Boyd K. Hartman

Purkinje Cell Expression of a Mutant Allele of SCA1 in Transgenic Mice Leads to Disparate Effects on Motor Behaviors, Followed by a Progressive Cerebellar Dysfunction and Histological Alterations

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurological disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract. Work presented here describes the behavioral and neuropathological course seen in mutant SCA1 transgenic mice. Behavioral tests indicate that at 5 weeks of age mutant mice have an impaired performance on the rotating rod in the absence of deficits in balance and coordination. In contrast, these mutantSCA1 mice have an increased initial exploratory behavior. Thus, expression of the mutant SCA1 allele within cerebellar Purkinje cells has divergent effects on the motor behavior of juvenile animals: a compromise of rotating rod performance and a simultaneous enhancement of initial exploratory activity. With age, these animals develop incoordination with concomitant progressive Purkinje neuron dendritic and somatic atrophy but relatively little cell loss. Therefore, the eventual development of ataxia caused by the expression of a mutant SCA1 allele is not the result of cell death per se, but the result of cellular dysfunction and morphological alterations that occur before neuronal demise.

Effect of decreasing afferent vagal activity with ondansetron on symptoms of bulimia nervosa: a randomised, double-blind trial

BACKGROUND Several lines of evidence have led us to postulate that afferent vagal hyperactivity could be an important factor in the pathophysiology of the eating disorder bulimia nervosa. Ondansetron is a peripherally active antagonist of the serotonin receptor 5-HT3, and is marketed for prevention of vagally-mediated emesis caused by cancer chemotherapeutic agents. We investigated the effects of ondansetron on bulimic behaviours in patients with severe and chronic bulimia nervosa in a randomised, double-blind, placebo-controlled study. METHODS We enrolled patients with severe bulimia nervosa (at least seven coupled binge/vomit episodes per week). The patients were otherwise healthy, their weight was normal, and they were not receiving medical or psychiatric treatment. During the first week of the study, patients recorded all eating-behaviour events to establish a baseline. In the second week, all patients received placebo, but were told that they were receiving either placebo or active drug. At the end of this single-blind phase, patients were randomly assigned placebo or ondansetron (24 mg daily) for a further 4 weeks. The primary outcome measure was the number of binge/vomit episodes per week. Data were analysed by intention to treat. FINDINGS 29 patients met the inclusion criteria, of whom 28 completed the baseline study, and 26 completed the single-blind placebo week. 12 patients were assigned placebo, and 14 ondansetron; one patient in the ondansetron group dropped out owing to accidental injury. During the 4th week of double-blind treatment, mean binge/vomit frequencies were 13.2 per week (SD 11.6) in the placebo group, versus 6.5 per week (3.9) in the ondansetron group (estimated difference 6.8 [95% CI 4.0-9.5]; p<0.0001). The ondansetron group also showed significant improvement, compared with the placebo group, in two secondary indicators of disease severity. The amount of time spent engaging in bulimic behaviours was decreased on average by 7.6 h per week in the ondansetron group, compared with 2.3 h in the placebo group (estimated difference 5.1 [0.6-9.7]). Similarly, the number of normal meals and snacks increased on average by 4.3 normal eating episodes without vomiting per week in the ondansetron group, compared with 0.2 in the placebo group (estimated difference 4.1 [1.0-7.2]). INTERPRETATION The decrease in binge-eating and vomiting under ondansetron treatment was not achieved by compensatory changes in eating behaviour such as by a smaller number of binges of longer duration, or by not eating, or by binge-eating without vomiting. Instead, our findings indicate a normalisation of the physiological mechanism(s) controlling meal termination and satiation. Since meal termination and satiety are mainly vagally mediated functions, since binge-eating and vomiting produce intense stimulation of vagal afferent fibres, and since ondansetron and other 5-HT3 antagonists decrease afferent vagal activity, the symptom improvement may result from a pharmacological correction of abnormal vagal neurotransmission.

Localization of NADPH diaphorase in neurons of the rostral ventral medulla: possible role of nitric oxide in central autonomic regulation and oxygen chemoreception

We studied whether neurons containing nitric oxide synthase (NOS) are localized to the rostral ventrolateral medulla (RVM) and, if so, whether they are distinct from the adrenergic neurons of the C1 group. NOS-containing neurons and/or C1 neurons were visualized using NADPH diaphorase histochemistry and phenylethanolamine N-methyltransferase (PNMT) immunohistochemistry, respectively. A column of NADPH diaphorase-positive neurons, extending 2 mm in the rostrocaudal plane, was observed lateral to the inferior olive and medial to the C1 neurons. Double labelling studies showed that NADPH diaphorase-positive neurons were not immunoreactive for PNMT, indicating that the two enzymes were localized in the different cells. Furthermore, only a small fraction of NADPH diaphorase neurons were retrogradely labelled after injections of fluorogold into the thoracic cord. We conclude that the RVM contains a well-defined group of neurons endowed with NOS that are distinct from the adrenergic neurons of the C1 group and have only limited monosynaptic projections to the spinal cord. Since the RVM is involved in the control of arterial pressure and in oxygen-conserving reflexes, the findings raise the possibility that nitric oxide participates in central autonomic regulation and oxygen chemoreception.

Functional neuroimaging of gastric distention.

This study aimed to measure brain activation during gastric distention as a way to investigate short-term satiety. We estimated regional cerebral blood flow with positron emission tomography (15O-water) during gastric balloon inflation and deflation in 18 healthy young women. The contrast between inflated minus deflated conditions showed activation in the following four key regions that were identified a priori: dorsal brain stem; left inferior frontal gyrus; bilateral insula; and right subgenual, anterior cingulate cortex. Extant neuroimaging literature provides context for these areas as follows: the brain stem represents vagal projection zones for visceral afferent processing; the inferior frontal gyrus serves as a convergence zone for processing food-related stimuli; and both the insula and subgenual anterior cingulate cortex respond to emotional stimulation. The identification of neural correlates of gastric distention is a key step in the discovery of new treatments for obesity. New therapies could intervene by modifying the perception of gastric distention, an important contributor to meal termination and short-term satiety. This first study of brain activation during nonpainful, proximal gastric distention provides the groundwork for future research to discover novel treatments for obesity.

Characterization of the extent of pontomesencephalic cholinergic neurons' projections to the thalamus: comparison with projections to midbrain dopaminergic groups.

We sought to determine whether pontomesencephalic cholinergic neurons which we have been shown previously to project to the substantia nigra and ventral tegmental area also contribute to the thalamic activation projection from the pedunculopontine and laterodorsal tegmental nuclei. Retrograde tracing, immunohistochemical localization of choline acetyltransferase and statistical methods were used to determine the full extent of the cholinergic projection from the pedunculopontine and laterodorsal tegmental nuclei to the thalamus. Progressively larger Fluoro-Gold injections in to the thalamus proportionally labeled increasing numbers of pontomesencephalic cholinergic cells both ipsi- and contralaterally in the pedunculopontine and laterodorsal tegmental nuclei. Multiple large thalamic injections left only a small fraction of the ipsilateral pontomesencephalic cholinergic group unlabeled. This small remainder did not correspond to the populations which project to the substantia nigra and ventral tegmental area, thereby indicating that substantia nigra- and ventral tegmental area-projecting cholinergic neurons must also project to the thalamus. We examined whether there existed any set of cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei which did not innervate a thalamic target. The distribution of descending projections of the pedunculopontine and laterodorsal tegmental nuclei demonstrated that the unlabeled remainder cannot correspond to a purely descending group. We also show that substance P-positive cholinergic cells in the laterodorsal tegmental nucleus project to the thalamus. Further studies demonstrated that the small population of cholinergic cells left unlabeled from the thalamus were the smallest sized cholinergic cells, and included two groups of small, light-staining cholinergic cells located in the parabrachial area and central gray, adjacent to the main pedunculopontine and laterodorsal tegmental nuclei cholinergic groups. These small cells, in contrast to thalamic-projecting cholinergic cells, did not stain positively for reduced nicotinamide adenine dinucleotide phosphate-diaphorase. Taken together, these results indicated that all of the reduced nicotinamide adenine dinucleotide phosphate diaphorase-positive/choline acetyltransferase-positive neurons of the pedunculopontine/laterodorsal tegmental nuclei ascend to innervate some portion of the thalamus, in addition to the other targets they innervate. These findings indicate that the diverse physiological and behavioral effects attributed to the activity of pontomesencephalic cholinergic neurons should not be dissociated from their activating effects in the thalamus.

Distinct choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) bipolar neurons project to local blood vessels in the rat cerebral cortex

Innervation of rat intracortical cerebral blood vessels by acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) remains largely unexplored and it is not known if the cells of origin are intra- or extracortical nor if perivascular fibers colocalize ACh and VIP. Cortical cholinergic innervation arises primarily from the basal forebrain and to a small extent from intrinsic bipolar ACh neurons thought to be the sole source of cortical VIP. In order to evaluate if intracortical perivascular ACh terminals could be distinguished from those of the basal forebrain by their colocalization with VIP, we performed a double immunofluorescence study and determined the percentage of colocalization of choline acetyltransferase (ChAT) and VIP in cortical neurons, as well as in terminal fields associated with intracortical blood vessels. From a total of 2103 cells examined in all cortical areas, VIP neurons accounted for the largest population (58.3%) followed by ChAT-positive cells (28.2%) with only 13.5% of cells being double-labelled for VIP and ChAT. Of the cortical ChAT-immunostained cells (n = 878), 32.3% colocalized VIP whereas only 18.8% of VIP neurons (n = 1509) also contained ChAT. In various cortical areas, ChAT cell bodies were seen to be contacted by VIP terminals which surrounded closely their cell soma and proximal dendrites. Perivascular fibers studied by double immunofluorescence and confocal microscopy were of three categories including cholinergic, VIPergic with a smaller population of fibers which costained for both ChAT and VIP. These results show that cortical VIP neurons are much more numerous than those containing ChAT, and that a majority of VIP neurons do not colocalize with ChAT. This observation indicates that ACh and VIP are primarily located in distinct neuronal populations and that VIP cannot be used as a marker of intracortical ACh neurons and terminals. Our results further suggest that intracortical blood vessels are primarily under the influence of distinct ChAT and VIP perivascular fibers. Also, the presence of a subset of VIP and ChAT/VIP fibers in association with intracortical blood vessels strongly suggests a role for cortical bipolar neurons in local cerebrovascular regulation. The origin of the perivascular ChAT fibers which do not colocalize VIP, however, remains unknown.

PACAP in the adrenal gland - relationship with choline acetyltransferase, enkephalin and chromaffin cells and effects of immunological sympathectomy

USING indirect immunohistochemistry and immuno- logical sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP- positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.

Transplantation of Fetal Neocortex Ameliorates Sensorimotor and Locomotor Deficits Following Neonatal Ischemic–Hypoxic Brain Injury in Rats

Ischemic brain injury in neonates can result in the degeneration of cortical and subcortical areas of brain and is associated with neurologic deficits. One approach to restoring function in conditions of ischemic brain injury is the use of neural transplants to repair damaged connections. This approach has been shown to reestablish neural circuitry and to ameliorate associated motor deficits in models of neonatal sensorimotor cortex damage. In this study, we utilized the Rice et al. rodent model of neonatal ischemic-hypoxic (IH) brain injury to assess whether transplantation of fetal neocortical tissue can promote functional recovery in tests of sensorimotor and locomotor ability throughout development and as adults. We show that animals that received neocortical grafts 3 days following the IH injury performed significantly better as adults on two measures of motor ability, the Rota-Rod treadmill and apomorphine-induced rotations, than did control animals that received sham transplants after the IH injury. Transplants were identifiable in 72% of the animals 10-12 weeks after implantation. Histochemical studies revealed that while the transplanted tissue did not establish normal cortical cytoarchitecture, cells and fibers within the grafts stained for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), choline acetyl transferase (ChAT), cholecystokinin (CCK), and glial fibrillary acidic protein (GFAP). These results suggest that transplantation of fetal neocortical tissue following IH injury in the neonatal period is associated with amelioration of motor deficits and that the grafted tissue demonstrated a neurochemical phenotype that resembled normal neocortex. This approach warrants continued investigation in light of potential therapeutic uses.

Effect of ondansetron, a 5-HT3 receptor antagonist, on the dynamic association between bulimic behaviors and pain thresholds.

Abstract Thresholds for detection of both pressure and thermal pain are elevated in patients with bulimia nervosa. The present study was aimed at determining (1) if pressure pain detection thresholds (PDT) varied dynamically with the primary disease symptoms of binge eating and vomiting and (2) if the elevation in PDT was effected by treatment with ondansetron (ONDAN), a 5‐HT3 receptor antagonist. PDT was defined as the mean of the minimal amount of pressure (measured in g) perceived as painful when exerted by a 1 mm2 blunted point onto the center of the ventral surface of the ungual phalanx of digits 2‐5 of the non‐dominant hand. Fourteen female patients with severe bulimia nervosa (currently >seven binge/vomit episodes per week;>2 years illness duration) served as participants. PDT were evaluated at weekly intervals during the course of ongoing treatment studies (double‐blind and ‘open’ label) investigating the therapeutic effects of ONDAN. Data were analyzed by random regression analyses, allowing for the repeated‐measures and non‐orthogonal design. Data collected from 14 patients under the no‐drug condition indicated that PDT increased over the interval between binge/vomit episodes, with significant elevations occurring at times when patients had naturally exceeded their average inter‐binge interval. Eleven of these 14 patients underwent 4 weeks of ONDAN treatment. Under this drug condition, the time since the last binge/vomit episode was no longer a significant predictor of PDT. These patients also experienced a significant reduction in the frequency of bulimic behaviors, a finding reported in detail elsewhere. The above finding from untreated patients support the involvement of a common underlying mechanism driving both the increase in pain detection thresholds and the occurrence of the next bulimic episode. This possibility is further supported by the findings that ONDAN treatment is associated with a significant moderation of both variables. The effect of ONDAN may be mediated by blockade of afferent vagal neurotransmission, although other mechanisms must be considered.

CHOLINE ACETYLTRANSFERASE IN THE RAT COCHLEAR NUCLEI: IMMUNOLOCALIZATION WITH A MONOCLONAL ANTIBODY

The cochlear nuclear complex (CoN) contains several populations of neurons that are highly diverse not only with respect to morphological features but also physiological responses to sound. With few exceptions, morphologically and functionally distinct cell types are differentially distributed in the anteroventral (AVCoN), posteroventral (PVCoN) and dorsal (DCoN) divisions of the CoN. With the advent of intracellular injection of tracers coupled with microelectrode recording, physiological response patterns can be more precisely correlated with individual cell classes within these subdivisions, increasing the need for extensive information on neurocytological features. In order to more fully describe the transformations of sound stimuli performed in the auditory centers, it is helpful to correlate the ultrastructural and chemical phenotypes of the neurons, especially those related to synapses, and the molecules involved in neurotransmission. Immunocytochemical methods have proven invaluable in this respect, because they afford the localization of special molecules not only at the regional level, as obtained with biochemical micromethods, but also at the cellular and subcellular levels.