Cholinergic neurons of the adult rat striatum are immunoreactive for glutamatergic N-methyl-d-aspartate 2D but not N-methyl-d-aspartate 2C receptor subunits.

Cholinergic neurons of the striatum play a crucial role in controlling output from this region. Their firing is under the control of a relatively limited glutamatergic input, deriving principally from the thalamus. Glutamate transmission is effected via three major subtypes of receptors, including those with affinity for N-methyl-d-aspartate (NMDA) and the properties of individual receptors reflect their precise subunit composition. We examined the distribution of NMDA2C and NMDA2D subunits in the rat striatum using immunocytochemistry and show that a population of large neurons is strongly immunoreactive for NMDA2D subunits. From their morphology and ultrastructure, these neurons were presumed to be cholinergic and this was confirmed with double immunofluorescence. We also show that NMDA2C is present in a small number of septal and olfactory cortical neurons but absent from the striatum. Receptors that include NMDA2D subunits are relatively insensitive to magnesium ion block making neurons more likely to fire at more negative membrane potentials. Their localization to cholinergic neurons may enable very precise regulation of firing of these neurons by relatively small glutamatergic inputs.

Nucleus accumbens nitric oxide immunoreactive interneurons receive nitric oxide and ventral subicular afferents in rats.

The nitric oxide generating neurons of the nucleus accumbens exert a powerful influence over striatal function, in addition, these nitrergic inputs are in a position to regulate the dopaminergic and glutamatergic inputs on striatal projection neurons. It was the aim of this study to establish the source of the glutamatergic drive to nitric oxide synthase interneurons of the nucleus accumbens. The nucleus accumbens nitric oxide-generating neurons receive asymmetrical, excitatory, presumably glutamatergic inputs. Possible sources of these inputs could be the limbic and cortical regions known to project to this area. To identify sources of the excitatory inputs to the nitric oxide synthase-containing interneurons of the nucleus accumbens in the rat we first examined the ultrastructural morphology of asymmetrical synaptic specializations contacting nitric oxide synthase-immunohistochemically labeled interneurons in the nucleus accumbens. Neurons were selected from different regions of the nucleus accumbens, drawn using camera lucida, processed for electron microscopic analysis, and the boutons contacting nitric oxide synthase-labeled dendrites were photographed and correlated to the drawings. Using vesicle size as the criterion the source was predicted to be either the prefrontal cortex or the ventral subiculum of the hippocampus. To examine this prediction, a further study used anterograde tracing from both the prefrontal cortex and the ventral subiculum, and nitric oxide synthase immunohistochemistry with correlated light and electron microscopy. Based on appositions by anterogradely labeled fibers, selected nitric oxide synthase-labeled neurons within the nucleus accumbens, were examined with electron microscopic analysis. With this technique we confirmed the prediction that subicular afferent boutons make synaptic contact with nitric oxide synthase interneurons, and demonstrated anatomically that nitric oxide synthase boutons make synaptic contact with the dendritic arbors of nitric oxide synthase interneurons. We suggest that the subicular input may excite the nitric oxide synthase neurons synaptically, while the nitric oxide synthase-nitric oxide synthase interactions underlie a nitric oxide signaling network which propagates hippocampal information, and expands the hippocampus's influence on 'gating' information flow across the nucleus accumbens.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine.

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphé nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

Sarcocystis spp. Infection in two Red Panda Cubs (Ailurus fulgens).

Two neonatal male red panda (Ailurus fulgens) littermates were submitted for necropsy examination. One animal was found dead with no prior signs of illness; the other had a brief history of laboured breathing. Post-mortem examination revealed disseminated protozoal infection. To further characterize the causative agent, transmission electron microscopy (TEM), immunohistochemistry (IHC), polymerase chain reaction (PCR) and amplification and nucleic acid sequencing were performed. IHC was negative for Toxoplasma gondii and Neospora caninum, but was positive for a Sarcocystis spp. TEM of cardiac muscle and lung revealed numerous intracellular apicomplexan protozoa within parasitophorous vacuoles. PCR and nucleic acid sequencing of partial 18S rRNA and the internal transcribed spacer (ITS)-1 region confirmed a Sarcocystis spp. that shared 99% sequence homology to Sarcocystis neurona and Sarcocystis dasypi. This represents the first report of sarcocystosis in red pandas. The histopathological, immunohistochemical, molecular and ultrastructural findings are supportive of vertical transmission resulting in fatal disseminated disease.

Effect of guar gum on hunger and satiety after meals of differing fat content: relationship with gastric emptying.

To determine whether the satiating effects of fiber are due to delaying gastric emptying or slowing absorption of meals, 3% guar gum was added to high- and low-fat soups and gastric emptying rate, hunger, and satiety were measured in eight male volunteers. Guar gum delayed the emptying of the low-fat soup but the small delays in the return of hunger and decline of fullness were significantly correlated with the gastric emptying, suggesting mediation by gastric mechanoreceptors. The high-fat soup also emptied more slowly but this had no effect on the return of hunger or the decline in fullness. The delays in the return of hunger and decline of fullness were far greater when guar gum was added to the fatty soup; these delays were not correlated with the small additional delay in gastric emptying. This is more compatible with slowed absorption and prolonged contact of nutrients with intestinal chemoreceptors.

Individual nucleus accumbens-projection neurons receive both basolateral amygdala and ventral subicular afferents in rats.

The nucleus accumbens is regarded as the limbic-motor interface, in view of its limbic afferent and somatomotor and autonomic efferent connections. Within the accumbens, there appear to be specific areas in which limbic afferent fibres, derived from the hippocampus and the amygdala, overlap. These afferent inputs have been suggested to converge monosynaptically on cells within the accumbens and are hypothesized to play a role in paradigms such as conditioned place preference. Convergence between inputs from basolateral amygdala and hippocampus can be demonstrated with electrophysiological recording methods, but these do not conclusively preclude polysynaptic mechanisms. We examined the synaptic input to the projection neurons of the accumbens, the medium-sized densely spiny neurons. We labelled the projection neurons with a small injection of biotinylated dextran amine into the accumbens, and the afferents from the basolateral amygdala and ventral subiculum of the hippocampus with injections of biotinylated dextran amine and Phaseolus vulgaris-leucoagglutinin respectively, and revealed the anterogradely labelled fibres with different chromogens. The labelled accumbens-projection neurons were studied with correlated light and electron microscopy for identified monosynaptic inputs. With this technique we have demonstrated anatomically that monosynaptic convergence between the ventral subicular region of the hippocampus and the basolateral region of the amygdala occurs at the level of the proximal as well as distal dendrites. Finally, we suggest that these anatomical arrangements may represent the framework for the integrative role that has been assigned to the accumbens.

Conditionally immortal neuroepithelial stem cell grafts reverse age-associated memory impairments in rats.

In order to investigate the effects of stem cell grafts on water maze deficits in aged (22-month-old) rats, three groups of aged rats, assigned by pre-training latency scores to unimpaired, impaired control and impaired grafted groups, were compared with young (five-month-old) controls, six to eight weeks after implantation of cells from the conditionally immortal Maudsley hippocampal stem cell line, clone 36 (MHP36 stem cell line), in the cortex, striatum and hippocampus. Grafted rats were substantially superior to their matched impaired aged controls, and learned to find the platform as rapidly as unimpaired aged rats, although young controls were more efficient than all aged groups in several measures of spatial search during training. On the probe trial, however, aged rats with grafts showed significantly better recall of the precise position of the platform than any other group, including young controls, possibly indicating some perseveration. A further comparison found that groups of unimpaired and moderately impaired aged rats showed far less improvement from water maze pre-training to acquisition phases than young controls, indicative of progressive deficits over time. Histological investigation showed that beta-galactosidase-positive MHP36 cells migrated widely from the implantation sites to infiltrate the striatal matrix, all hippocampal fields and areas of the cortex. Grafted cells showed both astrocytic and neuronal morphologies, with cells of pyramidal and granular appearance in appropriate hippocampal strata.Taken together, these results indicate that neuroepithelial stem cell grafts extensively colonize the aged rat brain and substantially reverse progressive cognitive decline associated with ageing.

Functional repair with neural stem cells.

Approval to commence phase I/II clinical trials with neural stem cells requires proof of concept in well-accepted animal models of human neurological disease or injury. We initially showed that the conditionally immortal MHP36 line of hippocampal origin (derived from the H-2Kb-tsA58 transgenic mouse) was effective in repopulating CA1 neurons in models of global ischaemia and repairing cognitive function, and have now shown that this line is multifunctional. MHP36 cells are effective in restoring spatial memory deficits in rats after excitotoxic lesions of the cholinergic projections to cortex and hippocampus and in rats showing cognitive impairments due to normal ageing. Moreover, grafts of MHP36 cells are effective in reversing sensory and motor deficits and reducing lesion volume as a consequence of occlusion of the middle cerebral artery, the major cause of stroke. In contrast, MHP36 cell grafts were unable to repair motor asymmetries in rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal dopamine system, the prototype rodent model of Parkinson's disease. These data show that conditionally immortal neuroepithelial stem cells are multifunctional, being able to repair diverse types of brain damage. However, there are limitations to this multifunctionality, suggesting that lines from different regions of the developing brain will be required to treat different brain diseases. ReNeuron is currently developing human neuroepithelial stem cell lines from different brain regions and with similar reparative properties to our murine lines.

Functional reconstruction of the hippocampus: fetal versus conditionally immortal neuroepithelial stem cell grafts.

Late fetal CA1 hippocampal grafts and stem cell grafts from the conditionally immortal MHP36 clonal line derived from the H-2Kb-tsA58 transgenic mouse neuroepithelium both improved spatial deficits in rats with ischaemic CA1 damage induced by four-vessel occlusion (4VO). However, the distribution of fetal and MHP36 grafts differed. Fetal cells lodged in clumps around the implant sites and along the corpus callosum, whilst MHP36 grafts infiltrated the area of CA1 ischaemic damage, achieving apparent architectural reconstruction of the hippocampus. The migration of MHP36 cells is damage-dependent. Few cells were found in intact brain; after 15 min of 4VO cells repopulated only the discrete area of CA1 cell loss, whereas with more extensive damage after 30 min occlusion cells migrated to all hippocampal fields and to cortex. A higher proportion of grafted MHP36 cells differentiated into neurons in the host CA1 field than grafts of striatal or cortical expanded cell populations. Cortical population grafts were as effective as MHP36 grafts in improving water maze learning, whereas striatal or ventral mesencephalic cells were ineffective, indicating a degree of stem cell specificity. The efficacy of MHP36 cells extends to primates. In marmosets with profound impairments in conditional discrimination tasks after lesions of the CA1 field, MHP36 cells improved performance as effectively as fetal grafts and migrated evenly through the CA1 field, in contrast to clustered fetal cells. These findings suggest that MHP36 stem cell grafts are as effective as fetal grafts in functional repair of hippocampal damage, and that their preference for areas of cell loss and adoption of appropriate morphologies is consistent with a point-to-point repair mechanism.

Hippocampal neurotrophin and trk receptor mRNA levels are altered by local administration of nicotine, carbachol and pilocarpine.

Cholinergic receptor agonists nicotine (nicotinic), carbachol (nicotinic/muscarinic) and pilocarpine (muscarinic) were administered into the hippocampus and mRNA levels of neurotrophins and their receptors determined using in situ hybridisation. Drug doses were carefully chosen to avoid the potentially confounding effects of seizure and cell death. Nicotine caused a long-lasting increase in nerve growth factor (NGF) mRNA in all subfields of the hippocampus. The increase was evident from 24 h up to 72 h after drug administration. This increase was dependent on excitatory amino acid neurotransmission as it was blocked by administration of an AMPA or NMDA receptor antagonist. In contrast, carbachol and pilocarpine produced a transient increase in NGF mRNA levels present 4-8 h after drug administration. Pilocarpine caused a transient increase in hippocampal brain-derived neurotrophic factor (BDNF) levels, with carbachol and nicotine showing the same trend. Nicotine and carbachol caused transient decreases in NT-3 mRNA levels in dentate gyrus and CA2 with pilocarpine showing a similar trend. Increases in mRNA encoding full-length trkB were seen 8 h after nicotine, with nicotine also causing elevations in a mRNA encoding a truncated isoform (trkB.T2). TrkC mRNA was not altered by any of the conditions used. The study suggests that muscarinic and nicotinic receptor activation in the hippocampus causes transient changes in all of the neurotrophins, but that NGF levels are selectively up-regulated by nicotinic receptor stimulation. The reciprocal interaction between NGF and ascending cholinergic systems may be a component of the cognitive enhancing effects of nicotine.

Oral, gastric and intestinal influences on human feeding.

Direct infusion of specific nutrients or foods into different areas of the gastrointestinal tract, and techniques to distend the stomach, are useful tools enabling eating behaviour to be studied without the influence of orosensory factors. Using these techniques, the role of nutrients on gastrointestinal mechanisms of satiation as well as the interactions between the various systems in the control of feeding can be examined. Recent research in humans investigating the relative contribution of signals arising from different areas of the gastrointestinal tract has demonstrated that optimal control of appetite occurs only when orosensory signals are coupled with signals arising from the stomach and intestine. Interactions between gastric and intestinal signals do however combine to produce a more potent suppression of appetite and food intake than when either of these sites is stimulated alone. Gastric distension probably exerts a predominate influence on appetite suppression compared with intestinal stimulation, whereas nutrient stimulation of the intestine may function to modulate the sensations arising from gastric distension to elicit a meal-like sensation of satiety. In addition, these studies have highlighted that previous hypotheses concerning differential fat and carbohydrate satiation may require an orosensory component and probably do not reflect an inherent difference in the physiological effects of these macronutrients at the level of the GI tract. The interaction of orosensory influences, such as palatability, with negative feedback signals arising from the GI tract can be further studied using a combination of these techniques with measurement of microstructure of feeding in humans.

Food intake responses to upper gastrointestinal lipid infusions in humans.

Previous studies in humans and animals have shown that the presence of lipid in the small intestine can reduce food intake. Studies that have combined intraduodenal lipid infusions with gastric distension produced a greater reduction in food intake than when these two stimuli were separated. In this study, subjects received duodenal lipid (Intralipid 20%) infusions for varying periods before and during the consumption of a liquid test meal. The aim of this procedure was to maximise the interaction between intestinal nutrient stimulation and gastric distension. A dose-dependent decrease in food intake was observed that corresponded to the duration of infusion; 90- (180 kcal) and 45-min (90 kcal) infusions but not 15-min (30 kcal) infusions significantly reduced intake compared to saline. These results show that concomitant intestinal nutrient stimulation and gastric distension is an effective test for the measurement of suppression of food intake by intestinal nutrients.

Maillard reaction induced lactose attachment to bovine beta-lactoglobulin: electrospray ionization and matrix-assisted laser desorption/ionization examination.

Nonenzymatic attachment of lactose to beta-lactoglobulin (beta-Lg) was investigated under different conditions. Solubilized conditions, dry environment, and a combination of dry and solubilized environments, were examined for their effects on lactosylation. Temperatures ranging from 50 to 65 degrees C and time intervals between 1 h and 4 days were used. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were implemented to examine the reaction products. Maximum attachment efficiency occurred at 65 degrees C held for 3 h in dry-way conditions. Incubations held for long periods of time under dry-way conditions suggest possible denaturation. Both ESI and MALDI data suggest beta-Lg removal in the solubilized samples held for long periods of time. A combination of solubilized and dry environments led to very similar mass spectrogram results over time.

The effect of a 5-HT3-antagonist on the ileal brake mechanism in the rat.

Studies have been carried out on 7 male adult rats to investigate how the action of the selective 5-HT3 receptor antagonist, granisetron, influences gastrointestinal transit under control conditions and when it is delayed by ileal infusion of lipid. Stomach to caecum transit time (SCTT) was measured using environmental hydrogen analysis. Subcutaneous administration of granisetron (BRL 43694, 40 micrograms kg-1) significantly delayed the passage of the head of the baked bean meal through the stomach and the small intestine under control conditions (P less than 0.05). The same compound, however, significantly reversed the delay in SCTT induced by ileal infusion of lipid (P less than 0.001). These apparently paradoxical results may be rationalized by postulating inhibition of receptors on afferent nerves initiating reflexes that both accelerate and delay transit.

Women gain weight and fat mass despite lipectomy at abdominoplasty and breast reduction.

OBJECTIVES: In animal models, fat removal results in compensatory weight gain. No study has reported measurement of weight following lipectomy in humans. We have examined changes in weight in patients who underwent lipectomy. METHODS: In a retrospective analysis, 16 patients who had abdominoplasty and 17 patients who underwent bilateral breast reduction were compared with 16 patients who had carpal tunnel syndrome release. Following this, a prospective study was carried out on 7 subjects awaiting abdominoplasty and 12 subjects awaiting bilateral breast reduction surgery. RESULTS: In the retrospective study, all three patient groups gained weight following surgery. The abdominoplasty group was heavier before surgery and showed greatest weight gain but there was no statistically significant difference in weight gain between the groups. In the prospective study, the abdominoplasty group had a mean fat removal of 1.77 kg and breast reduction group had a mean of 3.22 kg. Eighteen months following surgery the abdominoplasty group showed a significant mean increase in body weight (mean increase: 4.82 kg) and body mass index (BMI) (mean increase: 1.66 kg/m(2)). In the bilateral breast reduction group, there was a non-significant mean gain in weight (mean increase: 0.67 kg) and BMI (mean increase: 0.21 kg/m(2)). CONCLUSIONS: Patients undergoing lipectomy during abdominoplasty and bilateral breast reduction will gain weight in the long term. This weight gain probably reflects the expected gain in weight without surgery as a similar finding is observed in patients who have undergone surgery without lipectomy. These results highlight the limitation of lipectomy as a weight control measure.

The effects of specific nutrients on the regulation of feeding behaviour in human subjects.

Regulation of short-term energy intake involves the balance of positive drives to eat arising from the sight, smell and palatability of food with negative feedback signals from learned associations, gastrointestinal and metabolic signals. The stomach and small intestine are major sites in the feedback inhibition of food intake and subsequent period of appetite suppression. The present paper reviews the evidence that not only does the nature of the regulatory signal suppressing food intake depend on the type and energy content of nutrient consumed, but also the specific chemical composition of the nutrients and the site at which they are delivered. It is evident that feedback inhibition of feeding can be modulated by the particular chemical structure of nutrients (e.g. specific sugar or triacylglycerol structures). These differences in response are likely to be a consequence of differences in physical properties of particular nutrients depending on their chemical structure, and may also result from different receptor affinities for specific dietary structures. Moreover, the site of administration of nutrients can also profoundly affect the size and nature of the subsequent feeding response, suggesting that feed-forward interactions occur between the taste of foods and gastrointestinal stimulation.

Comparison of oral and gastric administration of sucrose and maltose on gastric emptying rate and appetite.

OBJECTIVES: To investigate mechanisms by which specific sugars affect feeding behaviour. DESIGN: In an initial study, gastric emptying rate and appetite were measured following ingestion of lemon flavoured solutions of sucrose, maltose (2160 kJ, 575 ml) and water control (67 kJ, 575 ml) given in randomised order on separate days to six male volunteers. In a second study, the effects of intragastric infusions of sucrose and maltose on appetite and gastric emptying were compared in six male volunteers. RESULTS: When given orally, both the sucrose and maltose solutions slowed gastric emptying compared with water, however sucrose emptied at a faster rate than maltose. The sucrose preload increased fullness and decreased prospective consumption during the following 3 h compared with maltose and water. When administered intragastrically, the gastric emptying rate of sucrose was again faster than that of maltose but there was no difference in ratings of hunger, fullness or prospective consumption for 3 h following the infusions. CONCLUSIONS: These results show that gastric emptying of sucrose is faster than that of maltose and suggest that gastric emptying rate and hence period of gastric distension is not the predominant factor regulating appetite by these sugars. The differences observed between oral and gastric delivery suggest that oro-sensory and cognitive factors, possibly stimulated by the sweetness of sucrose, were involved in the induction of satiety.

Adaptation to high-fat diet accelerates emptying of fat but not carbohydrate test meals in humans.

Previous work has shown that the gastric emptying rate in animals and humans can adapt due to previous dietary intake. The present study investigated whether adaptation in gastric emptying rate due to consumption of a high-fat diet (HFD) is nutrient specific in humans. Gastric emptying of high-fat and high-carbohydrate test meals was measured (using gamma scintigraphy) before and after consumption of an HFD for 14 days in eight free-living male volunteers. Visual analog ratings of appetite were recorded throughout each test. There was no effect of HFD on any parameters of gastric emptying rate (lag phase, half-emptying time, and linear emptying rate) measured for carbohydrate test meals. HFD led to an acceleration of the linear emptying rate of the high-fat test meal (0.36 vs. 0.47%/min; P < 0.05). All meals reduced appetite ratings, but there were no differences between tests. These results support our previous findings of accelerated gastric emptying of high-fat test meals following an HFD and show that these changes appear to be nutrient specific, confirming recent studies in rats.