Dynorphin inhibits basal forebrain cholinergic neurons by pre- and postsynaptic mechanisms.

KEY POINTS: The basal forebrain is an important component of the ascending arousal system and may be a key site through which the orexin neurons promote arousal. It has long been known that orexin-A and -B excite basal forebrain cholinergic neurons, but orexin-producing neurons also make the inhibitory peptide dynorphin. Using whole-cell recordings in brain slices, we found that dynorphin-A directly inhibits basal forebrain cholinergic neurons via κ-opioid receptors, and decreases afferent excitatory synaptic input to these neurons. While the effects of dynorphin-A and orexin-A desensitize over multiple applications, co-application of dynorphin-A and orexin-A produces a sustained response that reverses depending on the membrane potential of basal forebrain cholinergic neurons. At -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails. ABSTRACT: The basal forebrain (BF) is an essential component of the ascending arousal systems and may be a key site through which the orexin (also known as hypocretin) neurons drive arousal and promote the maintenance of normal wakefulness. All orexin neurons also make dynorphin, and nearly all brain regions innervated by the orexin neurons express kappa opiate receptors, the main receptor for dynorphin. This is remarkable because orexin excites target neurons including BF neurons, but dynorphin has inhibitory effects. We identified the sources of dynorphin input to the magnocellular preoptic nucleus and substantia innominata (MCPO/SI) in mice and determined the effects of dynorphin-A on MCPO/SI cholinergic neurons using patch-clamp recordings in brain slices. We found that the orexin neurons are the main source of dynorphin input to the MCPO/SI region, and dynorphin-A inhibits MCPO/SI cholinergic neurons through κ-opioid receptors by (1) activation of a G protein-coupled inwardly rectifying potassium current, (2) inhibition of a voltage-gated Ca(2+) current and (3) presynaptic depression of the glutamatergic input to these neurons. The responses both to dynorphin-A and to orexin-A desensitize, but co-application of dynorphin-A and orexin-A produces a sustained response. In addition, the polarity of the response to the co-application depends on the membrane potential of BF neurons; at -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails. This suggests that depending on their state of activation, BF cholinergic neurons can be excited or inhibited by signals from the orexin neurons.

Chronic sleep restriction induces long-lasting changes in adenosine and noradrenaline receptor density in the rat brain.

Although chronic sleep restriction frequently produces long-lasting behavioural and physiological impairments in humans, the underlying neural mechanisms are unknown. Here we used a rat model of chronic sleep restriction to investigate the role of brain adenosine and noradrenaline systems, known to regulate sleep and wakefulness, respectively. The density of adenosine A1 and A2a receptors and ß-adrenergic receptors before, during and following 5 days of sleep restriction was assessed with autoradiography. Rats (n = 48) were sleep-deprived for 18 h day(-1) for 5 consecutive days (SR1-SR5), followed by 3 unrestricted recovery sleep days (R1-R3). Brains were collected at the beginning of the light period, which was immediately after the end of sleep deprivation on sleep restriction days. Chronic sleep restriction increased adenosine A1 receptor density significantly in nine of the 13 brain areas analysed with elevations also observed on R3 (+18 to +32%). In contrast, chronic sleep restriction reduced adenosine A2a receptor density significantly in one of the three brain areas analysed (olfactory tubercle which declined 26-31% from SR1 to R1). A decrease in ß-adrenergic receptors density was seen in substantia innominata and ventral pallidum which remained reduced on R3, but no changes were found in the anterior cingulate cortex. These data suggest that chronic sleep restriction can induce long-term changes in the brain adenosine and noradrenaline receptors, which may underlie the long-lasting neurocognitive impairments observed in chronic sleep restriction.

Basal Forebrain Cholinergic Neurons Have Specific Characteristics during the Perinatal Period.

Cholinergic neurons of the basal forebrain represent the main source of cholinergic innervation of large parts of the neocortex and are involved in adults in the modulation of attention, memory, and arousal. During the first postnatal days, they play a crucial role in the development of cortical neurons and cortical cytoarchitecture. However, their characteristics, during this period have not been studied. To understand how they can fulfill this role, we investigated the morphological and electrophysiological maturation of cholinergic neurons of the substantia innominata-nucleus basalis of Meynert (SI/NBM) complex in the perinatal period in mice. We show that cholinergic neurons, whether or not they express gamma-aminobutyric acid (GABA) as a cotransmitter, are already functional at Embryonic Day 18. Until the end of the first postnatal week, they constitute a single population of neurons with a well developed dendritic tree, a spontaneous activity including bursting periods, and a short-latency response to depolarizations (early-firing). They are excited by both their GABAergic and glutamatergic afferents. During the second postnatal week, a second, less excitable, neuronal population emerges, with a longer delay response to depolarizations (late-firing), together with the hyperpolarizing action of GABAA receptor-mediated currents. This classification into early-firing (40%) and late-firing (60%) neurons is again independent of the coexpression of GABAergic markers. These results strongly suggest that during the first postnatal week, the specific properties of developing SI/NBM cholinergic neurons allow them to spontaneously release acetylcholine (ACh), or ACh and GABA, into the developing cortex.

Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain.

The expression of the FMR-1 gene, which is implicated in fragile-X syndrome was investigated in human fetuses by in situ hybridization. In 8 and 9 week-old fetuses, FMR-1 mRNAs are expressed in proliferating and migrating cells of the nervous system, in the retina, and in several non-nervous tissues. In the brain of 25 week-old fetuses, FMR-1 mRNAs are produced in all nearly differenciated structures, with the highest level in cholinergic neurons of the nucleus basalis magnocellularis and in pyramidal neurons of hippocampus. The early transcription of FMR-1 gene and the distribution of FMR-1 mRNAs in human fetuses suggest that alterations of FMR-1 gene expression may contribute to the pathogenesis of fragile-X syndrome and especially the mental retardation.

GABAergic neurons expressing p75 in rat substantia innominata and nucleus basalis.

In vitro findings suggested a role for the p75 neurotrophin receptor in the maturation of GABAergic neurons residing in the basal forebrain (BF), a brain area known to have p75 expression only on cholinergic neurons. We document here the presence of GABAergic neurons which express p75 in the BF in vivo. Colocalization of p75 with the cholinergic marker choline-acetyltransferase (ChAT) and/or the GABAergic marker glutamic acid decarboxylase-67 (GAD67) was investigated in the BF at birth, at two weeks, and in adulthood. A subset of GAD67(+) neurons was p75(+) (p75(+)/GAD67(+)) but ChAT(-) in the substantia innominata and nucleus basalis magnocellularis at birth, whereas all p75(+)/GAD67(+) neurons were also ChAT(+) from two weeks onward. These phenotypic features suggest that a subpopulation of GABAergic neurons could be sensitive to neurotrophins during brain maturation. To unravel this issue, we then pursued a functional analysis by assessing p75 expression profile, and its modulation by nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) in primary BF cell cultures. NGF increased p75 expression exclusively in cholinergic neurons, whereas BDNF induced p75 expression only in a subset of GABAergic neurons (p75(+)/GAD67(+)/ChAT(-)) through a p75- and tyrosine-kinase-dependent mechanism. The latter findings point to a selective role of BDNF in the induction of p75 expression in BF GABAergic neurons. Altogether these results confirm the role of neurotrophins in the developing and mature circuitry of GABAergic neurons in the BF regions.

Buprenorphine disrupts sleep and decreases adenosine concentrations in sleep-regulating brain regions of Sprague Dawley rat.

BACKGROUND: Buprenorphine, a partial µ-opioid receptor agonist and κ-opioid receptor antagonist, is an effective analgesic. The effects of buprenorphine on sleep have not been well characterized. This study tested the hypothesis that an antinociceptive dose of buprenorphine decreases sleep and decreases adenosine concentrations in regions of the basal forebrain and pontine brainstem that regulate sleep. METHODS: Male Sprague Dawley rats were implanted with intravenous catheters and electrodes for recording states of wakefulness and sleep. Buprenorphine (1 mg/kg) was administered systemically via an indwelling catheter and sleep-wake states were recorded for 24 h. In additional rats, buprenorphine was delivered by microdialysis to the pontine reticular formation and substantia innominata of the basal forebrain while adenosine was simultaneously measured. RESULTS: An antinociceptive dose of buprenorphine caused a significant increase in wakefulness (25.2%) and a decrease in nonrapid eye movement sleep (-22.1%) and rapid eye movement sleep (-3.1%). Buprenorphine also increased electroencephalographic delta power during nonrapid eye movement sleep. Coadministration of the sedative-hypnotic eszopiclone diminished the buprenorphine-induced decrease in sleep. Dialysis delivery of buprenorphine significantly decreased adenosine concentrations in the pontine reticular formation (-14.6%) and substantia innominata (-36.7%). Intravenous administration of buprenorphine significantly decreased (-20%) adenosine in the substantia innominata. CONCLUSIONS: Buprenorphine significantly increased time spent awake, decreased nonrapid eye movement sleep, and increased latency to sleep onset. These disruptions in sleep architecture were mitigated by coadministration of the nonbenzodiazepine sedative-hypnotic eszopiclone. The buprenorphine-induced decrease in adenosine concentrations in basal forebrain and pontine reticular formation is consistent with the interpretation that decreasing adenosine in sleep-regulating brain regions is one mechanism by which opioids disrupt sleep.

Cortical cholinergic dysfunction correlates with microglial activation in the substantia innominata in REM sleep behavior disorder.

INTRODUCTION: In vivo PET studies in patients with isolated REM sleep behavior disorder (iRBD) have shown presence of neuroinflammation (microglial activation) in the substantia nigra, and reduced cortical acetylcholinesterase activity, suggestive of cholinergic dysfunction, that was more widespread in patients with poorer cognitive performances. This study aimed to explore whether reduced cortical acetylcholinesterase activity in iRBD is linked to microglial activation in the substantia innominata (SI), the major source of cholinergic input to the cortex. METHODS: We used 11C(R)-PK11195 and 11C-Donepezil PET to assess levels of activated microglia and cholinergic function, respectively, in 19 iRBD patients. 11C(R)-PK11195 binding potential (BPND) and 11C-Donepezil distribution volume ratio (DVR) values were correlated using the Pearson statistic. RESULTS: We found that a lower cortical 11C-Donepezil DVR correlated with a higher 11C(R)-PK11195 BPND in the SI (r = -0.48, p = 0.04). At a voxel level, the strongest negative correlations were found in the frontal and temporal lobes. CONCLUSION: Our results suggest that reduced cortical acetylcholinesterase activity observed in our iRBD patients could be linked to the occurrence of neuroinflammation in the SI. Early modulation of microglial activation might therefore preserve cortical cholinergic functions in these patients.

Selective anterograde tracing of nonserotonergic projections from dorsal raphe nucleus to the basal forebrain and extended amygdala.

The dorsal raphe nucleus (DRN) contains both serotonergic and nonserotonergic projection neurons. Retrograde tracing studies have demonstrated that components of the basal forebrain and extended amygdala are targeted heavily by input from nonserotonergic DRN neurons. The object of this investigation was to examine the terminal distribution of nonserotonergic DRN projections in the basal forebrain and extended amygdala, using a technique that allows selective anterograde tracing of nonserotonergic DRN projections. To trace nonserotonergic DRN projections, animals were pretreated with nomifensine, desipramine and the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), 7 days prior to placing an iontophoretic injection of biotinylated dextran amine (BDA) into the DRN. In animals treated with 5,7-DHT, numerous nonserotonergic BDA-labeled fibers ascended to the basal forebrain in the medial forebrain bundle system. Some of these labeled fibers crossed through the lateral hypothalamus, bed nucleus of the stria terminalis, and substantial innominata. These fibers entered the amygdala through the ansa peduncularis and ramified within the central and basolateral amygdaloid nuclei. Other fibers entered the diagonal band of Broca and formed a dense plexus of labeled fibers in the dorsal half of the intermediate portion of the lateral septal nucleus and the septohippocampal nucleus. These findings demonstrate that the basal forebrain and extended amygdala receive a dense projection from nonserotonergic DRN neurons. Given that the basal forebrain plays a critical role in processes such as motivation, affect, and behavioral control, these findings support the hypothesis that nonserotonergic DRN projections may exert substantial modulatory control over emotional and motivational functions.

Nucleus basalis magnocellularis and substantia innominata corticopetal cholinergic lesions attenuate freezing induced by predator odor.

Previous studies have demonstrated that corticopetal cholinergic lesions applied to the nucleus basalis magnocellularis and substantia innominata (NBM/SI) attenuate operant suppression induced by aversive events. However, these lesions have no effect on open-arm behavior in the elevated plus-maze or changes in startle reactivity induced by bright light. This raises the possibility that NBM/SI corticopetal cholinergic lesions alter operant behavior and/or appetitive state, as opposed to the aversive state operant suppression is supposed to index. To address this concern, the authors documented the effect of NBM/SI corticopetal cholinergic lesions on freezing induced by a component of fox feces (2,4,5-trimethylthiazoline [TMT]), a paradigm that does not involve food deprivation or operant performance. TMT presentation induced freezing behavior, and this effect was attenuated by NBM/SI corticopetal cholinergic lesions. Because predator odor presentation, but not presentation of a predator, induces defense behaviors that are sensitive to anxiolytic drugs, the results of the study suggest that NBM/SI corticopetal cholinergic lesions attenuate anxiety-like states.

GABA receptor agonism in the sublenticular central extended amygdala impairs medial forebrain bundle self-stimulation but GABA blockade does not enhance it.

The sublenticular central extended amygdala (SLEAc), which is important in medial forebrain bundle (MFB) self-stimulation, is heavily populated with GABAergic medium spiny neurons that intercommunicate via local axon collaterals. This study examines the role of GABAergic communication in the SLEAc in MFB self-stimulation. Male Long-Evans rats were given unilateral MFB stimulation electrodes and bilateral injection guide cannulae aimed at the SLEAc. Changes in the stimulation's reward efficacy and in the animals' ability to respond for stimulation following 0.5 microl injections of saline, 50 ng of muscimol (GABA-A agonist), 75 ng of baclofen (GABA-B agonist), 25 ng of bicuculline methiodide (GABA-A antagonist), and 500 ng of phaclofen (GABA-B antagonist) were assessed using the rate-frequency curve shift paradigm. Injecting the receptor agonists ipsi- and contralateral to the stimulation site substantially decreased stimulation reward efficacy and responding. Baclofen's effects were more anatomically specific to the SLEAc and baclofen was less challenging to response rate than was muscimol. Ipsi- and contralateral injections of the receptor blockers and saline had no effect on either stimulation reward efficacy or response rate, although the blockers, especially phaclofen, sometimes enhanced response rate when injected into the globus pallidus or ventral pallidum. Comparing these results to others' observations about GABAergic communication in the basal forebrain suggests that muscimol may have inhibited endemic SLEAc neurons while baclofen may have impaired the release of glutamate into the SLEAc from cortical and amygdalar afferents. These results also suggest functional differences between the two basal forebrain macrostructures of striatopallidum and extended amygdala.

A Central Amygdala-Substantia Innominata Neural Circuitry Encodes Aversive Reinforcement Signals.

Aversive stimuli can impact motivation and support associative learning as reinforcers. However, the neural circuitry underlying the processing of aversive reinforcers has not been elucidated. Here, we report that a subpopulation of central amygdala (CeA) GABAergic neurons expressing protein kinase C-delta (PKC-δ+) displays robust responses to aversive stimuli during negative reinforcement learning. Importantly, projections from PKC-δ+ neurons of the CeA to the substantia innominata (SI) could bi-directionally modulate negative reinforcement learning. Moreover, consistent with the idea that SI-projecting PKC-δ+ neurons of the CeA encode aversive information, optogenetic activation of this pathway produces conditioned place aversion, a behavior prevented by simultaneous ablating of SI glutamatergic neurons. Taken together, our data define a cell-type-specific neural circuitry modulating associative learning by encoding aversive reinforcement signals.

Synaptologic and fine structural features distinguishing a subset of basal forebrain cholinergic neurons embedded in the dense intrinsic fiber network of the caudal extended amygdala.

Cholinergic basal forebrain neurons confined within the intrinsic connections of the extended amygdala in the caudal sublenticular region and anterior amygdaloid area (cSLR/AAA) differ from other basal forebrain cholinergic neurons in several morphological and neurochemical respects. These cSLR/AAA cholinergic neurons have been subjected to additional investigations described in this report. First, fibers traced anterogradely following injections of Phaseolus vulgaris-leucoagglutinin in the central amygdaloid nucleus were shown to contact cSLR/AAA cholinergic neurons and dendrites. Second, these neurons were shown to be contacted by numerous GABAergic boutons with symmetric synaptic specializations. Third, the numbers of synaptic densities of morphologically characterized symmetric contacts on the somata and proximal dendrites of cSLR/AAA cholinergic neurons were shown to significantly exceed those of extra-cSLR/AAA cholinergic neurons. Fourth, fine structural features distinguishing cSLR/AAA cholinergic neurons from other basal forebrain cholinergic neurons were revealed. Specifically, cSLR/AAA cholinergic neurons have less abundant cytoplasm and a less well-organized system of rough endoplasmic reticulum than their counterparts in other parts of the basal forebrain. Thus, morphologically and neurochemically distinct cSLR/AAA cholinergic neurons exhibit robust proximal inhibitory inputs, of which a significant number originate in the extended amygdala, while cholinergic neurons outside this region lack a substrate for strong proximal inhibitory input. The implications of these findings for interaction of fear, anxiety, and attention are considered.

Vesicular glutamate (VGlut), GABA (VGAT), and acetylcholine (VACht) transporters in basal forebrain axon terminals innervating the lateral hypothalamus.

The basal forebrain (BF) is known to play important roles in cortical activation and sleep, which are likely mediated by chemically differentiated cell groups including cholinergic, gamma-aminobutyric acid (GABA)ergic and other unidentified neurons. One important target of these cells is the lateral hypothalamus (LH), which is critical for arousal and the maintenance of wakefulness. To determine whether chemically specific BF neurons provide an innervation to the LH, we employed anterograde transport of 10,000 MW biotinylated dextran amine (BDA) together with immunohistochemical staining of the vesicular transporter proteins (VTPs) for glutamate (VGluT1, -2, and -3), GABA (VGAT), or acetylcholine (ACh, VAChT). In addition, we applied triple staining for the postsynaptic proteins (PSPs), PSD-95 with VGluT or Gephyrin (Geph) with VGAT, to examine whether the BDA-labeled varicosities may form excitatory or inhibitory synapses in the LH. Axons originating from BDA-labeled neurons in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) descended within the medial forebrain bundle and extended collateral varicose fibers to contact LH neurons. In the LH, the BDA-labeled varicosities were immunopositive (+) for VAChT ( approximately 10%), VGluT2 ( approximately 25%), or VGAT ( approximately 50%), revealing an important influence of newly identified glutamatergic together with GABAergic BF inputs. Moreover, in confocal microscopy, VGluT2+ and VGAT+ terminals were apposed to PSD-95+ and Geph+ profiles respectively, indicating that they formed synaptic contacts with LH neurons. The important inputs from glutamatergic and GABAergic BF cells could thus regulate LH neurons in an opposing manner to stimulate vs. suppress cortical activation and behavioral arousal reciprocally.

Adenosine inhibits basal forebrain cholinergic and noncholinergic neurons in vitro.

Adenosine has been proposed as a homeostatic "sleep factor" that promotes the transition from waking to sleep by affecting several sleep-wake regulatory systems. In the basal forebrain, adenosine accumulates during wakefulness and, when locally applied, suppresses neuronal activity and promotes sleep. However, the neuronal phenotype mediating these effects is unknown. We used whole-cell patch-clamp recordings in in vitro rat brain slices to investigate the effect of adenosine on identified cholinergic and noncholinergic neurons of the magnocellular preoptic nucleus and substantia innominata. Adenosine (0.5-100 microM) reduced the magnocellular preoptic nucleus and substantia innominata cholinergic neuronal firing rate by activating an inwardly rectifying potassium current that reversed at -82 mV and was blocked by barium (100 microM). Application of the A1 receptor antagonist 8-cyclo-pentyl-theophylline (200 nM) blocked the effects of adenosine. Adenosine was also tested on two groups of electrophysiologically distinct noncholinergic magnocellular preoptic nucleus and substantia innominata neurons. In the first group adenosine, via activation of postsynaptic A1 receptors, reduced spontaneous firing via inhibition of the hyperpolarization-activated cation current. Blocking the H-current with ZD7288 (20 microM) abolished adenosine effects on these neurons. The second group was not affected by adenosine. These results demonstrate that, in the magnocellular preoptic nucleus and substantia innominata region of the basal forebrain, adenosine inhibits both cholinergic neurons and a subset of noncholinergic neurons. Both of these effects occur via postsynaptic A1 receptors, but are mediated downstream by two separate mechanisms.

Increased brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer's disease.

In the last decade an important role for the progression of neuronal cell death in Alzheimer's disease (AD) has been ascribed to oxidative stress. trans-4-Hydroxy-2-nonenal, a product of lipid peroxidation, forms conjugates with a variety of nucleophilic groups such as thiols or amino moieties. Here we report for the first time the quantitation of glutathione conjugates of trans-4-hydroxy-2-nonenal (HNEGSH) in the human postmortem brain using the specific and very sensitive method of electrospray ionization triple quadrupole mass spectrometry (ESI-MS-MS). Levels of HNEGSH conjugates calculated as the sum of three chromatographically separated diastereomers were determined in hippocampus, entorhinal cortex, substantia innominata, frontal and temporal cortex, as well as cerebellum from patients with AD and controls matched for age, gender, postmortem delay and storage time. Neither age, nor postmortem delay, nor storage time did correlate with levels of HNEGSH conjugates which ranged between 1 and 500 pmol/g fresh weight in the brain areas examined. The brain specimen from patients with clinically and neuropathologically probable AD diagnosed according to criteria of the consortium to establish a registry for AD (CERAD) show increased levels of HNEGSH in the temporal and frontal cortex, as well as in the substantia innominata. Classification of disease severity according to Braak and Braak, which takes into consideration the amount of neurofibrillary tangles and neuritic plaques, revealed highest levels of HNEGSH in the substantia innominata and the hippocampus, two brain regions known to be preferentially affected in AD. These results substantiate the link between conjugates of glutathione with a product of lipid peroxidation and Alzheimer's disease and justify further studies to evaluate the role of HNE metabolites as potential biomarkers for disease progression in AD.

Organization of hypocretin/orexin efferents to locus coeruleus and basal forebrain arousal-related structures.

Hypocretin/orexin neurons give rise to an extensive projection system, portions of which innervate multiple regions associated with the regulation of behavioral state. These regions include the locus coeruleus, medial septal area, medial preoptic area, and substantia innominata. Evidence indicates that hypocretin modulates behavioral state via actions within each of these terminal fields. To understand better the circuitry underlying hypocretin-dependent modulation of behavioral state, the present study characterized the degree to which there exists: 1) lateralization of hypocretin efferents to basal forebrain and brainstem arousal-related regions, 2) topographic organization of basal forebrain- and brainstem-projecting hypocretin neurons, and 3) collateralization of individual hypocretin neurons to these arousal-related terminal fields. These studies utilized combined immunohistochemical identification of hypocretin neurons with single or double retrograde tracing from the locus coeruleus, medial preoptic area, medial septal area, and substantia innominata. Results indicate that approximately 80% of hypocretin efferents to basal forebrain regions project ipsilaterally, whereas projections to the locus coeruleus are more bilateral (65%). There was a slight preference for basal forebrain-projecting hypocretin neurons to be distributed within the medial half of the hypocretin cell group. In contrast, hypocretin neurons projecting to the locus coeruleus were located primarily within the dorsal half of the hypocretin cell group. Finally, a large proportion of hypocretin neurons appear to project simultaneously to at least two of the examined terminal fields. These latter observations suggest coordinated actions of hypocretin across multiple arousal-related regions.

Patterns of afferent projections to the dentate gyrus studied in organotypic co-cultures.

Cholinergic axons originating from the septum form a characteristic layer of preterminal axons and apparent termination in the molecular layer of the hippocampal dentate gyrus. The present study explored the specificity of this characteristic axonal pattern, through the use of organotypic slice co-cultures. Slices of hippocampus were co-cultured with a slice from one of a variety of other potential sources of afferents, and the afferent axons were labeled histochemically or immunocytochemically to determine which afferents distribute within the dentate molecular layer in a pattern similar to that formed by septal cholinergic projections. Acetylcholinesterase (AChE) histochemistry demonstrated that cholinergic axons from septum, substantia innominata, and striatum all consistently targeted the inner molecular layer of the dentate gyrus. AChE-labeled cholinergic axons from dorsal lateral pontine tegmentum and from spinal cord sometimes formed this pattern, while axons from the habenula failed to extend into the dentate gyrus. Immunocytochemically identified monoaminergic axons from the substantia nigra, locus coeruleus, and raphe extended into co-cultured hippocampus; each of these afferent systems displayed a prominent axonal plexus within the hilus of the dentate, but only the raphe axons projected prominently to the molecular layer. These data demonstrate that the molecular layer of the dentate gyrus provides an attractive target zone for some cholinergic and monoaminergic afferents, but not all. Commonalities between neuronal populations that preferentially project to the molecular layer in vitro may offer clues regarding the axon guidance mechanisms that normally direct cholinergic axons to target sites in the dentate gyrus molecular layer.

Dexmedetomidine-induced sedation does not mimic the neurobehavioral phenotypes of sleep in Sprague Dawley rat.

STUDY OBJECTIVES: Dexmedetomidine is used clinically to induce states of sedation that have been described as homologous to nonrapid eye movement (NREM) sleep. A better understanding of the similarities and differences between NREM sleep and dexmedetomidine-induced sedation is essential for efforts to clarify the relationship between these two states. This study tested the hypothesis that dexmedetomidine-induced sedation is homologous to sleep. DESIGN: This study used between-groups and within-groups designs. SETTING: University of Michigan. PARTICIPANTS: Adult male Sprague Dawley rats (n = 40). INTERVENTIONS: Independent variables were administration of dexmedetomidine and saline or Ringer's solution (control). Dependent variables included time spent in states of wakefulness, sleep, and sedation, electroencephalographic (EEG) power, adenosine levels in the substantia innominata (SI), and activation of pCREB and c-Fos in sleep related forebrain regions. MEASUREMENTS AND RESULTS: Dexmedetomidine significantly decreased time spent in wakefulness (-49%), increased duration of sedation (1995%), increased EEG delta power (546%), and eliminated the rapid eye movement (REM) phase of sleep for 16 h. Sedation was followed by a rebound increase in NREM and REM sleep. Systemically administered dexmedetomidine significantly decreased (-39%) SI adenosine levels. Dialysis delivery of dexmedetomidine into SI did not decrease adenosine level. Systemic delivery of dexmedetomidine did not alter c-Fos or pCREB expression in the horizontal diagonal band, or ventrolateral, median, and medial preoptic areas of the hypothalamus. CONCLUSIONS: Dexmedetomidine significantly altered normal sleep phenotypes, and the dexmedetomidine-induced state did not compensate for sleep need. Thus, in the Sprague Dawley rat, dexmedetomidine-induced sedation is characterized by behavioral, electrographic, and immunohistochemical phenotypes that are distinctly different from similar measures obtained during sleep.

Levels of NGF, p75NGFR and ChAT immunoreactivity in brain of adult and aged microencephalic rats.

Methylazoxymethanol (MAM)-induced microencephalic aged animals with reduced cortical mass and unmodified basal nucleus were used to study the relationship between cells that produce and cells that utilize NGF. Total cortical ChAT activity of MAM 2, 19 and 27 month old animals was reduced compared to their age-matched controls. To verify whether the reduction of enzyme activity can be ascribed to changes in or ablation of projecting neurons, we carried out immunohistochemical analysis of ChAT and low affinity NGF receptor (p75NGFR) in the basal nucleus of control and MAM-treated animals. ChAT and p75NGFR immunostaining of basal forebrain cholinergic neurons showed morphological changes in MAM animals, as revealed by cellular atrophy, reduced dendritic arborization and decreased staining intensity. In the cerebral cortex of microencephalic animals, reduced levels of NGF compared to controls were observed at all examined ages. These results suggest that MAM treatment induces long-lasting ablation of cortical NGF-synthesizing cells leading to reduced trophic support to basal forebrain cholinergic neurons, which might be responsible for the cellular atrophy observed in the basal nucleus.

A sex difference and no effect of ApoE type on the amount of cytoskeletal alterations in the nucleus basalis of Meynert in Alzheimer's disease.

In the nucleus basalis of Meynert (NBM) we studied the presence of early cytoskeletal alterations as shown by the antibody Alz-50 in ApoE-typed patients. Using an image analysis system, the area covered by Alz-50 staining and the percentage of neurons stained by Alz-50 were determined. There were no significant differences in the area covered by Alz-50 or in the proportion of Alz-50-stained neurons in the nucleus basalis of Meynert of Alzheimer's disease (AD) patients with one or two ApoE epsilon4 alleles as compared with those without any ApoE e4 allele. However, there was a significant sex difference in Alz-50 staining: female Alzheimer's disease patients showed more severe early cytoskeletal alterations than males. We also found a significant relationship between the number of Alz-50-stained neurons and the severity of dementia.