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Ibogaine is a tremorigenic hallucinogen that has been proposed for clinical use in treating addiction. We previously reported that ibogaine, administered systemically, produces degeneration of a subset of Purkinje cells in the cerebellum, primarily within the vermis. Ablation of the inferior olive affords protection against ibogaine-induced neurotoxicity leading to the interpretation that ibogaine itself is not directly toxic to Purkinje cells. We postulated that ibogaine produces sustained excitation of inferior olivary neurons that leads to excessive glutamate release at climbing fiber terminals, causing subsequent excitotoxic injury to Purkinje cells. The neuronal degeneration induced by ibogaine provides an animal model for studying excitotoxic injury in order to analyze the contribution of glutamate receptors to this injury and to evaluate neuroprotective strategies. Since non-N-methyl-d-aspartate (NMDA) receptors mediate Purkinje cell excitation by climbing fibers, we hypothesized that 1-4-aminophenyl-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466), which antagonizes non-NMDA receptors, may have a neuroprotective effect by blocking glutamatergic excitation at climbing fiber synapses. To test this hypothesis, rats were administered systemic ibogaine plus GYKI-52466 and the degree of neuronal injury was analyzed in cerebellar sections. The results indicate that the AMPA antagonist GYKI-52466 (10 mg/kg i.p.×3) does not protect against Purkinje cell injury at the doses used. Rather, co-administration of GYKI-52466 with ibogaine produces increased toxicity evidenced by more extensive Purkinje cell degeneration. Several hypotheses that may underlie this result are discussed. Although the reason for the increased toxicity found in this study is not fully explained, the present results show that a non-NMDA antagonist can produce increased excitotoxic injury under some conditions. Therefore, caution should be exercised before employing glutamate antagonists to reduce the risk of neuronal damage in human clinical disorders. Moreover, the contribution of different glutamate receptors to excitotoxic injury is complex and merits further analysis. [Copyright &y& Elsevier]

Background and Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target., Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously., Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control ( P  = .003 and P  = .0005, respectively)., Conclusion: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation., (© 2022 Published by Elsevier, Inc on behalf of the AGA Institute.)

Vesicular stomatitis (VS) is a vesicular disease of horses, cattle, and pigs in the Western Hemisphere caused by viruses in the genus Vesiculovirus. Disease manifests as vesicles and erosions on the oral mucosa, teats, prepuce, and coronary band, and is similar in presentation to foot-and-mouth disease. Laboratory confirmation is therefore required. Conventional assays include competitive (c)ELISA and complement fixation (CF). The cELISA provides more accurate herd-level detection of VSV-exposed cattle, but may lack the ability to capture fluctuating antibody levels in individual animals. The CF assay can confirm newly infected animals because of its ability to detect antigen-antibody complexes, thus is considered to be indicative of IgM. We evaluated the immune status of 2 herds affected by VSV in 2014 by testing sera collected in June 2015. Two conventional assays were compared to a novel IgM-IgG ELISA. When sampled in 2015, both herds had detectable VSV-specific antibodies; 18% and 36% of animals tested by cELISA and 2% and 8% of animals tested by CF were positive. The novel IgM-IgG assay exhibited fair agreement (adjusted kappa score of 48) with the conventional assays, and should be evaluated further to assess its ability to replace the 2 separate assays with a single assay system, or for its ability to replace the CF assay as a more sensitive method for defining newly exposed animals.

SCA12 is a late-onset, autosomal dominant, slowly progressive disorder. Action tremor is the usual presenting sign. Subsequent development of ataxia and hyperreflexia suggests spinocerebellar ataxia. In the index SCA12 kindred, which resides in North America and is of German ancestry, parkinsonism, anxiety, depression, and cognitive dysfunction are not uncommon. SCA12 is linked to a CAG repeat expansion mutation in exon 7 of PPP2R2B, a gene that encodes Bβ, a regulatory subunit of protein phosphatase 2A (PP2A). CAG repeats number 7-28 in normal individuals and 55-78 in SCA12 patients. The mechanism by which this mutation leads to SCA12 has not been determined. The CAG expansion in PPP2R2B has promoter function in vitro. CAG length correlates with increased Bβ expression. There is no evidence that this CAG expansion results in polyglutamine production. In addition to the North. American SCA12 kindred, multiple SCA12 families have been found in Northern India that are not related to the index SCA12 kindred. SCA12 has been reported, rarely, in Singapore and China. Action tremor, anxiety, and depression in SCA12 have responded to usual treatments for these disorders. SCA12 may be considered in patients who present with action tremor and later develop signs of cerebellar and cortical dysfunction., (2012 Elsevier B.V. All rights reserved.)

The authors performed a chart review to determine the frequency with which neurologists detect cognitive and psychiatric symptoms in patients with cerebellar degeneration. Psychopathology, including depression, personality change, cognitive impairment, anxiety, and psychosis was noted in 51% of 133 patients.

The most common form of autosomal dominant hereditary spastic paraparesis (HSP), SPG4, is caused by mutations in the spastin gene on chromosome 2p. This disease is characterized by intra- and interfamilial phenotypic variation. To determine the predictive values of clinical signs and symptoms in SPG4, we examined 43 members of a large pedigree with autosomal dominant HSP. We then identified the genetic etiology of the disorder in this family, a novel nonsense mutation in exon 1 of spastin, carried by 24 of the examined family members. The best clinical predictors of positive gene status were the presence of hyperreflexia in the lower extremities, >2 beats of ankle clonus, pes cavus, bladder symptoms and increased tone in the legs. The mean age of onset was 32.2 +/- 7.4 years, but the age of onset was earlier in children from 10 of 12 child-parent gene-positive pairs, with a mean difference of 10.8 +/- 3.3 years. The finding of leg weakness was especially common in older-onset affected family member with leg hyperreflexia. These results suggest that specific clinical signs and symptoms may be of value in differentiating individuals affected with SPG4 from family members with nonspecific neurological findings., (Copyright 2004 Movement Disorder Society)

To determine the specificity of cognitive impairments in patients with cerebellar degeneration (CD), the neuropsychological test performance of 31 CD patients was compared to that of 21 patients with Huntington's disease (HD) and 29 normal adults. The CD and HD groups did not differ in age, education, or duration of illness, and their overall severity on a quantified neurological examination was similar. Fifteen neuropsychological test variables were reduced to five underlying domains: motor, verbal, spatial, memory, and executive functioning. The CD patients had their greatest impairment in the executive domain and their least in the memory domain. In contrast, the HD patients had very substantial spatial deficits and significant memory impairment, in addition to executive dysfunction. The findings indicate that 1) the cognitive impairment in CD is not as severe as in HD, and 2) the pattern of deficits in CD, while consistent with a subcortical dementia, differs in important ways from that in HD. These differences may reflect the involvement of the cerebellar dentate nucleus and the striatal nuclei in separate "loops" or closed circuits, linking them with specific areas of cerebral neocortex.

Huntington's disease-like 2 is an autosomal dominantly inherited disorder due to an expansion of trinucleotide repeats. It resembles classic Huntington's disease in clinical phenotype, inheritance pattern, and neuropathological features. We highlight the clinical features of this disorder, including chorea, dystonia, parkinsonism, and cognitive deficits., (Copyright 2003 Movement Disorder Society)

Purpose: Penclomedine (PEN), a multichlorinated alpha-picoline derivative which is metabolized to highly reactive alkylating species, was selected for clinical development due to its prominent activity against a wide range of human tumor xenografts when administered either parentally or orally. Its principal dose-limiting toxicity in preclinical and clinical studies has been neurocerebellar toxicity, which has been related to the magnitude of peak plasma PEN concentrations, but not to plasma concentrations of its putative principal alkylating metabolite, 4,o-demethylpenclomedine (DMPEN). These observation, as well as PEN's toxicologic, pharmacologic, and tissue distribution profiles, have suggested that the parent compound is primarily responsible for cerebellar toxicity. The studies described in this report were undertaken to characterize the neuropathology of PEN neurotoxicity, with a long-term goal of developing strategies to maximize its therapeutic index., Design: Male Sprague-Dawley rats were treated with therapeutically relevant doses of PEN, orally and intraperitoneally (i.p.), on various administration schedules, and DMPEN administered i.p. The animals were monitored for neurotoxicity, and brain sections were examined for neuropathology, particularly Purkinje cell loss and neuronal injury. Brain sections were stained using standard histochemical techniques and immunostained with OX-42 to detect microglial cells that are activated following neuronal damage, and calbindin D(28K), a calcium-binding protein expressed by cerebellar Purkinje cells., Results: Dose-related neurocerebellar toxicity associated with parasagittal bands of Purkinje cell degeneration and microglial activation in the cerebellar vermis were evident in rats treated with PEN 100-400 mg/kg i.p. as a single dose. Neuronal injury was not observed in other regions of the brain. Furthermore, neither clinical nor histopathological evidence of cerebellar toxicity was apparent in rats treated with similar total doses of PEN administered i.p. on a dailyx5-day dosing schedule. Similar histological findings, in an identical neuroanatomical distribution, were observed in rats treated with PEN orally; however, the magnitude of the neuronal toxicity was much less than in animals treated with equivalent doses of PEN administered i.p. Although acute lethality occurred in some rats treated with equimolar doses of DMPEN as a single i.p. treatment, surviving animals exhibited neither signs nor histopathological evidence of neurocerebellar toxicity., Conclusions: PEN produces selective dose- and schedule-dependent Purkinje cell degeneration in the cerebellar vermis of rats, whereas therapeutically relevant doses of PEN administered orally are better tolerated and produce less neurocerebellar toxicity. In addition, roughly equivalent, albeit intolerable, doses of the major active metabolite DMPEN, which was lethal to some animals, produced neither clinical manifestations of neurocerebellar toxicity nor Purkinje cell loss. These results support a rationale for investigating whether PEN administered orally, which may undergo significant first-pass metabolism to DMPEN and other less toxic intermediates, or treatment with DMPEN, itself, may result in less neurocerebellar toxicity and superior therapeutic indices than PEN administered parenterally.

Objective: This study estimated the psychiatric morbidity of patients with degenerative cerebellar diseases., Method: The study included a series of 31 patients with degenerative cerebellar diseases, compared with 21 patients with Huntington's disease and 29 neurologically healthy comparison subjects. Comprehensive psychiatric evaluations, including the Structured Clinical Interview for DSM-IV and psychopathology rating scales, were administered., Results: The overall rate of noncognitive psychiatric disorders was 77% in the patients with degenerative cerebellar diseases, nearly identical to that in the patients with Huntington's disease (81%) and about double that seen in the neurologically healthy subjects (41%). There were high rates of all mood disorders in both the degenerative cerebellar diseases group (68%) and the Huntington's disease group (43%); the rate in the degenerative cerebellar diseases group was significantly higher than that in the neurologically healthy subjects (31%). The frequency of personality change in the three groups was striking: change was present in 26% of the degenerative cerebellar diseases patients, 48% of the Huntington's disease patients, and none of the neurologically healthy comparison subjects. A total of 19% of the degenerative cerebellar diseases subjects and 71% of the Huntington's disease subjects met DSM-IV criteria for either cognitive disorder or dementia., Conclusions: The high rate of psychiatric and cognitive disorders in the patients with degenerative cerebellar diseases suggests that many, if not most, patients with degenerative cerebellar diseases may benefit from psychiatric interventions. These results also support previous findings that the cerebellum may have a role in modulating emotion and cognition.

The experiments strongly suggested that the reason why Purkinje cells die so easily after global brain ischemia relates to deficiencies in aldolase C and EAAT4 that allow them to survive pathologically intense synaptic input from the inferior olive after the restoration of blood flow. This conclusion is based on: (a) the remarkably tight correspondence between the regional absence of aldolase C and EAAT4 in Purkinje cells and the patterned loss of Purkinje cells after a bout of global brain ischemia; (b) the necessity of the olivocerebellar pathway for the ischemic death of Purkinje cells; and (c) the build-up of pathologically synchronous and high-frequency burst activity within the inferior olive during recovery from ischemia. Indeed, the correspondence between the absence of aldolase C and EAAT4 to sensitivity to ischemia could be demonstrated for zones of Purkinje cells as small as two neurons. A second finding was that Purkinje cells are not uniformly sensitive to transient ischemia, since they die most frequently in zones where aldolase C and EAAT4 are absent. One implication of the experiment is that factors beyond the unique synaptic and membrane properties of Purkinje cells play an important role in determining this neuron's high sensitivity to ischemia. The data strongly imply that two properties of Purkinje cells that make them susceptible to ischemic death are their reduced capability to sequester glutamate and reduced ability to generate energy during anoxia. The patterned death of Purkinje cells is sufficient to induce a form of audiogenic myoclonus, as determined with a neurotoxic dose of ibogaine. Ibogaine-induced myoclonus is recognized behaviorally as a reduced ability to habituate to a startle stimulus and resembles the myoclonic jerk of rats during recovery from a prolonged bout of global brain ischemia. Commonalities of ischemia and ibogaine-induced neurodegeneration are the intricately striped Purkinje cell loss in the posterior lobe and a nearly complete deafferentation of the lateral aspect of the fastigial nucleus from the cerebellar cortex, in particular the dorsolateral protuberance. Thus, the data point strongly to a cerebellar contribution to audiogenic myoclonus. Single-neuron electrophysiology experiments in monkeys have demonstrated that the evoked activity in the deep cerebellar nuclei occurs too late to initiate the startle response (60) and electromyography of the postischemic myoclonus of rats corroborates this view (see Chapter 31) (20). However, the nearly complete loss of GABAergic terminals in the dorsolateral protuberance after Purkinje cell death would be expected to dramatically increase its tonic firing and the background excitation of the brain-stem structures that it innervates. The fastigial nucleus innervates a large number of autonomic and motor structures in the brainstem and diencephalon, including the ventrolateral nucleus of the thalamus and the gigantocellular reticular nucleus in the medulla--structures that have been implicated in human posthypoxic myoclonus (6, 7). We propose that the posthypoxic myoclonic jerk of rats is, at least in part, due to disinhibition of the fastigial nucleus produced by patterned Purkinje cell death in the vermis. The argument is as follows: the loss of GABAergic inhibition in the fastigial nucleus after ischemia leads to diaschisis of the motor thalamus and reticular formation which, in turn, is responsible for enhanced motor excitability and myoclonus. That the audiogenic myoclonus after global brain ischemia in the rat gradually resolves over a period of 2 to 3 weeks is consistent with this view, as restoration of background excitability after CNS damage in rats has been documented to occur within this time-frame (61). Our view brings together the physiologic finding that posthypoxic myoclonus appears to originate in the sensory-motor cortices and/or reticular formation with the consistent anatomical finding of Purkinje cell loss after ischemia, and explains the puzzle of Marsden's unique cases of myoclonus associated with coeliac disease (1). Moreover, our argument is consistent with findings both in rats (62, 63) and humans (64) that damage to the vermis impairs the long-term habituation of the startle reflex. It remains to be determined whether the pathologically enhanced startle responses after vermal damage resemble brain-stem reticular or cortical myoclonus at the electrophysiologic level of analysis. What is the purpose of the regional expression of aldolase C and EAAT4 in Purkinje cells? The close correspondence between the spatial distribution of aldolase C and the parasagittal anatomy of the cerebellum (48) has led to the view that aldolase C may help specify connectivity during development. While the present experiments do not address this issue, they underscore the fact that aldolase plays a fundamental role in metabolism. Because Purkinje cells have a repressed expression of aldolase A (31), whatever role the absence of aldolase C may play during development comes at the price of metabolic frailty later in adulthood. From another point of view, aldolase C and EAAT4 appear to confer upon Purkinje cells the ability to survive their own climbing fiber. Indeed, climbing fibers form a distributed synapse that synchronously releases glutamate (or aspartate) at all levels of the dendritic tree simultaneously (65, 66). Such synchronous activation triggers calcium influx throughout the Purkinje cell dendrites at a magnitude that is unparalleled in the nervous system (12), and, thus, places an extraordinarily high metabolic demand on the Purkinje cell. The apparently reduced level of aldolase in a subpopulation of Purkinje cells provides the condition for energy failure and death during anoxia so long as the climbing fibers are intact or when climbing fiber activation is pharmacologically enhanced under normoxic conditions, such as after ibogaine (53-56). Lastly, the argument that diaschisis produced by patterned cerebellar degeneration leads to thalamo-cortical and reticular hyperexcitability agrees with C. David Marsden and his colleagues' bold demonstration of an inhibitory influence of cerebellar cortex on motor cortex in humans (67). Our anatomic data indicate that the spatially distinct zones of Purkinje cells, which are killed by global brain ischemia, may be the origin of such inhibition.

We recently described a disorder termed Huntington disease-like 2 (HDL2) that completely segregates with an unidentified CAG/CTG expansion in a large pedigree (W). We now report the cloning of this expansion and its localization to a variably spliced exon of JPH3 (encoding junctophilin-3), a gene involved in the formation of junctional membrane structures.

Ibogaine, an indole alkaloid that causes hallucinations, tremor, and ataxia, produces cerebellar neurotoxicity in rats, manifested by degeneration of Purkinje cells aligned in narrow parasagittal bands that are coextensive with activated glial cells. Harmaline, a closely related alkaloid that excites inferior olivary neurons, causes the same pattern of Purkinje cell degeneration, providing a clue to the mechanism of toxicity. We have proposed that ibogaine, like harmaline, excites neurons in the inferior olive, leading to sustained release of glutamate at climbing fiber synapses on Purkinje cells. The objective of this study was to test the hypothesis that increased climbing fiber activity induced by ibogaine mediates excitotoxic Purkinje cell degeneration. The inferior olive was pharmacologically ablated in rats by a neurotoxic drug regimen using 3-acetylpyridine, and cerebellar damage attributed to subsequent administration of ibogaine was analyzed using immunocytochemical markers for neurons and glial cells. The results show that ibogaine administered after inferior olive ablation produced little or no Purkinje cell degeneration or glial activation. That a lesion of the inferior olive almost completely prevents the neurotoxicity demonstrates that ibogaine is not directly toxic to Purkinje cells, but that the toxicity is indirect and dependent on integrity of the olivocerebellar projection. We postulate that ibogaine-induced activation of inferior olivary neurons leads to release of glutamate simultaneously at hundreds of climbing fiber terminals distributed widely over the surface of each Purkinje cell. The unique circuitry of the olivocerebellar projection provides this system with maximum synaptic security, a feature that confers on Purkinje cells a high degree of vulnerability to excitotoxic injury.

Ibogaine, an indole alkaloid, has been proposed for treatment of drug addiction, yet its mechanism, site of action, and possible neurotoxicity have not been determined. Since neuronal injury is known to activate neurologlial cells, we investigated potential neurotoxic effects of this drug in rats by examining expression of specific glial markers. After treatment with ibogaine (100 mg kg-1 i.p.; 1-3 doses), we observed increased cytochemical markers in both microglia (OX-6, OX-42, W3/25) and astrocytes (GFAP), associated with striking morphologic changes in these cells. Activated glial cells were restricted to longitudinally oriented, parasagittal stripes within the vermis of cerebellar cortex. The ibogaine-induced activation of cerebellar glial cells is highly suggestive of neuronal degeneration, most likely of Purkinje cells.

The cerebral cortex of the rat and other mammals is innervated by two morphologically distinct classes of serotoninergic (5-HT) axon terminals: fine axons with minute varicosities and beaded axons characterized by large, spherical varicosities. Fine and beaded 5-HT axons exhibit different regional and laminar distributions in forebrain and arise from separate brainstem nuclei, the dorsal and median raphe nuclei, respectively. The present neuroanatomic study, based on immunocytochemical methods to visualize 5-HT axons, demonstrates that the two axon types differ markedly in their vulnerability to the neurotoxic amphetamine derivatives, methylenedioxyamphetamine (MDA), and p-chloroamphetamine (PCA). While both drugs cause extensive degeneration of fine 5-HT axons throughout forebrain, beaded 5-HT axons are consistently spared. Fine 5-HT axons, which richly innervate most regions of dorsal forebrain in control rats, are rarely seen 2 weeks after treatment with MDA or PCA; this loss of fine axons reflects a marked denervation that persists for months after drug administration. The serotoninergic axon terminals remaining after MDA or PCA administration are almost entirely of the beaded type and appear to be unaffected by both drugs. Over a wide range of doses (2.5-40 mg/kg PCA) and survival times (2 weeks to 2 months), these spared 5-HT axons with large, spherical varicosities cannot be distinguished from the normal, beaded 5-HT axons in control rats by morphologic criteria. Moreover, beaded 5-HT axons exhibit a highly characteristic regional distribution which is the same in control as in MDA- and PCA-treated rats: these axons innervate specific zones or layers within parietal and occipital cortex, hippocampus, cingulate cortex, entorhinal cortex, and the olfactory bulb, among other forebrain areas, and they form a dense plexus lining the ventricular system. Taken together, the results of this study demonstrate that fine 5-HT axons are highly vulnerable to the neurotoxic effects of the amphetamine derivatives MDA and PCA, while beaded 5-HT axons are markedly resistant. These findings are consistent with the hypothesis that there are two anatomically and functionally distinct sets of serotoninergic neurons projecting to forebrain. While both of these neuronal systems utilize 5-HT as a neurotransmitter, they differ in several features: 1) origin from separate nuclei in the brainstem (the dorsal and median raphe), 2) two types of morphologically distinct axon terminals, 3) markedly different distribution and innervation patterns in forebrain, and 4) dissimilar pharmacological properties. The results further suggest that psychotropic amphetamine derivatives have a selective action upon fine serotoninergic axons that arise from the dorsal raphe nucleus.

The cytotoxic effects of amphetamine derivatives were studied by immunocytochemistry to identify the cellular compartments affected by these drugs, to obtain morphologic evidence of neuronal degeneration, and to assess the potential for regeneration. The substituted amphetamines, MDA, MDMA, PCA, and fenfluramine, all release serotonin and cause acute depletion of 5-HT from most axon terminals in forebrain. (1) Unequivocal signs of axon degeneration were seen at 36-48 hour survivals: 5-HT axons exhibited increased caliber, huge, swollen varicosities, fragmentation, and dilated proximal axon stumps. (2) Fine 5-HT axon terminals were persistently lost after drug administration, while beaded axons and raphe cell bodies were spared. These two types of 5-HT axons, which arise from separate raphe nuclei and form distinct ascending projections, are differentially vulnerable to psychotropic drugs. (3) From 2-8 months after treatment, there was progressive serotonergic re-innervation of neocortex along a fronto-occipital gradient. Longitudinal 5-HT axons grew into layers I and VI from rostral to caudal, before sprouting into middle cortical layers; this bilaminar pattern of growth simulates perinatal development of 5-HT innervation. This study demonstrates differential vulnerability of 5-HT projections, evidence for axonal degeneration, and sprouting of 5-HT axons leading to re-innervation of forebrain. While the sprouting axons are anatomically similar to the type that was damaged, it is not known whether a normal pattern of innervation is re-established.

Retrograde transport of a fluorescent dye was employed to study the projections from raphe nuclei to neocortex in the rat. The spatial distributions of labeled raphe cells were analyzed quantitatively to determine whether the nuclei are topographically organized with respect to different cortical targets. The dorsal raphe nucleus (DRN), exclusive of the lateral wing regions, has a predominantly (3:1) ipsilateral projection with decreasing numbers of cells projecting to frontal, parietal, and occipital cortex. Overlapping cell groups within the DRN project differentially to these three cortical areas: DRN cells innervating frontal cortex extend more rostrally and laterally than those to either parietal or occipital cortex. The medium raphe and B9 projections are bilaterally symmetric, with equal cell numbers projecting to frontal, parietal, and occipital cortex. The rostro-caudal distributions of cells that project to disparate cortical areas differ in B9 but not in MR. The percentage of cortically projecting cells that are serotonergic is 80% for the DRN, 60% in the MR and 33% in the B9 cell group. The dorsal raphe nucleus and the B9 cell group are organized heterogeneously, and overlapping sets of neurons project differentially upon particular areas of neocortex. In contrast, the median raphe nucleus projects uniformly upon the neocortex and does not exhibit topographic organization. The three rostral raphe nuclei (DR, MR and B9) are each organized according to different rules with regard to their efferent projections to cortex. The differential organization of the raphe nuclei suggests that groups of cells within these three raphe nuclei are likely to innervate different combinations of cortical targets and thus to have different functional effects.

Although bronchoalveolar lavage (BAL) has been used as a research tool for over a decade, the technique of lavage has varied markedly between laboratories. For example, lavage instillate volumes from 50 to 300 ml have been used, and yet the influence of the variable of total lavage volume on subsequent protein recovery is uncertain. We performed sequential BAL (50 ml/aliquot; total volume, 300 ml) of the right middle lobe of 14 normal volunteers and separately processed and analyzed recovered aliquots for the absolute and relative concentrations of several protein substances. These proteins include free secretory component and secretory IgA, which emanate from airway secretions, and IgG, which is thought to transude from more distal alveolar sites. Analysis of these data showed a marked decrease in the absolute concentration of all proteins measured in serial aliquots. Analysis of protein ratios in sequential aliquots, however, revealed no significant change from the first to the fifth recovered aliquot. Finally, we analyzed the influence of the size of the first recovered aliquot on absolute and relative concentrations of proteins. Here there seemed to be a trend indicating preferential recovery of airway proteins in smaller aliquots. This was significant for the ratio of free secretory component to albumin (p less than 0.05). We conclude that lung proteins are efficiently and homogeneously sampled with 100 ml of lavage instillate. Larger volumes will add more protein but not alter protein ratios. Lavage with smaller volumes may preferentially sample airway proteins.