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We have carried out the largest randomised trial to date of newly diagnosed myeloma patients, in which lenalidomide has been used as an induction and maintenance treatment option and here report its impact on second primary malignancy (SPM) incidence and pathology. After review, 104 SPMs were confirmed in 96 of 2732 trial patients. The cumulative incidence of SPM was 0.7% (95% confidence interval (CI) 0.4–1.0%), 2.3% (95% CI 1.6–2.7%) and 3.8% (95% CI 2.9–4.6%) at 1, 2 and 3 years, respectively. Patients receiving maintenance lenalidomide had a significantly higher SPM incidence overall (P=0.011). Age is a risk factor with the highest SPM incidence observed in transplant non-eligible patients aged >74 years receiving lenalidomide maintenance. The 3-year cumulative incidence in this group was 17.3% (95% CI 8.2–26.4%), compared with 6.5% (95% CI 0.2–12.9%) in observation only patients (P=0.049). There was a low overall incidence of haematological SPM (0.5%). The higher SPM incidence in patients receiving lenalidomide maintenance therapy, especially in advanced age, warrants ongoing monitoring although the benefit on survival is likely to outweigh risk.

Repeated injection of urethane (ethyl carbamate) is carcinogenic in susceptible strains of mice. Most recent cancer studies involving urethane-induced tumor formation use p53(+/-) mice, which lack one copy of the p53 tumor suppressor gene. In contrast, the same protocol elicits at most a single tumor in wildtype C57BL/6 mice. The effect of repeatedly injecting urethane as a component of a ketamine-xylazine anesthetic mixture in the highly prevalent mouse strain C57BL/6 is unknown. Male C57BL/6J mice (n = 30; age, 3 mo) were anesthetized once monthly for 4 mo by using 560 mg/kg urethane, 28 mg/kg ketamine, and 5.6 mg/kg xylazine. The physical health of the mice was evaluated according to 2 published scoring systems. The average body condition score (scale, 1 to 5; normal, 3) was 3.3, 3.3, and 3.4 after the 2nd, 3rd, and 4th injections, respectively. The visual assessment score was 0 (that is, normal) at all time points examined. Within 1 wk after the 4th injection, the mice were euthanized, necropsied, and evaluated histopathologically. No histopathologic findings were noteworthy. We conclude that repeated monthly injection with urethane as a component of an anesthetic cocktail does not cause clinically detectable abnormalities or induce neoplasia in C57BL/6J mice. These findings are important because urethane combined with low-dose ketamine, unlike other anesthetic regimens, allows for accurate recording of neuronal activity in both the brain and retina. Longitudinal neuronal recordings minimize the number of mice needed and improve the analysis of disease progression and potential therapeutic interventions.

Objective Pathological gambling (PG) is a disabling disorder experienced by approximately 1% of adults and for which few empirically validated treatments exist. This study examined the efficacy and tolerability of the opioid antagonist naltrexone in adults with PG who have urges to gamble. Method An 18-week, double-blind, placebo-controlled trial was conducted to evaluate the safety and efficacy of 3 doses of oral naltrexone for PG. Seventy-seven individuals with DSM-IV-TR PG were randomly assigned to naltrexone (50 mg/day, 100 mg/day, or 150 mg/day) or placebo. Subjects were assessed with the Pathological Gambling Adaptation of the Yale-Brown Obsessive Compulsive Scale (PG-YBOCS), the urge and behavior subscales of the PG-YBOCS, the Gambling Symptom Assessment Scale (G-SAS), the Clinical Global Impressions-Severity of Illness scale (CGI-S), and measures of depression, anxiety, and psychosocial functioning. Data were collected from September 2002 to June 2005. Results Outcomes did not significantly differ between the various doses of naltrexone. Subjects assigned to naltrexone had significantly greater reductions in PG-YBOCS total scores (p = .0094), gambling urges (p = .0053), and gambling behavior (p = .0134) compared to subjects assigned to placebo. Subjects assigned to naltrexone also had greater improvement in overall gambling severity (reflected in the CGI-S scores) (p = .0080) and in psychosocial functioning (p = .0177) than subjects assigned to placebo. A complete analysis (N = 49) demonstrated significantly greater improvement on all variables for subjects assigned to naltrexone. A sex analysis demonstrated that men and women did not differ significantly in their response to naltrexone. Conclusion Subjects assigned to naltrexone demonstrated statistically significant reductions in gambling urges and behavior in PG. Low-dose naltrexone (50 mg/day) appeared as efficacious as higher doses (100 mg/day and 150 mg/day), and all doses were well tolerated.

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.

Background: Inclusion of obsessive–compulsive disorder (OCD) as an anxiety disorder in DSM-IV assumes that anxiety is the primary symptom of OCD; however, persuasive empirical evidence in support of this view has not been presented yet. In the present study we hypothesized that provoked anxiety symptoms respond better to intravenous diazepam than would provoked obsessions. We, therefore, reasoned that anxiety symptoms are secondary symptoms of OCD. Methods: To test the hypothesis we designed a double-blind, randomized, placebo-controlled crossover study. Patients underwent four experimental conditions in which the sequence of symptom provocation and IV injection of (placebo or diazepam) were alternated. Baseline and IV injection-induced symptom changes were assessed using visual analogs. Results: Obsessions and anxiety correlated strongly for all four experimental conditions in which the sequence of the symptom provocation and diazepam IV injections was alternated. IV diazepam injection before and after symptom provocation failed to preferentially modulate anxiety symptoms over obsessions. Unexpectedly, in the group in which IV diazepam injection preceded the symptom provocation, reduction of mean obsessions was even more pronounced. Conclusions: Strong correlations between anxiety and obsessions at baseline, during symptom provocation, and after IV diazepam infusion suggest that anxiety and obsessions are tightly coupled phenomena in OCD.

Using indirect immunohistochemistry and immunological 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.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

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.

Diet-induced changes in serum lipoproteins are a major risk factor for the development of atherosclerosis, the leading cause of mortality in Westernized countries. Atherosclerosis is now appreciated to be a systemic inflammatory disease where increased synthesis of inducible proteins by the liver, such as C-reactive protein (CRP) and others, may play roles in accelerating the disease process. To systematically investigate the genetic response of the liver to diet-induced atherosclerosis, we applied high-density microarray technology in a mouse model of atherosclerosis (LDLR−/−mouse). LDLR−/−mice and congenic (LDLR+/+) controls were placed on low-fat (LF) or high-fat (HF) Western-style diets. The Western diet produced sustained elevations in total cholesterol (2.5-fold for LDLR+/+, 5.0-fold LDLR−/−) relative to the respective LF groups. Tissues were harvested after 12 wk when significant atherosclerotic lesion development was first detectable by en face histomorphometry of oil red O-stained aortas. Diet, rather than genotype, was most highly associated with development of atherosclerotic lesions. Liver mRNA expression profiles of triplicate animals from each group were determined by high-density oligonucleotide microarrays; and genes with transcript levels influenced by genotype and diet were identified by two-way ANOVA. Approximately one-third of the 102 genes identified to be altered by diet [Pr(F) < 0.0005] were involved in lipid metabolism. In addition, we identified components of the alternative complement pathway, including C3, properdin, and factor D, for which mRNA levels were independently confirmed by quantitative real-time RT-PCR analysis, and C3 protein was demonstrated in aortic lesions by immunostaining. These findings suggest that induction of the alternative complement pathway may be an additional mechanism by which a high-fat/Western diet accelerates atherosclerosis.

Serotonin 5-HT(3) antagonists have been suggested for treatment of several disorders involving altered gastrointestinal (GI) function. CCK also has well documented GI actions on both food intake and vago-vagal reflexes. To evaluate potential interactions, the effect of a 5-HT(3) antagonist, ondansetron, on exogenous CCK induced satiety and c-fos activation was determined. Ondansetron reduced both actions of CCK by approximately 50%. The reduction in c-fos was localized to a specific subregion of the dorsal medulla, suggesting that a distinct subpopulation of CCK receptive fibers are modulated by 5-HT(3) ligands. Treatments using 5-HT(3) antagonists also may affect endogenous CCK functions.

Using a monoclonal antibody against rat brain choline acetyltransferase (ChAT) the cholinergic innervation of the adult rat adrenal gland was visualized. Almost all ChAT-positive fibres contained nitric oxide synthase (NOS), whereas enkephalin (ENK) was exclusively found in ChAT fibres among adrenaline chromaffin cells. The ChAT/NOS/ENK fibres disappeared after immunological sympathectomy, indicating a preganglionic origin. ChAT was not found in the superficial peptide- and NOS containing fibre plexus in the adrenal cortex or in small or large intra-adrenal ganglion neurones under control conditions. Even after colchicine treatment only one single ChAT-positive small ganglion neurone was found. It is possible, therefore that some small intra-adrenal ganglion neurones, which express NOS- and VIP-like immunoreactivities, are noncholinergic, nonadrenergic neurones.

Locations of pontomesencephalic cholinergic projection neurons from the laterodorsal tegmental (LDTg) and pedunculopontine tegmental (PPTg) nuclei to midbrain dopaminergic nuclei were mapped. Stereotaxic microinjections of Fluoro-Gold- or rhodamine-labeled microspheres were made either to substantia nigra (SN) or ventral tegmental area (VTA) in rat. Choline acetyltransferase was visualized immunohistochemically. Labeled cells were digitally mapped at multiple levels of the nuclei using an interactive computer/microscope system. SN-projecting neurons were distributed predominantly ipsilaterally in distinct regions of the PPTg: either at its rostral pole or caudally in an area ventromedial to the superior cerebellar peduncle. Few SN-projecting neurons were found in LDTg. VTA-projecting neurons were distributed bilaterally throughout the cholinergic group, primarily in the densest regions of the LDTg and caudal PPTg. Neurons were not strictly segregated into these patterns. Scattered cells belonging to either projection could be found throughout the cholinergic group on either side. Hierarchical log- linear analysis showed these differences in topographic distribution to be statistically significant. Subtraction of cell density images demonstrated well delineated regions of the cholinergic group where the projections were predominately either to SN or VTA. These data indicate a high degree of internal organization within the pontomesencephalic cholinergic group based on the location of efferent projections to SN or VTA. These findings support the concept that this cholinergic group is functionally organized in a manner which selectively innervates motor (SN) and limbic (VTA) dopaminergic nuclei.

This study was aimed at characterizing the ultrastructural morphology of the normal acetylcholine (ACh) innervation in adult rat parietal cortex. After immunostaining with a monoclonal antibody against purified rat brain choline acetyltransferase (ChAT), more than 100 immunoreactive axonal varicosities (terminals) from each layer of the Par 1 area were photographed and examined in serial thin sections across their entire volume. These varicosities were relatively small, averaging 0.6 μm in diameter, 1.6 μM2 in surface, and 0.12 μM3 in volume. In every layer, a relatively low proportion exhibited a synaptic membrane differentiation (10% in layer I, 14% in II–III, 11% in IV, 21% in V, 14% in VI), for a I–VI average of 14%. These synaptic junctions were usually single, symmetrical (>99%), and occupied a small portion of the surface of varicosities (85% of its varicosities), and the composition of the microenvironment of its varicosities suggests some randomness in their distribution at the microscopic level. It is unlikely that these ultrastructural characteristics are exclusive to the parietal region. Among other functional implications, they suggest that this system depends predominantly on volume transmission to exert its modulatory effects on cortical activity. © 1994 Wiley-Liss, Inc.

Innervation of rat intracortical cerebral blood vessels by acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) remains largely unexplored and it is known if the cells of origin are intra- or extracortical nor 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 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 hat 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.

A sensitive immunofluorescence technique was used to describe systematically the distrubution of dopamine-beta-hydroxylase (DBH)-containing cell bodies, non-terminal fiber pathways, and terminal fields in the brain of the male albino rat. DBH is the enzyme that catalyzes the conversion of dopamine to noradrenaline, and as such is useful as an anatomical marker for noradrenaline and possibly adrenaline neurons. The enzyme is not present in dopamine- or indolamine-containing neurons. Ten micron frozen sections (1-in 20 series) were prepared in the frontal, sagittal, and horizontal planes from the olfactory bulb to the upper cervical segments of the spinal cord; adjacent sections in each plane were stained for DBH and for cells (toluidine blue=azure II). An atlas consisting of 40 projection drawings of selected frontal sections illustrates the results of the investigation. DBH perikarya are confined to three groups in the pons and medulla: the well defined locus coeruleus, a more diffuse but continuous subcoeruleus group that arches through the pons and ventral medulla, and a third dorsal medullary group centered in the dorsal motor nucleus of the vagus. A single principal adrenergic fiber system distributes a great many of the axons from these neuron groups to a majority of nuclear areas in the brain. In the pons and medulla two components of the fiber system may be distinguished. A medullary branch may be followed from the posterior aspect of the subcoeruleus group dorsally and then anteriorly through the lateral tegmental field and ventral aspect of the vestibular complex to a position subjacent to the locus coeruleus, where it is joined by a subcoeruleus branch consisting of a large number of fibers coursing among cells along the length of the subcoeruleus group, and by fibers arising from the locus coeruleus. Anterior to the locus coeruleus the principal adrenergic bundle courses as a single fiber tract immediately ventrolateral to the central gray in the mesencephalon and in the zona incerta and substantia innominata in the diencephalon. At the level of the septal area separate bundles reach the cortex dorsally over the genu of the corpus calosum via the medial septal-diagonal band nuclei and the lateral septum and ventrally between the olfactory tubercle and caudate-putamen. In the medulla and pons adrenergic fibers undoubtedly course in both directions. Anterior to the most rostral pontine cell bodies, however, all fibers presumably ascend. Along the course of the bundle distinct branches emerge to innervate circumscribed terminal fields. In addition, certain regions of the brain such as the reticular formation and pontine gray receive diffuse DBH innervation derived from less clearly defined pathways. A small number of areas in the brain contain little or no detectable DBH. These include the caudate-putamen, nucleus accumbens, globus pallidus, olfactory tubercle, subthalamic nucleus, substantia nigra, pretectal area, third, fourth and sixth cranial verve nuclei, and the trapezoid body nucleus.

The Sternberg fixed-set memory-search paradigm was used to assess the relative vulnerability of hypothetical stages of information processing to an oral dose of secobarbital (2.9 mg/kg). D-amphetamine (15 mg, oral dose) was intended to serve as an active placebo. However, since the amphetamine produced a slight, non-significant reduction in choice reaction time (RT), the principal analysis of secobarbital effects was conducted between drug and baseline conditions. Secobarbital slowed choice RT by 60 msec. and did not increase errors significantly. The results, as interpreted within Sternberg's model, suggest that input processes, e.g., stimulus preprocessing-encoding, are particularly sensitive to the effects of the barbiturate. There was no evidence of a drug effect on cognitive processes associated with serial comparison, binary decision, or translation-response organization (response selection). In contrast, earlier studies have indicated that another CNS depressant, alcohol, interferes with both speed and accuracy of output processes, viz., the response selection stage.

Rabbit anti-bovine myo-inositol-1-phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion-fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo-inositol-1-phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo-inosose reductase and myo-inositol-1-phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo-inositol-1-phosphate synthase in brain raises new questions about the mechanism by which myo-inositol is concentrated to such high cellular levels in the principal substance of that organ.

Immunohistochemistry was used to map the distribution of four neuropeptides in song control regions of two songbird species, the European starling (Sturnus vulgaris) and the song sparrow (Melospiza melodia). We searched for positively stained cell bodies or apparent terminals containing vasoactive intestinal peptide (VIP), methionine-enkephalin (MET), cholecystokinin (CCK), and substance P (SUB P). Intraventricular colchicine pretreatment was administered to enhance the visualization of peptide-containing cell bodies. Four areas implicated in the central control of song were examined. Three of these areas are sexually dimorphic telencephalic nuclei characteristic of songbirds: the caudal nucleus of the ventral hyperstriatum (HVc), the robust nucleus of the archistriatum (RA), and the magnocellular nucleus of the anterior neostriatum (MAN). The fourth region is the mesencephalic nucleus intercollicullaris (ICo), common to all birds, which contains the dorsomedial nucleus (DM) that appears to be specifically involved in the motor control of song. The pattern of neuropeptide localization was similar between the two species. However, the neuropeptides were heterogeneously dispersed among the four areas. VIP and MET were the most widely distributed, whereas CCK and SUB P were seen only in DM. MAN and HVc revealed remarkably similar patterns of staining for both MET and VIP. Fine varicosities immunolabeled for both these peptides appear to encircle nonreactive somata. In both these nuclei positively stained somata were observed for MET but not for VIP. In RA there was a dense accumulation of MET-positive multipolar cell bodies. VIP-containing neurons were seen in the surrounding archistriatum and caudal neostriatum but not in RA itself. Cell bodies and fibers for all four peptides were observed in DM; in no case were they limited to this subregion, but rather seemed to encompass the surrounding intercollicular area as well. The widespread distribution of VIP and MET strongly suggests a role for these peptides in the acquisition or production of passerine song.

An intravenous injection method to measure cerebral vascular extraction fractions of highly diffusible substances in the rat is described. The brain extraction fractions of 3H-labeled water ( Ew) and ethanol ( Ee) were defined as the ratio of either of those tracers to the freely diffusible reference tracer, 14C-butanol, in the brain, divided by the ratio of the tracers available for the extraction during the time between simultaneous intravenous injection of the tracers and decapitation of the rat. Ew and Ee were measured in five regions of brain, including brainstem and cerebellum, under Paco2 conditions ranging from 15 to 85 mm Hg. The extraction fractions for both test tracers were shown to vary with Paco2-induced flow changes according to the equation, ln(1 – E) = − PS/ F. When PS/ F values calculated from regional measurements of Ew and Ee were plotted versus Paco2, least squares regression equations of the plots could be used to compare permeabilities of both tracers at any given Paco2 value. Ratios of the permeabilities of water and ethanol varied regionally but were relatively constant in a given region under different flow states. This intravenous injection method allows for accurate measurement of the extraction fractions of even highly diffusible tracers under varied flow conditions in all brain regions regardless of arterial blood supply.

Chronic treatment of rats with amitriptyline (AMI) induced a persistent increase in the diffusibility of water into the brain (EW). This effect was observable 12 h after the last dose. AMI induces this alteration at plasma drug concentrations of 71 +/- 13 ng/ml (the therapeutic range for man is 100--250 ng/ml). Furthermore, chronic treatment potentiated the increase observed after acute drug administration and resulted in a 350% enhancement in the permeability of water across the cerebral capillary. Thus, long-term antidepressant administration can chronically influence cerebral function by affecting capillary permeability.

Mild acute hypertension was induced in rats pharmacologically and non-pharmacologically with metaraminol, angiotensin II or bilateral vagotomy. The extraction fraction of water (Ew) was measured in both hypertensive and normotensive animals. It was demonstrated that cerebral blood flow did not change during hypertensive states. Under constant flow conditions Ew becomes a direct index of the cerebral capillary permeability to water. Ew was significantly decreased (P less than 0.001) in all brain regions examined in the hypertensive animals as compared to normotensive controls. However, the decrease Ew induced by hypertension was not observed if the animal had previously received bilateral carotid sinus denervation. This observation indicated that the capillary permeability response induced by hypertension is mediated by a neurogenic mechanism involving information transfer from the peripheral baroreceptors in the carotid sinus along afferent connections into the central nervous system.

The aim of this study was to examine human physiological sleep profiles, including the amount and distribution of electroenoephalographic (EEG) stages of sleep, variations in specific frequency bands in the EEG spectrum and certain phasic phenomena such as movement arousals, sigma spindles and rapid eye movements, following oral administration of a moderate dose (150 mg) of chlorpromazine (CPZ) to 12 young male volunteers. At this dose level the drug had few systematic effects on sleep, although it did reduce the latency of onset of stage REM and the number of movement arousals, while increasing the amount of slow-wave (SW) sleep. These effects persisted during the post-medication recovery night, but at no time was there any systematic change in the total amount or percent of REM sleep, the duration of the REM-to-REM cycle, the average length of REM episodes or the density of rapid eye movements during stage REM. Frequency analysis of EEG revealed that CPZ produced a trend toward increased fast (beta) activity recorded from pre-central placements during stage REM, and reduced density of sigma spindles in stage 2 sleep. Thus, for the most part, a single moderate dose of CPZ left the tonic, phasic and sequential properties of the sleep cycle unaltered. These results confirm previous investigations showing that for small to moderate clinical doses, CPZ invariably enhances SW sleep and reduces the frequency of movement arousals. On the other hand, the effect of the drug on stage REM apparently depends on dose. Small doses potentiate REM sleep or accelerate its onset, whereas larger doses either reduce stage REM or leave it unaffected. Several authors have pointed out that most hypnotic agents cause substantial alterations of the sleep profile, and that their withdrawal can cause profound disruption of sleep and marked clinical disturbance. It also has been suggested that there exists a relation between drug dependency and the degree of initial REM suppression caused by a drug. The finding confirmed by the present study that clinical doses of CPZ cause mild sedation, and enhanced SW sleep without any significant modification of REM, sleep, indicates that CPZ has features which may recommend it as a standard hypnotic.

Sleep physiological patterns were examined following a single oral dose (500 mg) of the hypnotic drug alpha chloralose. The drug increased SW sleep and decreased REM sleep without affecting total sleep time or the amount of stage 2. These changes were accompanied by a shift to slower frequencies and greater EEG synchrony, as well as a decrease in the number of spontaneous arousals in all stages of sleep, and throughout the night of medication. Except for a slight decrease in eye movement density, the drug had no systematic effects on phasic phenomena such as electrodermal or cardio-respiratory fluctuations, nor was there a systematic change in basal heart and breathing rates.

Oral administration of L-tryptophan increases brain serotonin levels by following normal 5-hydroxyindole pathways (Moir & Eccleston, 1968). Since recent studies (Jouvet, 1969) suggest that serotonin is important in controlling sleep mechanisms, L-tryptophan provides a useful method for investigating further the role of serotonin in sleep. In two experiments, EEG sleep patterns from 21 young adult males were examined following moderate (7.5 g) and high (12 g) oral doses of L-tryptophan. Moderate doses produced sedative effects (reports of extreme drowsiness and reduced time awake) accompanied by increased slow-wave sleep. The only effects on REM parameters were a trend (in some Ss) to early onset of stage REM, a small decrease in the period of the REM cycle, and decreased density of rapid eye movements. With the high dose, Ss again reported extreme drowsiness, and time to sleep onset was decreased. However, changes in SW sleep and waking time appeared only as non-significant trends. In the high-dose group, percent of REM sleep was markedly increased, due to early onset of stage REM and to increased duration of REM episodes during the second half of the night. EEG sleep patterns on recovery nights following large doses of tryptophan were not systematically different from baseline nights. These results indicate that the changes in sleep patterns produced by L-tryptophan, presumably acting through 5-hydroxyindole pathways, are dependent on dose. The findings are consistent with the idea that serotonin, or one of its metabolites, is involved in the mechanisms controlling both SW and REM sleep.

The noradrenergic innervation of the area postrema has been studied by means of immunofluorescent localization of dopamine-β-hydroxylase and with electron microscopy. This type of correlative study is effective because the immunofluorescent localization is highly specific and electron microscopy affords a resolution not possible with the fluorescent technique alone. Two distinct groups of noradrenergic cell bodies are found to be associated with the area postrema. The bulk of noradrenergic innervation appears to derive from those DBH containing cell bodies immediately adjacent to the area postrema. The distribution of axons originating from the other group of neurons, which are located within the area postrema, cannot be determined by these techniques since there are too many intertwined fibers in this part of the tissue. The noradrenergic nerve terminals assume two distinct patterns. In the ventral and lateral parts, the synapses are typical of the central nervous system, being axosomatic or axodendritic. The terminals of the superior margins are related to non-neural structures as in the peripheral nervous system and involve ependymal cells and perivascular smooth muscle.

Polycythemia and hyperhomocysteinemia are risk factors for thrombosis. Since red blood cells actively metabolize methionine to homocysteine, we investigated whether or not patients with polycythemia have increased plasma levels of homocysteine, which might contribute to their increased thrombotic risk. In ten patients with polycythemia, the plasma homocysteine levels were measured before phlebotomy, three days after the procedure and 1-2 months later. The baseline mean plasma homocysteine levels in patients (9.7 +/- 1.6 mumol/L [+/-SD]) did not differ significantly from that found in 30 sex- and age-matched healthy controls (12.2 +/- 6.9). Despite a fall in the patients' mean [+/-SD] hematocrit from 0.50 +/- 0.02 at baseline to 0.47 +/- 0.03 three days after phlebotomy (significant at 95%) and to 0.48 +/- 0.02 after 1 to 2 months (not significant), the mean plasma homocysteine levels did not change significantly (9.9 +/- 2.3 mumol/L at 3 days and 9.7 +/- 2.1 mumol/L at 1-2 months). It is unlikely that high plasma homocysteine levels contribute to the increased thrombotic risk of polycythemic patients.

Summary Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) was localized using fluorescence immunohistochemistry in rat liver and kidney and in rat, chinchilla, and bovine brains. In the brain, specific fluorescence was visable only in non-neuronal cellular elements in all three species. Ventricular ependymal cells and cells of the choroid plexuses exhibited the greatest intensity of immunofluorescence. Glial immunofluorescence appeared prominently in large myelinated fiber tracts. Interfascicular and perineuronal satellite oligodendrocytes as well as fibrous astrocytes were immunoreactive, though myelin itself did not exhibit a positive reaction. Bergmann glial cells in the cerebellum also stained brightly for COMT. Although this study indicates that the predominant localization of COMT is non-neuronal, it is important to note that the presence of small quantities of this enzyme in neurons cannot be excluded. The patterns of localization observed in the non-neuronal elements suggest that this enzyme may function as a barrier to free diffusion of catechol compounds within the central nervous system.

The anatomical characteristics of the accumulation of dopamine-β-hydroxylase (DBH) that occurs after ligation of rat sciatic nerve were examined in detail using a recently developed specific immunofluorescence method. Although rapid accumulation of DBH occurred showing the presence of axonal transport, the anatomical observations indicated that the build-up was considerably more complex than the simple "blockade" model frequently used as a basis for computation of transport rates from quantitative estimates of accumulation. During the early time period (7 hr) the situation was complicated by a substantial amount of accumulation of DBH distal to the ligation. By 24 hr after ligation the distal accumulation was small compared to that present proximally. The size of the discrete areas covered by DBH fluorescence at 24 hr indicated that axonal membranes may have already started to rupture, thus releasing vesicles into the extracellular space. By 48 hr after ligation the DBH was no longer confined to the area near the ligation, but extended 3.6 mm proximally. By 96 hr after ligation there had been a net loss of DBH fluorescence indicating that degradation and clearance of accumulated DBH had occurred.

Two experiments were conducted to determine the effect of T-2 toxin on brain biogenic monoamines and their metabolites. Male rats (180 g) and cockerels (28 day, 300 g) were orally dosed with T-2 toxin at 2.5 mg kg-1 body weight. In the first experiment, whole brains were collected 2, 6, 12, 24 and 48 h postdosing and analyzed for monoamines by high performance liquid chromatography with electro-chemical detection. T-2 toxin did not influence whole brain concentrations of monoamines in either species. In the second experiment, brains were collected 24 h postdosing, dissected into five brain regions, and analyzed for monoamines. T-2 toxin treatment resulted in increased serotonin and 5-hydroxy-3-indoleacetic acid in all brain regions of the rat. However, this was not seen in poultry where T-2 toxin treatment resulted in an increase in 5-hydroxy-3-indoleacetic acid, no alteration in serotonin concentration and a decrease in regional norepinephrine and dopamine concentrations. These results suggest that T-2 toxin influences brain biogenic amine metabolism and that there is an intraspecies difference in the central effects of this mycotoxin.

Sleep patterns of 14 male Ss were examined following a single oral dose (200 mg) of the barbiturate secobarbital. Compared to baseline, medication caused definite changes in the amount and distribution of the EEG stages of sleep. With the drug, a decrease in percent stage REM and an increase in percent stage 2 were accompanied by fewer body movements and a trend toward less waking. A more striking effect was the drug-induced redistribution of EEG stages with slow-wave sleep potentiated during the first half of the night but virtually eliminated during the last half. Stage REM, on the other hand, was inhibited in the first half but returned to baseline levels in the last half of the night. Recent evidence suggests that such effects could result from modulation of brain levels of the monoamines. Within the stages of sleep the amount of fast EEG activity was increased by the drug, with a tendency toward desynchrony. Pre-central beta activity became especially prominent in stages REM and 2 (low-voltage phases), and this change was associated with inhibition of such phasic events as eye movements during REM sleep, sigma spindles during stage 2 and spontaneous electrodermal responses in slow-wave sleep. Thus, it appears that secobarbital potentiates tonic and suppresses phasic phenomena during sleep. A possible interpretation of these results is that secobarbital enhances electrical activity in forebrain structures (Routtenberg's arousal system II) while inhibiting the reticular activating system (arousal system I) causing a reduction of phasic variability in all stages of sleep.

Recent studies of the effects of reserpine on human sleep have reported increased rapid eye movement (REM) sleep, and decreased slow-wave (SW) sleep. These results are relevant to theories linking serotonin and the catecholamines to the control of different stages of sleep. However, since reserpine causes release and subsequent depletion of both monoamines, it is difficult to relate changes in sleep profiles to specific alterations in one or the other amine system. The results to be reported here, when compared to those obtained with other treatments which affect the biogenic amines, encourage the view that level and turnover of serotonin are the primary mediators for reserpine-induced modifications of sleep. In two separate experiments, EEG sleep patterns from 20 male Ss were examined following single and repeated oral doses (1 mg) of reserpine. In the single-dose study, reserpine caused increased REM, and decreased SW sleep, effects which became statistically significant on the post-medication (P-M) recovery session. These changes were accompanied by reduced frequency per minute of sigma spindles (stage 2) decreased eye-movement density (stage REM) and a tendency toward increased brief arousals, especially during stage REM. Examination of parameters of the REM cycle revealed that the potentiation of REM sleep was due to its reduced latency of onset, and more frequent cyclic occurrence, not to increased duration of REM episodes. The results of the repeated-dose study replicated and amplified those of the first experiment, showing that medication caused a progressive increase in the amount of stage REM, accompanied by a simultaneous loss of SW sleep. The increase in REM was again due to acceleration of its cycle rather than lengthening of its episodes. During medication, epochs of stage REM were increasingly interrupted by brief arousals, with a simulteneous decline in the density of rapid eye movements. Most of these reserpine effects persisted into the P-M recovery session.

In two separate experiments, sleep patterns of 17 young male adults were examined following single and repeated doses (0.9 g/kg body weight) of alcohol. A third study of 10 additional Ss measured the rate of elimination of alcohol from blood during sleep and waking. With a single dose of alcohol, onset of sleep was brisk, the latency of SW sleep (stages 3+4) was reduced and the first episode of stage REM was shortened. These transient alterations were accompanied by loss of high-frequency beta rhythms in the EEG and a gain in abundance and synchrony of activity in the alpha-rhythm range.

PROBLEM ADDRESSED: Adults with intellectual and developmental disabilities (IDD), a group with complex health problems and inequities in access to health care, look to family physicians for primary care. OBJECTIVE OF PROGRAM: To enable residents to learn and demonstrate competencies that are unique to the care of adults with IDD with minimal extra time and resources required of the residency program. PROGRAM DESCRIPTION: In their regular family medicine teaching practices, residents undertake planned encounters with adults with IDD involving comprehensive health assessments with physical examinations. Tools to implement the Canadian guidelines for primary care of adults with IDD are available to support the residents in their encounters. Background information in the form of self-learning and small group learning resources, field notes with rubrics to assess residents’ development of competencies, and faculty development resources are also available. CONCLUSION: It is important to include such planned clinical experiences in family medicine residency curricula because people with IDD have special needs that are difficult to learn about in other settings. It is a benefit to residents to have patients and families actively contributing to teaching.