Copper regulates rest-activity cycles through the locus coeruleus-norepinephrine system.

The unusually high demand for metals in the brain, along with insufficient understanding of how their dysregulation contributes to neurological diseases, motivates the study of how inorganic chemistry influences neural circuitry. We now report that the transition metal copper is essential for regulating rest-activity cycles and arousal. Copper imaging and gene expression analysis in zebrafish identifies the locus coeruleus-norepinephrine (LC-NE) system, a vertebrate-specific neuromodulatory circuit critical for regulating sleep, arousal, attention, memory and emotion, as a copper-enriched unit with high levels of copper transporters CTR1 and ATP7A and the copper enzyme dopamine ß-hydroxylase (DBH) that produces NE. Copper deficiency induced by genetic disruption of ATP7A, which loads copper into DBH, lowers NE levels and hinders LC function as manifested by disruption in rest-activity modulation. Moreover, LC dysfunction caused by copper deficiency from ATP7A disruption can be rescued by restoring synaptic levels of NE, establishing a molecular CTR1-ATP7A-DBH-NE axis for copper-dependent LC function.

Axonal projection-specific differences in somatodendritic α2 autoreceptor function in locus coeruleus neurons.

The locus coeruleus (LC) contains the majority of central noradrenergic neurons sending wide projections throughout the entire CNS. The LC is considered to be essential for multiple key brain functions including arousal, attention and adaptive stress responses as well as higher cognitive functions and memory. Electrophysiological studies of LC neurons have identified several characteristic functional features such as low-frequency pacemaker activity with broad action potentials, transient high-frequency burst discharges in response to salient stimuli and an apparently homogeneous inhibition of firing by activation of somatodendritic α2 autoreceptors (α2AR). While stress-mediated plasticity of the α2AR response has been described, it is currently unclear whether different LC neurons projecting to distinct axonal targets display differences in α2AR function. Using fluorescent beads-mediated retrograde tracing in adult C57Bl6/N mice, we compared the anatomical distributions and functional in vitro properties of identified LC neurons projecting either to medial prefrontal cortex, hippocampus or cerebellum. The functional in vitro analysis of LC neurons confirmed their mostly uniform functional properties regarding action potential generation and pacemaker firing. However, we identified significant differences in tonic and evoked α2AR-mediated responses. While hippocampal-projecting LC neurons were partially inhibited by endogenous levels of norepinephrine and almost completely silenced by application of saturating concentrations of the α2 agonist clonidine, prefrontal-projecting LC neurons were not affected by endogenous levels of norepinephrine and only partially inhibited by saturating concentrations of clonidine. Thus, we identified a limited α2AR control of electrical activity for prefrontal-projecting LC neurons indicative of functional heterogeneity in the LC-noradrenergic system.

Chemogenetic locus coeruleus activation restores reversal learning in a rat model of Alzheimer's disease.

See Grinberg and Heinsen (doi:10.1093/brain/awx261) for a scientific commentary on this article. Clinical evidence suggests that aberrant tau accumulation in the locus coeruleus and noradrenergic dysfunction may be a critical early step in Alzheimer's disease progression. Yet, an accurate preclinical model of these phenotypes that includes early pretangle tau accrual in the locus coeruleus, loss of locus coeruleus innervation and deficits locus coeruleus/norepinephrine modulated behaviours, does not exist, hampering the identification of underlying mechanisms and the development of locus coeruleus-based therapies. Here, a transgenic rat (TgF344-AD) expressing disease-causing mutant amyloid precursor protein (APPsw) and presenilin-1 (PS1ΔE9) was characterized for histological and behavioural signs of locus coeruleus dysfunction reminiscent of mild cognitive impairment/early Alzheimer's disease. In TgF344-AD rats, hyperphosphorylated tau was detected in the locus coeruleus prior to accrual in the medial entorhinal cortex or hippocampus, and tau pathology in the locus coeruleus was negatively correlated with noradrenergic innervation in the medial entorhinal cortex. Likewise, TgF344-AD rats displayed progressive loss of hippocampal norepinephrine levels and locus coeruleus fibres in the medial entorhinal cortex and dentate gyrus, with no frank noradrenergic cell body loss. Cultured mouse locus coeruleus neurons expressing hyperphosphorylation-prone mutant human tau had shorter neurites than control neurons, but similar cell viability, suggesting a causal link between pretangle tau accrual and altered locus coeruleus fibre morphology. TgF344-AD rats had impaired reversal learning in the Morris water maze compared to their wild-type littermates, which was rescued by chemogenetic locus coeruleus activation via designer receptors exclusively activated by designer drugs (DREADDs). Our results indicate that TgF344-AD rats uniquely meet several key criteria for a suitable model of locus coeruleus pathology and dysfunction early in Alzheimer's disease progression, and suggest that a substantial window of opportunity for locus coeruleus/ norepinephrine-based therapeutics exists.

Inorganic mercury in human astrocytes, oligodendrocytes, corticomotoneurons and the locus ceruleus: implications for multiple sclerosis, neurodegenerative disorders and gliomas.

Neurotoxic metals have been implicated in the pathogenesis of multiple sclerosis, neurodegenerative disorders and brain tumours but studies of the location of heavy metals in human brains are rare. In a man who injected himself with metallic mercury the cellular location of mercury in his brain was studied after 5 months of continuous exposure to inorganic mercury arising from metallic mercury deposits in his organs. Paraffin sections from the primary motor and sensory cortices and the locus ceruleus in the pons were stained with autometallography to detect inorganic mercury and combined with glial fibrillary acidic protein immunohistochemistry to identify astrocytes. Inorganic mercury was found in grey matter subpial, interlaminar, protoplasmic and varicose astrocytes, white matter fibrous astrocytes, grey but not white matter oligodendrocytes, corticomotoneurons and some locus ceruleus neurons. In summary, inorganic mercury is taken up by five types of human brain astrocytes, as well as by cortical oligodendrocytes, corticomotoneurons and locus ceruleus neurons. Mercury can induce oxidative stress, stimulate autoimmunity and damage DNA, mitochondria and lipid membranes, so its location in these CNS cells suggests it could play a role in the pathogenesis of multiple sclerosis, neurodegenerative conditions such as Alzheimer's disease and amyotrophic lateral sclerosis, and glial tumours.

Monosynaptic glutamatergic activation of locus coeruleus and other lower brainstem noradrenergic neurons by the C1 cells in mice.

The C1 neurons, located in the rostral ventrolateral medulla (VLM), are activated by pain, hypotension, hypoglycemia, hypoxia, and infection, as well as by psychological stress. Prior work has highlighted the ability of these neurons to increase sympathetic tone, hence peripheral catecholamine release, probably via their direct excitatory projections to sympathetic preganglionic neurons. In this study, we use channelrhodopsin-2 (ChR2) optogenetics to test whether the C1 cells are also capable of broadly activating the brain's noradrenergic system. We selectively expressed ChR2(H134R) in rostral VLM catecholaminergic neurons by injecting Cre-dependent adeno-associated viral vectors into the brain of adult dopamine-ß-hydroxylase (DßH)(Cre/0) mice. Most ChR2-expressing VLM neurons (75%) were immunoreactive for phenylethanolamine N-methyl transferease, thus were C1 cells, and most of the ChR2-positive axonal varicosities were immunoreactive for vesicular glutamate transporter-2 (78%). We produced light microscopic evidence that the axons of rostral VLM (RVLM) catecholaminergic neurons contact locus coeruleus, A1, and A2 noradrenergic neurons, and ultrastructural evidence that these contacts represent asymmetric synapses. Using optogenetics in tissue slices, we show that RVLM catecholaminergic neurons activate the locus coeruleus as well as A1 and A2 noradrenergic neurons monosynaptically by releasing glutamate. In conclusion, activation of RVLM catecholaminergic neurons, predominantly C1 cells, by somatic or psychological stresses has the potential to increase the firing of both peripheral and central noradrenergic neurons.

Reduction of the nitro group to amine by hydroiodic acid to synthesize o-aminophenol derivatives as putative degradative markers of neuromelanin.

Neuromelanin (NM) is produced in dopaminergic neurons of the substantia nigra (SN) and in noradrenergic neurons of the locus coeruleus (LC). The synthesis of NM in those neurons is a component of brain aging and there is the evidence that this pigment can be involved in the pathogenesis of neurodegenerative diseases such as Parkinson's disease. NM is believed to derive from the oxidative polymerization of dopamine (DA) or norepinephrine (NE) with the participation of cysteine, dolichols and proteins. However, there are still unknown aspects in the chemical structure of NM from SN (SN-NM) and LC (LC-NM). In this study, we designed a new method to synthesize o-aminophenol compounds as putative degradation products of catecholamines and their metabolites which may be incorporated into NM. Those compounds are aminohydroxyphenylethylamine (AHPEA) isomers, aminohydroxyphenylacetic acid (AHPAA) isomers and aminohydroxyethylbenzene (AHEB) isomers, which are expected to arise from DA or NE, 3,4-dihydroxyphenylacetic acid (DOPAC) or 3,4-dihydroxyphenylmandelic acid (DOMA) and 3,4-dihydroxyphenylethanol (DOPE) or 3,4-dihydroxyphenylethyleneglycol (DOPEG), respectively. These o-aminophenol compounds were synthesized by the nitration of phenol derivatives followed by reduction with hydroiodic acid (HI), and they could be identified by HPLC in HI hydrolysates of SN-NM and LC-NM. This degradative approach by HI hydrolysis allows the identification of catecholic precursors unique to SN-NM and LC-NM, which are present in catecholaminergic neurons.

Structural basis for noradrenergic regulation of neural circuits in the mouse olfactory bulb.

Olfactory input is processed in the glomerulus of the main olfactory bulb (OB) and relayed to higher centers in the brain by projection neurons. Conversely, centrifugal inputs from other brain regions project to the OB. We have previously analyzed centrifugal inputs into the OB from several brain regions using single-neuron labeling. In this study, we analyzed the centrifugal noradrenergic (NA) fibers derived from the locus coeruleus (LC), because their projection pathways and synaptic connections in the OB have not been clarified in detail. We analyzed the NA centrifugal projections by single-neuron labeling and immunoelectron microscopy. Individual NA neurons labeled by viral infection were three-dimensionally traced using Neurolucida software to visualize the projection pathway from the LC to the OB. Also, centrifugal NA fibers were visualized using an antibody for noradrenaline transporter (NET). NET immunoreactive (-ir) fibers contained many varicosities and synaptic vesicles. Furthermore, electron tomography demonstrated that NET-ir fibers formed asymmetrical synapses of varied morphology. Although these synapses were present at varicosities, the density of synapses was relatively low throughout the OB. The maximal density of synapses was found in the external plexiform layer; about 17% of all observed varicosities contained synapses. These results strongly suggest that NA-containing fibers in the OB release NA from both varicosities and synapses to influence the activities of OB neurons. The present study provides a morphological basis for olfactory modulation by centrifugal NA fibers derived from the LC.

Glutamate signaling proteins and tyrosine hydroxylase in the locus coeruleus of alcoholics.

It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

Imprinted Nesp55 influences behavioral reactivity to novel environments.

Genomic imprinting results in parent-of-origin-dependent monoallelic expression of selected genes. Although their importance in development and physiology is recognized, few imprinted genes have been investigated for their effects on brain function. Gnas is a complex imprinted locus whose gene products are involved in early postnatal adaptations and neuroendocrine functions. Gnas encodes the stimulatory G-protein subunit Gsalpha and two other imprinted protein-coding transcripts. Of these, the Nesp transcript, expressed exclusively from the maternal allele, codes for neuroendocrine secretory protein 55 (Nesp55), a chromogranin-like polypeptide associated with the constitutive secretory pathway but with an unknown function. Nesp is expressed in restricted brain nuclei, suggesting an involvement in specific behaviors. We have generated a knockout of Nesp55 in mice. Nesp55-deficient mice develop normally, excluding a role of this protein in the severe postnatal effects associated with imprinting of the Gnas cluster. Behavioral analysis of adult Nesp55 mutants revealed, in three separate tasks, abnormal reactivity to novel environments independent of general locomotor activity and anxiety. This phenotype may be related to prominent Nesp55 expression in the noradrenergic locus coeruleus. These results indicate a role of maternally expressed Nesp55 in controlling exploratory behavior and are the first demonstration that imprinted genes affect such a fundamental behavior.

Visualization of neuromelanin in the Substantia nigra and locus ceruleus at 1.5T using a 3D-gradient echo sequence with magnetization transfer contrast.

Recent technical advances have enabled the visualization of neuromelanin in the substantia nigra pars compacta (SNc) and locus ceruleus (LC) by 3-tesla (T) magnetic resonance imaging in vivo. In the present study, we successfully detected neuromelanin in the SNc and LC of 6 healthy volunteers at 1.5T using a 3D gradient echo sequence with off-resonance magnetization transfer contrast.

Compensatory changes in the noradrenergic nervous system in the locus ceruleus and hippocampus of postmortem subjects with Alzheimer's disease and dementia with Lewy bodies.

In Alzheimer's disease (AD), there is a significant loss of locus ceruleus (LC) noradrenergic neurons. However, functional and anatomical evidence indicates that the remaining noradrenergic neurons may be compensating for the loss. Because the noradrenergic system plays an important role in learning and memory, it is important to determine whether compensation occurs in noradrenergic neurons in the LC and hippocampus of subjects with AD or a related dementing disorder, dementia with Lewy bodies (DLB). We observed profound neuronal loss in the LC in AD and DLB subjects with three major changes in the noradrenergic system consistent with compensation: (1) an increase in tyrosine hydroxylase (TH) mRNA expression in the remaining neurons; (2) sprouting of dendrites into peri-LC dendritic zone, as determined by alpha2-adrenoreceptors (ARs) and norepinephrine transporter binding sites; and (3) sprouting of axonal projections to the hippocampus as determined by alpha2-ARs. In AD and DLB subjects, the postsynaptic alpha1-ARs were normal to elevated. Expression of alpha1A- and alpha2A-AR mRNA in the hippocampus of AD and DLB subjects were not altered, but expression of alpha1D- and alpha2C-AR mRNA was significantly reduced in the hippocampus of AD and DLB subjects. Therefore, in AD and DLB subjects, there is compensation occurring in the remaining noradrenergic neurons, but there does appear to be a loss of specific AR in the hippocampus. Because changes in these noradrenergic markers in AD versus DLB subjects were similar (except neuronal loss and the increase in TH mRNA were somewhat greater in DLB subjects), the presence of Lewy bodies in addition to plaques and tangles in DLB subjects does not appear to further affect the noradrenergic compensatory changes.

Effects of (-)-epigallocatechin gallate on the development of morphine-induced physical dependence.

Among the various nervous systems and signaling components involved in the development of morphine withdrawal symptoms, sensitization of the brain dopaminergic nervous system and an increase in the cAMP levels in the locus coeruleus are believed to be the most important cellular events. This study tested the effects of (-)-epigallocatechin gallate (EGCG), a major compound of green tea, on the development of morphine-induced withdrawal symptoms. All the naloxone-precipitated withdrawal symptoms in morphine-dependent animals were inhibited by an EGCG pretreatment in a dose-dependent manner, being forepaw tremor, rearing, teeth chattering, urination, and wet dog shake were more sensitive than jumping and ptosis. In addition, EGCG showed moderate inhibitory effects on the morphine-induced increase in the cAMP levels in the locus coeruleus at 100 mg/kg and the signaling of the dopamine D2 receptor at 100 microM. Effects of EGCG on the sequestration of D2 receptor were inconclusive. These results suggest that EGCG has strong pharmacological activity against the development of morphine dependence, which can be partly explained by its inhibitory effects on the morphine-induced increase in the cAMP levels in the locus coeruleus and the signaling of the dopamine D2 receptor.

Modular organization of the brainstem noradrenaline system coordinates opposing learning states.

Noradrenaline modulates global brain states and diverse behaviors through what is traditionally believed to be a homogeneous cell population in the brainstem locus coeruleus (LC). However, it is unclear how LC coordinates disparate behavioral functions. We report a modular LC organization in rats, endowed with distinct neural projection patterns and coding properties for flexible specification of opposing behavioral learning states. LC projection mapping revealed functionally distinct cell modules with specific anatomical connectivity. An amygdala-projecting ensemble promoted aversive learning, while an independent medial prefrontal cortex-projecting ensemble extinguished aversive responses to enable flexible behavior. LC neurons displayed context-dependent inter-relationships, with moderate, discrete activation of distinct cell populations by fear or safety cues and robust, global recruitment of most cells by strong aversive stimuli. These results demonstrate a modular organization in LC in which combinatorial activation modes are coordinated with projection- and behavior-specific cell populations, enabling adaptive tuning of emotional responding and behavioral flexibility.

Locus coeruleus: From global projection system to adaptive regulation of behavior.

The brainstem nucleus locus coeruleus (LC) is a major source of norepinephrine (NE) projections throughout the CNS. This important property was masked in very early studies by the inability to visualize endogenous monoamines. The development of monoamine histofluorescence methods by Swedish scientists led to a plethora of studies, including a paper published in Brain Research by Loizou in 1969. That paper was highly cited (making it a focal point for the 50th anniversary issue of this journal), and helped to spark a large and continuing set of investigations to further refine our understating of the LC-NE system and its contribution to brain function and behavior. This paper very briefly reviews the ensuing advances in anatomical, physiological and behavioral aspects of the LC-NE system. Although its projections are ubiquitously present throughout the CNS, recent studies find surprising specificity within the organizational and operational domains of LC neurons. These and other findings lead us to expect that future work will unmask additional features of the LC-NE system and its roles in normative and pathological brain and behavioral processes. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

GABA(A) receptor epsilon and theta subunits display unusual structural variation between species and are enriched in the rat locus ceruleus.

Previously, GABA(A) receptor epsilon and theta subunits have been identified only in human. Here, we describe properties of the epsilon and theta subunit genes from mouse and rat that reveal an unusually high level of divergence from their human homologs. In addition to a low level of amino acid sequence conservation ( approximately 70%), the rodent epsilon subunit cDNAs encode a unique Pro/Glx motif of approximately 400 residues within the N-terminal extracellular domain of the subunits. Transcripts of the rat epsilon subunit were detected in brain and heart, whereas the mouse theta subunit mRNA was detectable in brain, lung, and spleen by Northern blot analysis. In situ hybridization revealed a particularly strong signal for both subunit mRNAs in rat locus ceruleus in which expression was detectable from the first postnatal day. Lower levels of coexpression were also detected in other brainstem nuclei and in the hypothalamus. However, the expression pattern of theta subunit mRNA was more widespread than that of epsilon subunit, being found also in the cerebral cortex of rat pups. In contrast to primate brain, neither subunit was expressed in the hippocampus or substantia nigra. The results indicate that GABA(A) receptor epsilon and theta subunits are evolving at a much faster rate than other known GABA(A) receptor subunits and that their expression patterns and functional properties may differ significantly between species.

Do substance P and the NK1 receptor have a role in depression and anxiety?

Research on Substance P (SP) has, until recently, focused on its role in pain and inflammation. However, a report that NK(1) receptor antagonists have utility in the treatment of depression has stimulated research into the function of SP and the NK(1)receptor in anxiety and depression. The distribution of SP and the NK(1) receptor in brain areas implicated in anxiety and depression is initially reviewed. This is followed by evaluation of the preclinical data obtained for SP and NK(1) receptor antagonists in behavioral models of depression as well as the phenotype of genetically modified animals lacking the genes encoding for the NK(1) receptor or for SP. The weight of the evidence supports antidepressant and anxiolytic activity of NK(1) receptor antagonists. However, many of the studies do not control for nonspecific effects of the compounds, and when enantiomers that lack activity at the NK(1) receptor are included, the results, in some cases, suggest that blockade of NK(1) receptors does not account for the observed behavioral activity. Finally, clinical studies in depressed patients assessing SP levels in plasma and cerebrospinal fluid as well as the effect of NK(1) receptor antagonists are reviewed. The clinical studies are a mixture of positive, failed and negative studies on the antidepressant activity of NK(1) receptor antagonists, not unlike the early clinical results obtained with selective serotonin reuptake inhibitors.

Effects of long-term cigarette smoking on the human locus coeruleus.

BACKGROUND: It has been hypothesized that cigarette smoking among subjects with major depression is a form of self-medication. To explore a possible biological basis for this hypothesis, noradrenergic proteins in the locus coeruleus (LC) were measured in long-term cigarette smokers and in nonsmokers. The LC was studied because elevated amounts of alpha2-adrenoceptors and tyrosine hydroxylase have been observed postmortem in the LCs of subjects with major depression or who commit suicide, and because long-term administration of antidepressant drugs to rats down-regulates these proteins in the LC. METHODS: Postmortem LCs were obtained from long-term cigarette smokers (n=7) and from nonsmokers (n = 9), all of whom lacked diagnoses of major depression. Amounts of tyrosine hydroxylase immunoreactivity and radioligand binding to the norepinephrine transporter, monoamine oxidase A, and alpha2-adrenoceptors were measured. RESULTS: Amounts of tyrosine hydroxylase immunoreactivity and radioligand binding to alpha2-adrenoceptors were significantly lower (approximately 60% and 40%, respectively) along the axis of the LCs of long-term smokers compared with nonsmokers. Smoking had no statistically significant effects on binding to monoamine oxidase A or to the norepinephrine transporter. CONCLUSION: This is the first demonstration that cigarette smoking affects noradrenergic proteins in the LC. The direction of these changes is opposite to that observed when comparing subjects who have major depression with normal controls and the same as that produced by long-term antidepressant treatment in animals. If the present observations reflect long-term effects of smoking on premortem noradrenergic biochemistry, smoking-induced changes in LC biochemistry may strengthen the smoking habit among subjects with major depression.

CRH Engagement of the Locus Coeruleus Noradrenergic System Mediates Stress-Induced Anxiety.

The locus coeruleus noradrenergic (LC-NE) system is one of the first systems engaged following a stressful event. While numerous groups have demonstrated that LC-NE neurons are activated by many different stressors, the underlying neural circuitry and the role of this activity in generating stress-induced anxiety has not been elucidated. Using a combination of in vivo chemogenetics, optogenetics, and retrograde tracing, we determine that increased tonic activity of the LC-NE system is necessary and sufficient for stress-induced anxiety and aversion. Selective inhibition of LC-NE neurons during stress prevents subsequent anxiety-like behavior. Exogenously increasing tonic, but not phasic, activity of LC-NE neurons is alone sufficient for anxiety-like and aversive behavior. Furthermore, endogenous corticotropin-releasing hormone(+) (CRH(+)) LC inputs from the amygdala increase tonic LC activity, inducing anxiety-like behaviors. These studies position the LC-NE system as a critical mediator of acute stress-induced anxiety and offer a potential intervention for preventing stress-related affective disorders.

Unbiased estimation of neuronal numbers in the human nucleus coeruleus during aging.

The total number of the neuromelanin-containing neurons of the nucleus coeruleus was determined by means of a newly developed unbiased stereological counting scheme and a low-cost apparative set-up. The individuals (n = 20, age from 49-98 years) included in this study were carefully examined for absence of neurological or psychiatric disorders. However, minor Alzheimer's disease-related neurofibrillary changes occurred in some of the individuals of higher age and these changes were staged. The mean number of neurons per side of the nucleus coeruleus was 15,731 +/- 3,408 SD with a range from 11,737 to 25,319. In three individuals, we compared the left and right nuclei and did not observe significant side differences between the numbers of neurons. There was no correlation between the age of the individuals and the cell number. Also, no correlation was detected between the cell number and the staged occurrence of minor neurofibrillary changes of the Alzheimer type. Due to the novel counting method, the determination of the total cell number took less than 2 h per case.

Spatial, temporal and numeric analysis of Alzheimer changes in the nucleus coeruleus.

The distribution of neurofibrillary tangles in the nucleus coeruleus was topographically and quantitatively analyzed. The topographical analysis showed statistically significant differences with regard to the distribution of neurofibrillary tangles in the dorsal-ventral and medial-lateral axes. More neurofibrillary tangles were found to be located in the dorsal and medial regions than in ventral and lateral areas. No significant difference in neurofibrillary tangle content was found between the rostral and the caudal areas of the nucleus coeruleus. Neurofibrillary tangle formation begins in the central parts of the nucleus coeruleus. The total number of neuromelanized neurons in the nucleus coeruleus was determined using a modern, unbiased sampling scheme and related to the cortical stage of Alzheimer's disease-related neurofibrillary changes present. A statistically significant reduction (50%) in nucleus coeruleus neurons was evident only in cases meeting the histopathological criteria for Alzheimer's disease. The extent of reduction in the total number of neurons in the nucleus coeruleus did not correlate with the number of neurofibrillary tangles observed. Our data suggest that despite the relatively early susceptibility of the nucleus coeruleus to neurofibrillary tangle formation, significant neuronal loss appears to occur much later, with an estimated average delay time of at least 25 years. Nonetheless, comparison of the topographical pattern of neurofibrillary tangle formation and cell loss indicates that neuronal loss is tangle-related.