Chronic Fragmentation of the Daily Sleep-Wake Rhythm Increases Amyloid-beta Levels and Neuroinflammation in the 3xTg-AD Mouse Model of Alzheimer's Disease.

Fragmentation of the daily sleep-wake rhythm with increased nighttime awakenings and more daytime naps is correlated with the risk of development of Alzheimer's disease (AD). To explore whether a causal relationship underlies this correlation, the present study tested the hypothesis that chronic fragmentation of the daily sleep-wake rhythm stimulates brain amyloid-beta (Aß) levels and neuroinflammation in the 3xTg-AD mouse model of AD. Female 3xTg-AD mice were allowed to sleep undisturbed or were subjected to chronic sleep fragmentation consisting of four daily sessions of enforced wakefulness (one hour each) evenly distributed during the light phase, five days a week for four weeks. Piezoelectric sleep recording revealed that sleep fragmentation altered the daily sleep-wake rhythm to resemble the pattern observed in AD. Levels of amyloid-beta (Aß40 and Aß42) determined by ELISA were higher in hippocampal tissue collected from sleep-fragmented mice than from undisturbed controls. In contrast, hippocampal levels of tau and phospho-tau differed minimally between sleep fragmented and undisturbed control mice. Sleep fragmentation also stimulated neuroinflammation as shown by increased expression of markers of microglial activation and proinflammatory cytokines measured by q-RT-PCR analysis of hippocampal samples. No significant effects of sleep fragmentation on Aß, tau, or neuroinflammation were observed in the cerebral cortex. These studies support the concept that improving sleep consolidation in individuals at risk for AD may be beneficial for slowing the onset or progression of this devastating neurodegenerative disease.

Effects of acute lying and sleep deprivation on metabolic and inflammatory responses of lactating dairy cows.

Dairy cows that are restricted from lying down have a reduced ability to sleep. In other species, sleep loss is a key risk factor for disease, mediated by changes in metabolic and inflammatory responses. The cumulative effect of lying and sleep deprivation on cow health is unknown. The objective was to determine the effects of lying and sleep deprivation on metabolic and inflammatory responses of dairy cows. Data were collected from 8 multiparous and 4 primiparous lactating cows (199 ± 44 d in milk, 77 ± 30 d pregnant; mean ± standard deviation) enrolled in a study using a crossover design. Each cow was exposed to 2 treatments meant to induce sleep loss: (1) human disturbance (imposed by researchers making noise or physical contact when the cow's posture suggested sleep) and (2) lying deprivation (imposed by a wooden grid placed on the pen floor). Cows experienced a 24-h baseline period (d -1) followed by a 24-h treatment period (d 0), with a 12-d washout period between treatments. Baseline and treatment periods were imposed from 2100 to 2059 h. Cows were housed in individual pens during the acclimation period (d -3 and -2), d -1, and d 0. Nonesterified fatty acid and glucose concentrations were measured at 0300, 0900, 1500, and 2059 h on d -1 and 0. Proinflammatory cytokine mRNA [tumor necrosis factor (TNF), interleukin-1B (IL1B), and interleukin-6 (IL6)] abundance in whole-blood leukocytes, both nonstimulated and stimulated with lipopolysaccharide, were assessed at 2059 h on d -1 (end of baseline) and d 0 (end of treatment). Nonesterified fatty acids and glucose varied by time of day but were not affected by treatment or day. The abundances of TNF and IL1B from both stimulated and nonstimulated cells were higher following 24 h of lying deprivation (d 0) compared with baseline (d -1). Abundance of IL6 was increased in nonstimulated cells after lying deprivation compared with baseline. In contrast, human disturbance for 24 h did not alter TNF, IL1B, or IL6 abundance relative to baseline levels. These results suggest that a short period of lying deprivation generally increases inflammatory responses but not metabolic responses.

Development of a noninvasive system for monitoring dairy cattle sleep.

Limited research has been conducted to assess sleep in production livestock primarily because of limitations with monitoring capabilities. Consequently, biological understanding of production circumstances and facility options that affect sleep is limited. The objective of this study was to assess if data collected from a proof-of-concept, noninvasive 3-axis accelerometer device are correlated with sleep and wake-like behaviors in dairy cattle. Four Holstein dairy cows housed at the University of Kentucky Coldstream Dairy in September 2013 were visually observed for 2 consecutive 24-h periods. The accelerometer device was attached to a harness positioned on the right side of each cow's neck. Times of classified behaviors of wake (standing, head up, alert, eyes open) or sleep-like behaviors (lying, still, head resting on ground, eyes closed) were recorded continuously by 2 observers who each watched 2 cows at a time. The radial signal was extracted from 3 different axes of the accelerometer to obtain a motion signal independent of direction of movement. Radial signal features were examined for maximizing the performance of detecting sleep-like behaviors using a Fisher's linear discriminant analysis classifier. The study included 652min of high-activity wake behaviors and 107min of sleep-like behavior among 4 cows. Results from a bootstrapping analysis showed an agreement between human observation and the linear discriminant analysis classifier, with an accuracy of 93.7±0.7% for wake behavior and 92.2±0.8% for sleep-like behavior (±95% confidence interval).This prototype shows promise in measuring sleep-like behaviors. Improvements to both hardware and software should allow more accurate determinations of subtle head movements and respiratory movements that will further improve the assessment of these sleep-like behaviors, including estimates of deep, light, and rapid eye movement sleep. These future studies will require simultaneous electroencephalography and electromyography measures and perhaps additional measures of arousal thresholds to validate this system for measuring true sleep.

Increased fragmentation of sleep-wake cycles in the 5XFAD mouse model of Alzheimer's disease.

Sleep perturbations including fragmented sleep with frequent night-time awakenings and daytime naps are common in patients with Alzheimer's disease (AD), and these daily disruptions are a major factor for institutionalization. The objective of this study was to investigate if sleep-wake patterns are altered in 5XFAD mice, a well-characterized double transgenic mouse model of AD which exhibits an early onset of robust AD pathology and memory deficits. These mice have five distinct human mutations in two genes, the amyloid precursor protein (APP) and Presenilin1 (PS1) engineered into two transgenes driven by a neuron-specific promoter (Thy1), and thus develop severe amyloid deposition by 4 months of age. Age-matched (4-6.5 months old) male and female 5XFAD mice were monitored and compared to wild-type littermate controls for multiple sleep traits using a non-invasive, high throughput, automated piezoelectric system which detects breathing and gross body movements to characterize sleep and wake. Sleep-wake patterns were recorded continuously under baseline conditions (undisturbed) for 3 days and after sleep deprivation of 4h, which in mice produces a significant sleep debt and challenge to sleep homeostasis. Under baseline conditions, 5XFAD mice exhibited shorter bout lengths (14% lower values for males and 26% for females) as compared to controls (p<0.001). In females, the 5XFAD mice also showed 12% less total sleep than WT (p<0.01). Bout length reductions were greater during the night (the active phase for mice) than during the day, which does not model the human condition of disrupted sleep at night (the inactive period). However, the overall decrease in bout length suggests increased fragmentation and disruption in sleep consolidation that may be relevant to human sleep. The 5XFAD mice may serve as a useful model for testing therapeutic strategies to improve sleep consolidation in AD patients.

Acetylcholine becomes the major excitatory neurotransmitter in the hypothalamus in vitro in the absence of glutamate excitation.

Glutamate and GABA are two major fast neurotransmitters (excitatory and inhibitory, respectively) in the CNS, including the hypothalamus. They play a key role in the control of excitation/inhibition balance and determine the activity and excitability of neurons in many neuronal circuits. Using neuronal cultures, whole-cell recording, Ca(2+) imaging, and Northern blots, we studied the compensatory regulation of neuronal activity during a prolonged decrease in glutamate excitation. We report here that after a chronic (6-17 d) blockade of ionotropic glutamate receptors, neurons in hypothalamic cultures revealed excitatory electrical and Ca(2+) synaptic activity, which was not elicited in the control cultures that were not subjected to glutamate blockade. This activity was suppressed with acetylcholine (ACh) receptor antagonists and was potentiated by eserine, an inhibitor of acetylcholinesterase, suggesting its cholinergic nature. The upregulation of ACh receptors and the contribution of ACh to the control of the excitation/inhibition balance in cultures after a prolonged decrease in glutamate activity were also demonstrated. Enhanced ACh transmission was also found in chronically blocked cerebellar but not cortical cultures, suggesting the region-specific character of glutamate-ACh interactions in the brain. We believe that in the absence of glutamate excitation in the hypothalamus in vitro, ACh, a neurotransmitter normally exhibiting only weak activity in the hypothalamus, becomes the major excitatory neurotransmitter and supports the excitation/inhibition balance. The increase in excitatory ACh transmission during a decrease in glutamate excitation may represent a novel form of neuronal plasticity that regulates activity and excitability of neurons during the glutamate/GABA imbalance.

HSP70 expression is increased during the day in a diurnal animal, the golden-mantled ground squirrel Spermophilus lateralis.

Heat shock protein 70 (HSP70) gene expression was studied in a seasonal hibernator, the diurnal ground squirrel, Spermophilus lateralis. RNA transcripts of 2.7 and 2.9 kb hybridizing to an HSP70 cDNA were expressed in both brain and peripheral tissues of pre-hibernation euthermic animals; higher levels of expression were observed during the day than during nighttime samples. A decline in the expression of both transcripts occurred in all tissues examined during hibernation that remained low throughout the hibernation season, including the interbout euthermic periods and regardless of time of day. Quantitative comparisons showed pre-hibernation nighttime HSP70 expression to be as low as that observed during hibernation, despite the drastic increase in metabolic state and nearly 30 degrees C difference in body temperature. In contrast to HSP70, some mRNAs, such as beta-actin and HSP60, remained relatively constant, while others, such as glyceraldehyde 3-phosphate dehydrogenase, increased in specific tissues during the hibernation season. These results indicate that the expression of a highly conserved gene involved in protection from cellular stress, HSP70, can vary with an animal's arousal state.

Gene expression in the brain across the hibernation cycle.

The purpose of this study was to characterize changes in gene expression in the brain of a seasonal hibernator, the golden-mantled ground squirrel, Spermophilus lateralis, during the hibernation season. Very little information is available on molecular changes that correlate with hibernation state, and what has been done focused mainly on seasonal changes in peripheral tissues. We produced over 4000 reverse transcription-PCR products from euthermic and hibernating brain and compared them using differential display. Twenty-nine of the most promising were examined by Northern analysis. Although some small differences were observed across hibernation states, none of the 29 had significant changes. However, a more direct approach, investigating expression of putative hibernation-responsive genes by Northern analysis, revealed an increase in expression of transcription factors c-fos, junB, and c-Jun, but not junD, commencing during late torpor and peaking during the arousal phase of individual hibernation bouts. In contrast, prostaglandin D2 synthase declined during late torpor and arousal but returned to a high level on return to euthermia. Other genes that have putative roles in mammalian sleep or specific brain functions, including somatostatin, enkephalin, growth-associated protein 43, glutamate acid decarboxylases 65/67, histidine decarboxylase, and a sleep-related transcript SD464 did not change significantly during individual hibernation bouts. We also observed no decline in total RNA or total mRNA during torpor; such a decline had been previously hypothesized. Therefore, it appears that the dramatic changes in body temperature and other physiological variables that accompany hibernation involve only modest reprogramming of gene expression or steady-state mRNA levels.

Developmental changes in nicotinic receptor mRNAs and responses to nicotine in the suprachiasmatic nucleus and other brain regions.

Our previous studies demonstrated that nicotine induces c-fos expression in the suprachiasmatic nucleus (SCN) of the rat during a narrow developmental window occurring in the perinatal period. We have extended these observations by showing that c-fos cannot be induced in the adult SCN by nicotine even during the subjective night, when phase shifts do occur. In contrast to the SCN, significant induction of c-fos and NGFI-A was observed in the medial habenula and paraventricular nucleus at all circadian times. In the fetal rat SCN we show that NGFI-A and junB are also induced by nicotine, but not c-jun. To investigate whether changes in nicotinic acetylcholine receptor (nAChR) expression in the SCN may underlie this change in sensitivity during the perinatal period, we examined nAChR mRNAs across this developmental period. By Northern analyses, alpha2, alpha3 and alpha4 subunit mRNAs are relatively abundant in the fetal SCN but decline substantially in the adult. alpha7 mRNA increases substantially while beta2 mRNA is relatively abundant throughout development. We also examine expression in the whole mouse brain beginning at embryonic day 11. Many mRNA sizes for nAChR subunits in both the rat and mouse are characterized here for the first time by Northern analyses and some show very large changes in expression across development. In particular, a small 1.4 kb alpha2-related mRNA is highly expressed during early development, perhaps indicating an important novel function for this subunit.

Characterization of the circadian system of NGFI-A and NGFI-A/NGFI-B deficient mice.

The genes NGFI-A (also known as EGR-1, zif/268, and Krox-24) and NGFI-B (nur/77) have previously been shown to be induced in the SCN of rats and hamsters by photic stimulation during the subjective night. The purpose of this study is to determine whether these genes are also induced in the SCN of mice and, if so, to characterize the circadian system of animals in which either NGFI-A or both NGFI-A and NGFI-B were eliminated by homologous recombination. In wildtype mice, NGFI-A mRNA was found to be induced in the SCN as in other rodent species. Therefore, wheel-running activity was recorded from null mutants and wildtype controls under LD 12:12 and DD conditions. Mice of all three strains appeared to entrain normally to LD 12:12 and could re-entrain to both phase advances and phase delays of the light cycle. The response of the circadian pacemaker of all three genotypes to acute light pulses appeared to be normal. The retinal innervation of the SCN in NGFI-A-/- mice and the photic induction of Fos in the SCN of both NGFI-A-/- and NGFI-A-/-/B-/- mice were indistinguishable from wildtype mice. These results indicate that induction of NGFI-A and NGFI-B is not required for photic entrainment or phase shifting of the mouse circadian system.

Nicotine and nicotinic receptors in the circadian system.

Considerable data support a role for cholinergic influences on the circadian system. The extent to which these influences are mediated by nicotinic acetylcholine receptors (nAChRs) has been controversial, as have the specific actions of nicotine and acetylcholine in the suprachiasmatic nucleus (SCN) of the hypothalamus. In this article we review the existing literature and present new data supporting an important role for nAChRs in both the developing and adult SCN. Specifically, we present data showing that nicotine is capable of causing phase shifts in the circadian rhythms of rats. Like light and carbachol, nicotine appears to cause phase delays in the early subjective night and phase advances in the late subjective night. In the isolated SCN slice, however, only phase advances are seen, and, surprisingly, nicotine appears to cause the inhibition rather than the excitation of neurons. Among nAChR subunit mRNAs, alpha 7 appears to be the most abundant subunit in the adult SCN, whereas in the perinatal period, the more typical nAChRs with higher affinity for nicotine predominate in the SCN. This developmental change in subunit expression may explain the dramatic sensitivity of the perinatal SCN to nicotine that we have previously observed. The effects of nicotine on the SCN may contribute to alterations caused by nicotine in other physiological systems. These effects might also contribute to the dependence properties of nicotine through influences on arousal.

Daily variation of CNS gene expression in nocturnal vs. diurnal rodents and in the developing rat brain.

Expression of c-fos has been shown to vary throughout the brain over the course of the 24-h day. The magnitude of these changes appear to be similar in a light:dark (LD) cycle or in constant dark (DD). To further examine whether the diurnal and circadian changes in c-fos and other immediate-early gene (IEG) expression in brain are related to waking behaviors such as locomotor activity, we conducted three experiments using Northern analysis. First, we compared IEG expression in nocturnal vs. diurnally active species. Second, we investigated IEG expression in a hibernating species during its active and inactive phases. Third, we examined the development of IEG expression in the young post-natal rat. As a comparison to results obtained in extra-SCN brain regions, we also examined IEG and vasopressin expression in the SCN itself across the circadian cycle. Animals maintained under a 12:12-h LD cycle were sacrificed in the morning (10:00-11:00 h, ZT2-ZT3) or night (22:00-23:00 h, ZT14-ZT15) or at the corresponding circadian times (CT) when kept in DD. Rats sacrificed in the morning always showed lower c-fos expression than at night in all brain areas examined while the reverse pattern was seen in squirrels under both LD and DD conditions, suggesting a direct correlation between c-fos message and activity. The cerebellum displayed the greatest magnitude change between morning and night (often reaching 10-fold). Among other IEGs examined, the expression of NGFI-A and junB are similar to c-fos, but of lesser magnitude, whereas c-jun appears to be invariant in the rat but is increased during the active phase in squirrels. During the hibernation season, squirrels have lower levels of c-fos consistent with their low levels of activity even during their euthermic interbout periods. c-fos expression in the cerebellum and rest of brain of 1-week-old rats sacrificed at ZT3 and ZT15 showed low levels at both timepoints whereas 2- and 3-week-old animals had higher levels at night as do adults. Among other IEGs, junB and NGFI-A again were similar to c-fos while c-jun and junD were more constant. Our observations support the idea of a diurnal rhythm of IEG expression in the CNS that is related to waking behaviors. Among IEGs, c-fos exhibits the greatest daily variation in expression.

Sequence and tissue distribution of a candidate G-coupled receptor cloned from rat hypothalamus.

We have used RT-PCR with degenerate transmembrane primers to clone members of the G-coupled protein receptor family from rat hypothalamic suprachiasmatic nuclei. We report here a novel clone, UHR-1, which encodes a candidate receptor that is most similar to the neuropeptide receptor family, including the tachykinins, somatostatins, and opioids. Message for this putative receptor is expressed in several brain regions, with the highest levels in pituitary, cerebellum, and hypothalamus. No message was detected in peripheral tissues. Southern blot analysis suggests that UHR-1 is likely a member of a multigene family. The natural ligand for this novel receptor is unknown, but based on sequence homology and structural features is likely to be a peptide.

GABAA, GABAC, and NMDA receptor subunit expression in the suprachiasmatic nucleus and other brain regions.

Identification of the neurotransmitter receptor subtypes within the suprachiasmatic nuclei (SCN) will further understanding of the mechanism of the biological clock and may provide targets to manipulate circadian rhythms pharmacologically. We have focused on the ionotropic GABA and glutamate receptors because these appear to account for the majority of synaptic communication in the SCN. Of the 15 genes known to code for GABA receptor subunits in mammals we have examined the expression of 12 in the SCN, neglecting only the alpha 6, gamma 3, and rho 2 subunits. Among glutamate receptors, we have focused on the five known genes coding for the NMDA receptor subunits, and two subunits which help comprise the kainate-selective receptors. Expression was characterized by Northern analysis with RNA purified from a large number of mouse SCN and compared to expression in the remaining hypothalamus, cortex and cerebellum. This approach provided a uniform source of RNA to generate many replicate blots, each of which was probed repeatedly. The most abundant GABA receptor subunit mRNAs in the SCN were alpha 2, alpha 5, beta 1, beta 3, gamma 1 and gamma 2. The rho 1 (rho 1) subunit, which produces GABAC pharmacology, was expressed primarily in the retina in three different species and was not detectable in the mouse SCN despite a common embryological origin with the retina. For several GABA subunits we detected additional mRNA species not previously described. High expression of both genes coding for glutamic acid decarboxylase (GAD65 and GAD67) was also found in the SCN. Among the NMDA receptor subunits, NR1 was most highly expressed in the SCN followed in order of abundance by NR2B, NR2A, NR2C and NR2D. In addition, both GluR5 and GluR6 show clear expression in the SCN, with GluR5 being the most SCN specific. This approach provides a simple measure of receptor subtype expression, complements in situ hybridization studies, and may suggest novel isoforms of known subunits.

Nicotine administration differentially affects gene expression in the maternal and fetal circadian clock.

Exposure to nicotine by active and passive cigarette smoke is a common public health problem. Recent studies have demonstrated that human fetuses are also exposed to significant levels of nicotine and that there is a five-fold increase in the incidence of Sudden Infant Death Syndrome among infants born to smoking mothers. We examined the effect of nicotine administration and expression of the immediate early gene c-fos in the maternal and fetal rat brain by in situ hybridization. Nicotine injection (1 mg/kg s.c.) on embryonic day 20 (E20) induced detectable c-fos mRNA in the maternal habenula and hypothalamic paraventricular nucleus whereas, in the fetal brain, c-fos was induced in both these structures and also in the suprachiasmatic nucleus (SCN). Nicotine-induced c-fos expression in the fetal SCN was confirmed by Northern analysis and found to return to near basal levels by 3 h post-injection. These responses were blocked by pre-administration of mecamylamine, indicating that the effect of nicotine is mediated through the cholinergic system. Investigation of the development of this response revealed that nicotine failed to induce c-fos expression in the SCN on E16, caused minimal expression on E18, robust expression on E20 and postnatal day 0 (P0), and no expression on P2 or thereafter. These observations suggest that an alteration in the composition of the nicotinic receptors (nAChR), or the subsequent intracellular responses leading to c-fos expression, occurs in the SCN during the perinatal period. Induction of c-fos mRNA in the SCN by light has been associated with phase-shifts of the circadian system, however, the behavioral consequences of the transient sensitivity of the fetal and neonatal SCN to nicotine administration and the consequences for maternal-fetal entrainment remain to be directly determined.

Proenkephalin transgenic mice: a short promoter confers high testis expression and reduced fertility.

The regulation and possible function of the preproenkephalin gene in testis were studied in vivo in transgenic mice containing: (1) bases -193 to +210 of the human proenkephalin gene and an additional one kilobase of 3' proenkephalin flanking sequence driving expression of bacterial chloramphenicol acetyltransferase (CAT), and (2) the same promoter and flanking sequences driving expression of a rat proenkephalin cDNA. Five lines of mice, designated HEC1-5, expressed the first construct and 10, HER1-10, the second. Each HEC male and many HER males showed dramatic expression of the transgene in the testis, although much lower expression was observed in the brain and other enkephalin-producing tissues. High levels of expression in testis can thus be achieved with a very short promoter region and do not require intron A sequences previously considered necessary. Altered enkephalin expression may affect testicular function. One founder, HER8, displayed grossly abnormal testicular morphology and was completely infertile. A second founder, HER6, had low sperm motility. Two offspring from other lines also displayed subnormal fertility. These studies support a role for specific promoter sequences in testis expression and may further support a significant role for proenkephalin in testicular function.

C-fos mRNA increases in the ground squirrel suprachiasmatic nucleus during arousal from hibernation.

During hibernation the body temperature of the golden-mantled ground squirrel, Spermophilus lateralis, may drop below 5 degrees C for a few hours to a week or more. Animals cycle between euthermia and deep hibernation many times over the course of the hibernation season. Expression of the transcription factor c-fos increased in the suprachiasmatic nucleus (SCN) of the hypothalamus, the mammalian circadian clock, during deep hibernation and peaked during the arousal from hibernation. The pattern of increase in c-fos messenger RNA seen in the SCN by in situ hybridization was similar to that seen by Northern blot analysis of total hypothalamic RNA. The induction of c-fos may reflect a wake-up signal, increasing transcription of genes required in the euthermic state.

Brain transcription factor expression: effects of acute and chronic amphetamine and injection stress.

Amphetamine influences behaviors and the expression of transcription factor genes in the central nervous system (CNS). A single d-amphetamine dose (7.5 mg/kg, i.p.) enhances behavioral stereotypy and augments brain expression of c-fos, fos-B, fra-1, zif268, jun-B, and c-jun by 2-11 fold. When the single amphetamine dose is preceded by 28 saline injections over 14 days, it is half as effective in enhancing expression of these genes. Rats injected with 7.5 mg/kg i.p. twice daily for 2 weeks and sacrificed after the last injection reveal further attenuation or abolition of the amphetamine-induced mRNA upregulation. These stigmata of 'tolerance' in gene expression display partial overlap with behavioral tolerance, manifest as changes in locomotor activity. Rats receiving low (2 mg/kg) amphetamine challenge doses following the 2-week 7.5 mg/kg b.i.d. amphetamine treatment show tolerance to the locomotor activating effects of the drug; no tolerance is evident following a high (7.5 mg/kg) challenge dose. These data suggest that amphetamine-induced alterations in brain transcription factor gene expression can display 'tolerance' and possibly 'cross-tolerance' with the stress caused by i.p. injection.

Expression and novel subunit isoforms of glutamate receptor genes GluR5 and GluR6.

Molecular heterogeneity of kainate-selective glutamate receptor subunits GluR5 and GluR6 was revealed by identification of a human cDNA, GluR5-1d, and a murine cDNA, GluR6-2, that each encode subunits with novel carboxy-terminal sequences. Both GluR5-1d and GluR6-2 appear to be generated by alternative splicing at analogous sites 14 codons following the fourth putative transmembrane segment. The principal transcripts of GluR5 and GluR6 were detected by Northern analyses of several regions of mammalian brains as 4 and 6 kb bands, respectively. Potential roles for these receptors in development are indicated by detection of their mRNAs in mouse embryos of 11 days gestation. These findings add to the description of the remarkable diversity of glutamate receptor gene expression.

Dopamine D2 receptor RFLPs, haplotypes and their association with substance use in black and Caucasian research volunteers.

Alleles of the dopamine D2 receptor gene are distinguished by polymorphic A and B TaqI sites approximately 10 kb 3' to the final exon and bordering the second exon, respectively. These alleles have been reported to be more prevalent in heavy substance users than in control populations in several, although not all studies. Meta-analyses of combined data from available work support significant association. Two competing hypotheses could explain this association: (1) the A1 and B1 RFLPs are in linkage disequilibrium with a functional allelic determinant that in some way influences behavior; (2) the affected subjects are drawn disproportionately from populations stratified on the basis of, for example, ethnicity that happen to have higher A1 and B1 RFLP frequencies. We report here data collected from 616 substance-abusing and control individuals that document substantial differences in A1 RFLP frequencies between white and black Americans, the almost exclusive presence of the A3 RFLP in blacks, and low frequencies of rare A4 and B3 RFLPs. In blacks, neither the A1 nor B1 RFLPs display association with substance use, while white individuals display significant association with polysubstance use. When expressed as a percent of the maximum possible disequilibrium, both white and black individuals display strong linkage disequilibrium between these loci, although blacks display many more A1/B2 chromosomes. These racial differences in TaqI RFLP haplotypes underscore the need for caution in interpreting allelic associations when careful matching for ethnicity has not been performed.

Chromosomal localization of glutamate receptor genes: relationship to familial amyotrophic lateral sclerosis and other neurological disorders of mice and humans.

Receptors for the major excitatory neurotransmitter glutamate may play key roles in neurodegeneration. The mouse Glur-5 gene maps to chromosome 16 between App and Sod-1. The homologous human GLUR5 gene maps to the corresponding region of human chromosome 21, which contains the locus for familial amyotrophic lateral sclerosis. This location, and other features, render GLUR5 a possible candidate gene for familial amyotrophic lateral sclerosis. In addition, dosage imbalance of GLUR5 may have a role in the trisomy 21 (Down syndrome). Further characterization of the murine glutamate receptor family includes mapping of Glur-1 to the same region as neurological mutants spasmodic, shaker-2, tipsy, and vibrator on chromosome 11; Glur-2 near spastic on chromosome 3; Glur-6 near waltzer and Jackson circler on chromosome 10; and Glur-7 near clasper on chromosome 4.