest that at least thirteen BRN can be distinguished in the medulla. Some medullary BRN contain neurons whose dendritic arborizations (DA) are radially symmetric (e.g., nucleus reticularis (NR) ventralis pars beta (RVb), NR gigantocellularis (RGc) and nucleus raphe magnus (RaM]. Some BRN contain neurons whose DA exhibit a pronounced dorsomedial to ventrolateral slant (e.g., NR dorsalis (RD) and NR parvocellularis (RPc). The DA of NR paragigantocellularis dorsalis (RPgcd) neurons tend to course dorsally. The DA of nucleus raphe obscurus (RaO) neurons course vertically, while those of NR magnocellularis pars alpha (RMca) and NR magnocellularis pars beta (RMcb) course horizontally. The DA of NR ventralis pars alpha (RVa) may be oriented horizontally also, but sometimes slant from dorsolateral to ventromedial. The DA of NR paramedianus neurons (RPm) are cruciform. The neurons of NR paragigantocellularis lateralis (RPgcl) and of the nucleus raphe pallidus (RaP) exhibit a variety of DA patterns. The neurons of RD, RVa, RMcb and RMca project to the spinal cord with a strong ipsilateral predominance, while those of RVb, RPgcl and RGc project to the spinal cord with a weak ipsilateral predominance. The axons of RPc, RaO, RaP, and RaM neurons exhibit no lateral predominance. RPm neurons project to the cord with a weak contralateral predominance, and RPgcd neurons project to the cord with a strong contralateral predominance. Most medullary BRN project to the spinal cord via the medial longitudinal fasciculus (mlf) and sulcomarginal fasciculus. However, RPgcl, RMca and RMcb also project to the spinal cord via the lateral funiculus. The neurons of RD, RPm and RaM project to the spinal cord exclusively via the lateral or dorsolateral funiculus. Since the various medullary BRN are distinguishable on the basis of neuronal morphology, they may play distinct roles in brainstem modulation of spinal motor, sensory or autonomic activity.

Distinguishing rat brainstem reticulospinal nuclei by their neuronal morphology. II. Pontine and mesencephalic nuclei.

The purpose of this study was to determine whether pontine and mesencephalic reticulospinal nuclei like those of the medulla, can be differentiated on the basis of neuronal morphology. Accordingly, neurons of the various pontine and mesencephalic brainstem reticulospinal nuclei (BRN) of adult albino or hooded rats were either backfilled with horseradish peroxidase (HRP) from spinal injections, stained with a Nissl method or impregnated with a Golgi-Kopsch variant. The results suggest that at least 13 BRN can be distinguished in the pons and mesencephalon of the rat on the basis of neuronal morphology. The dendritic arborizations (DA) of neurons in nucleus reticularis (NR) pedunculopontinus pars compacta (RPpc), NR pedunculopontinus pars dissipatus (RPpd), NR cuneiformis (RCf) and NR subcuneiformis (RScf) are radially symmetrical. The DA of neurons in NR pontis caudalis pars beta (RPoCb) and alpha (RPoCa), as well as those of NR subcoeruleus (RSc) and the A5 cell group, exhibit a pronounced dorsomedial to ventrolateral slant. The dendrites of locus coeruleus (LC) neurons slant from dorsolateral to ventromedial. The DA of NR pontis oralis pars medialis (RPoOm) neurons course medially, while those of NR pontis oralis pars lateralis (RPoOl) course laterally. The DA of Kolliker-Fuse neurons (KF) course horizontally. Finally, the DA of the nucleus raphe dorsalis (RaD) are either symmetrically multipolar or fusiform. The neurons of RPoCb, RPpc, KF and RCf project to the spinal cord with a strong ipsilateral predominance, while those of LC, RSc, RPoOm and RPpd project to the spinal cord with a weak ipsilateral predominance. The axons of A5, RPoOl and RaD neurons exhibit no lateral predominance in their spinal projections. Finally, RPoCa neurons, as well as neurons in RScf, project to the spinal cord with a strong contralateral predominance. The neurons of RPoCb, RPoCa, RPoOl, RPpd and RScf project to the spinal cord via the mlf and sulcomarginal fasciculus. The neurons of RSc, KF, RPoOm, RPpc and RCf project to the spinal cord via the lateral funiculus.

The sources of supraspinal afferents to the spinal cord in a variety of limbed reptiles. I. Reticulospinal systems.

Horseradish peroxidase was injected into various levels of the spinal cord of turtles (Pseudemys and Chrysemys), lizards (Tupinambis, Iquana, Gekko, Sauromelus, and Gerrhonotus), and a crocodilian (Caiman). The results suggest that brainstem reticulospinal projections in limbed reptiles rival mammalian reticulospinal systems in complexity. The reptilian myelencephalic reticular formation can be divided into four distinct reticulospinal nuclei. Reticularis inferior pars dorsalis (RID) contains multipolar neurons which project bilaterally to the spinal cord. Reticularis inferior pars ventralis (RIV), which is only found in lizards and crocodilians, contains fusiform neurons with horizontally running dendrites and it projects ipsilaterally to the spinal cord. Reticularis ventrolateralis (RVL), which is found only in field lizards, contains triangular neurons whose dendrites parallel the ventrolateral edge of the brainstem and it projects ipsilaterally to the spinal cord. The myelencephalic raphe (RaI) varies considerably. RaI of turtles contains large reticulospinal neurons which form a continuous population with more laterally situated RID cells. RaI of lizards contains a few small reticulospinal neurons. RaI of the crocodilian Caiman contains giant reticulospinal neurons with laterally directed dendrites. The caudal metencephalic reticular formation of reptiles can be divided into two distinct reticulospinal nuclei. Reticularis medius (RM) contains large neurons with long, ventrally directed dendrites; it projects ipsilaterally to the spinal cord. Reticularis medius pars lateralis (RML) contains small neurons with laterally directed dendrites; it projects contralaterally to the spinal cord. The rostral mesencephalic and caudal mesencephalic reticular formation of reptiles can be divided into three distinct reticulospinal nuclei. Reticularis superior pars medialis (RSM) consists mostly of small, spindle-shaped neurons which project bilaterally to the spinal cord. In the lizard Tupinambis, however, large multipolar, ipsilaterally projecting neurons are occasionally seen in RSM. Reticularis superior pars lateralis (RSL) contains large, ipsilaterally projecting neurons with long, ventrolaterally directed dendrites. SRL in lizards can be divided into a dorsomedial portion, which projects ipsilaterally to the spinal cord, and a ventrolateral portion which projects contralaterally. The locus ceruleus-subceruleus field (LC-SC) contains small spindle-shaped neurons which project bilaterally to the spinal cord. Labelled reticulospinal neurons were also observed in the rostral metencephalic raphe (RaS) of the turtle brainstem. These cells are small, spindle-shaped neurons which resemble the small cells of the adjacent RSM field.

The organization of the reptilian formation: a comparative study using Nissl and Golgi techniques.

The brainstem reticular formation has been studied in 16 genera representing 11 families of reptiles. Measurements of Nissl-stained reticular neurons revealed that they are distributed along a continuum, ranging in length from 10 micrometer to 95 micrometer. Reticular neurons in crocodilians and snakes tend to be larger than those found in lizards and turtles. Golgi studies revealed that reticular neurons possess long, rectilinear, sparsely branching dendrites. Small reticular neurons (less than 31 micrometers length) possess fusiform or triangular somata which bear two or three primary dendrites. These dendrites have a somewhat simpler ramification pattern when compared with those of large reticular neurons ( greater than 30 micrometers length). Large reticular neurons generally possess perikarya which are triangular or polygonal in shape. The somata of large reticular neurons bear an average of four primary dendrites. The dendrites of reptilian reticular neurons ramify predominantly in the transverse plane and are devoid of spines or excrescences. The dendritic ramification patterns observed in the various repitilian reticular nuclei were correlated with known input and output connections of these nuclei. Nissl and golgi techniques were used to divide the reticular formation into seven nuclei. A nucleus reticularis inferior (RI) is found in the myelencephalon, a reticularis medius (RM) in the caudal two-thirds of the metencephalon, and a reticularis superior (RS) in the rostral metencephalon and caudal mesencephalon. Reticularis inferior can be subdivided into a dorsal portion (RID) and a ventral portion (RIV). All reptilian groups possess RID and RM but RIV is lacking in turtles. Reticularis superior can be subdivided into a large-celled lateral portion (RSL) and a small-celled medial portion (RSM). All reptilian groups possess RSM and RSL, but RSL is quite variable in appearance, being best developed in snakes and crocodilians. The myelencephalic raphe nucleus is also quite variable in its morphology among the different reptilian families. A seventh reticular nucleus, reticularis ventrolateralis (RVL), is found only in snakes and in teiid lizards. It was noted that the reticular formation is simpler (fewer numbers of nuclei) in the representative of older reptilian lineages and more complex (greater numbers of nuclei) in the more modern lineages. Certain reticular nuclei are present or more extensive in those families which have prominent axial musculature.

Brain stem origins of spinal projections in the lizard Tupinambis nigropunctatus.

In order to study brainstem origins of spinal projections, ten Tegu lizards (Tupinambis nigropunctatus) received complete or partial hemisections of the spinal cord at the first or second cervical segment. Their brains were processed for conventional Nissl staining. The sections were surveyed for the presence or absence of retrograde chromatolysis. Based on analysis and comparison of results from lesions in the various spinal cord funiculi, the following conclusions were reached: The interstitial nucleus projects ipsilaterally to the spinal cord via the medial longitudinal fasciculus, as does the middle reticular field of the metencephalon. The red nucleus and dorsal vagal motor nucleus both project contralaterally to the spinal cord via the dorsal part of the lateral funiculus. The superior reticular field in the rostral metencephalon and the ventrolateral vestibular nucleus project ipsilaterally to the spinal cord via the ventral funiculus. The dorsolateral metencephalic nucleus and the ventral part of the inferior reticular nucleus of the myelencephalon both project ipsilaterally to the spinal cord via the dorsal part of the lateral funiculus. Several brainstem nuclei in Tupinambis project bilaterally to the spinal cord. The ventrolateral metencephalic nucleus, for example, projects ipsilaterally to the cord via the medial longitudinal fasciculus and contralaterally via the dorsal part of the lateral funiculus. The dorsal part of the inferior reticular nucleus projects bilaterally to the spinal cord via the dorsal part of the lateral funiculus. The nucleus solitarius complex projects contralaterally via the dorsal part of the lateral funiculus but ipsilaterally via the middle of the lateral funiculus. The inferior raphe nucleus projects bilaterally to the spinal cord via the middle part of the lateral funiculus. These data suggest that supraspinal projections in reptiles, especially reticulospinal systems, are more highly differentiated than previously thought. On the other hand, recent findings in cat, opossum, and monkey reveal that the organization of supraspinal pathways in the Tegu lizard bears a striking resemblance to that observed in mammals.