Cytotoxic screening and antibacterial activity of Withaferin A.

Cancer and bacterial infections are among the leading causes of death worldwide. Plant-derived bioactive compounds constitute promising alternatives for development of new therapeutics. This study aimed at evaluating the biological activity of Withaferin A using 6 tumor cell lines: A549 (lung cancer), U87MG (glioblastoma), SH-SY5Y (neuroblastoma), B16-F10 (mouse melanoma), HeLa (uterine colon cancer) and K562 (chronic myeloid leukemia). In addition, 17 other standard bacterial strains and several multidrug resistant bacteria (MDR) clinical isolates were examined. Cell viability was assessed using the following assays: MTT, neutral red, and dsDNA PicoGreen®. Further, oxidative stress was measured by quantification of reactive oxygen species (ROS) production. The activity against bacteria was determined by the minimum inhibitory concentration (MIC), minimum bacterial concentration (CBM) and antibiofilm activity in the production of strains. Withaferin A was effective, as evidenced by its cytotoxic activity in tumor cell lines, enhanced ROS production in tumor cells and bactericidal and antibiofilm activity. Data demonstrated that Withaferin A may be therapeutically considered as an antitumor and antibacterial agent.

Oxidation-Induced Increase In Photoreactivity of Bovine Retinal Lipid Extract.

The mammalian retina contains a high level of polyunsaturated fatty acids, including docosahexaenoic acid (22:6) (DHA), which are highly susceptible to oxidation. It has been shown that one of the products of DHA oxidation-carboxyethylpyrrole (CEP), generated in situ, causes modifications of retinal proteins and induces inflammation response in the outer retina. These contributing factors may play a role in the development of age-related macular degeneration (AMD). It is also possible that some of the lipid oxidation products are photoreactive, and upon irradiation with blue light may generate reactive oxygen species. Therefore, in this work we analysed oxidation-induced changes in photoreactivity of lipids extracted from bovine neural retinas. Lipid composition of bovine neural retinas closely resembles that of human retinas making the bovine tissue a convenient model for studying the photoreactivity and potential phototoxicity of oxidized human retinal lipids. Lipid composition of bovine neural retinas Folch' extracts (BRex) was determined by gas chromatography (GC) and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS) analysis. Liposomes prepared from BRex, equilibrated with air, were oxidized in the dark at 37 °C for up to 400 h. The photoreactivity of BRex at different stages of oxidation was studied by EPR-oximetry and EPR-spin trapping. Photogeneration of singlet oxygen (1O2, 1Δg) by BRex was measured using time-resolved detection of the characteristic phosphorescence at 1270 nm. To establish contribution of lipid components to the analysed photoreactivity of Folch' extract of bovine retinas, a mixture of selected synthetic lipids in percent by weight (w/w %) ratio resembling that of the BRex has been also studied. Folch's extraction of bovine neural retinas was very susceptible to oxidation despite the presence of powerful endogenous antioxidants such as α-tocopherol and zeaxanthin. Non-oxidized and oxidized BRex photogenerated singlet oxygen with moderate quantum yield. Blue-light induced generation of superoxide anion by Folch' extract of bovine neural retinas strongly depended on the oxidation time. The observed photoreactivity of the studied extract gradually increased during its in vitro oxidation.

A randomized phase II trial of ridaforolimus, dalotuzumab, and exemestane compared with ridaforolimus and exemestane in patients with advanced breast cancer.

PURPOSE: To evaluate whether adding humanized monoclonal insulin growth factor-1 receptor (IGF-1R) antibody (dalotuzumab) to mammalian target of rapamycin (mTOR) inhibitor (ridaforolimus) plus aromatase inhibitor (exemestane) improves outcomes in patients with estrogen receptor (ER)-positive advanced/metastatic breast cancer. METHODS: This randomized, open-label, phase II trial enrolled 80 postmenopausal women with high-proliferation (Ki67 index staining ≥15%), ER-positive breast cancer that progressed after a non-steroidal aromatase inhibitor (NCT01605396). Randomly assigned patients were given oral ridaforolimus 10 mg QD 5 ×/week, intravenous dalotuzumab 10 mg/kg/week, and oral exemestane 25 mg/day (R/D/E, n = 40), or ridaforolimus 30 mg QD 5 ×/week and exemestane 25 mg/day (R/E; n = 40). Primary end point was progression-free survival (PFS). RESULTS: Median PFS was 23.3 weeks for R/D/E versus 31.9 weeks for R/E (hazard ratio 1.18; 80% CI 0.81-1.72; P = 0.565). Grade 3-5 adverse events were reported in 67.5% of patients in the R/E arm and 59.0% in the R/D/E arm. Stomatitis (95.0 vs. 76.9%; P = 0.021) and pneumonitis (22.5 vs. 5.1%; P = 0.027) occurred more frequently in the R/E than the R/D/E arm; hyperglycemia (27.5 vs. 28.2%) occurred at a similar rate. CONCLUSIONS: R/D/E did not improve PFS compared with R/E. Because the PFS reported for R/E was similar to that reported for everolimus plus exemestane in patients with advanced breast cancer, it is possible that lower-dose ridaforolimus in the R/D/E arm (from overlapping toxicities with IGF1R inhibitor) contributed to lack of improved PFS.

A phase II study of combined ridaforolimus and dalotuzumab compared with exemestane in patients with estrogen receptor-positive breast cancer.

PURPOSE: Combining the mTOR inhibitor ridaforolimus and the anti-IGFR antibody dalotuzumab demonstrated antitumor activity, including partial responses, in estrogen receptor (ER)-positive advanced breast cancer, especially in high proliferation tumors (Ki67 > 15%). METHODS: This randomized, multicenter, international, phase II study enrolled postmenopausal women with advanced ER-positive breast cancer previously treated with a nonsteroidal aromatase inhibitor (NCT01234857). Patients were randomized to either oral ridaforolimus 30 mg daily for 5 of 7 days (once daily [qd] × 5 days/week) plus intravenous dalotuzumab 10 mg/kg/week or oral exemestane 25 mg/day, and stratified by Ki67 status. Due to a high incidence of stomatitis in the ridaforolimus-dalotuzumab group, two sequential, nonrandomized, reduced-dose cohorts were explored with ridaforolimus 20 and 10 mg qd × 5 days/week. The primary endpoint was progression-free survival (PFS). RESULTS: Median PFS was 21.4 weeks for ridaforolimus 30 mg qd × 5 days/week plus dalotuzumab 10 mg/kg (n = 29) and 24.3 weeks for exemestane (n = 33; hazard ratio = 1.00; P = 0.5). Overall survival and objective response rates were similar between treatment arms. The incidence of drug-related, nonserious, and serious adverse events was higher with ridaforolimus/dalotuzumab (any ridaforolimus dose) than with exemestane. Lowering the ridaforolimus dose reduced the incidence of grade 3 stomatitis, but overall toxicity remained higher than acceptable at all doses without improved efficacy. CONCLUSIONS: The combination of ridaforolimus plus dalotuzumab was no more effective than exemestane in patients with advanced ER-positive breast cancer, and the incidence of adverse events was higher. Therefore, the combination is not being further pursued.

Comparison of skeletal muscle strength between cardiac patients and age-matched healthy controls.

The purpose of the present study was to compare muscular strength of knee extensors and arm flexor muscles of cardiac patients (n = 638) and healthy controls (n = 961) in different age groups. Isometric torques were measured in a sitting position with the elbow, hip, and knee flexed to 90(0). For statistical analysis, age groups were pooled in decades from the age of 30 to 90 years. Additionally, the influence of physical lifestyle prior to disease on muscular strength was obtained in the patients. For statistical analysis three-way ANOVA (factors age, gender, and physical activity level) was used.Both in patients and in controls a significant age-dependent decline in maximal torque could be observed for arm flexors and knee extensors. Maximal leg extensor muscle showed statistically significant differences between healthy controls and cardiac patients as well as between subgroups of patients: Physically inactive patients showed lowest torques (male: 148 +/- 18 Nm; female: 82 +/- 25 Nm) while highest values were measured in control subjects (male: 167 +/- 16 Nm; female: 93 +/- 17 Nm). In contrast, arm flexor muscles did not show any significant influence of health status or sports history.This qualitative difference between weight-bearing leg muscles and the muscle group of the upper extremity suggest that lower skeletal muscle strength in heart patients is mainly a consequence of selective disuse of leg muscles rather than any pathological skeletal muscle metabolism. Since a certain level of skeletal muscle strength is a prerequisite to cope with everyday activities, strength training is recommended as an important part of cardiac rehabilitation.

Epstein-Barr virus is associated with grey matter atrophy in multiple sclerosis.

OBJECTIVE: The aim of the study was to determine whether the presence of anti-Epstein-Barr virus (EBV) antibodies is associated with MRI measures of brain injury and neurodegeneration in patients with multiple sclerosis (MS). METHODS: 135 patients with MS (86 women, 49 men) underwent brain MRI and testing for antibodies against EBV. MRI measurements included gadolinium enhancing lesion volume, T1 and T2 lesion volumes and fractions of whole brain parenchyma (BPF), white matter and grey matter (GMF). The anti-EBV panel included anti-EBV early antigen IgG, anti-EBV nuclear antigen IgG and anti-EBV viral capsid antigen (VCA) IgG levels. The relationships between antibody levels and MRI measurements were assessed in regression analysis. Repeat measurements of anti-EBV VCA IgG and MRI measures were available for a subset of 50 patients after a mean follow-up of 3.1 years. RESULTS: GMF (R(2) = 0.24 for overall model, p = 0.002) and BPF (R(2) = 0.39 for overall model, p<0.001) showed negative associations with anti-EBV-VCA IgG levels. A greater decline in BPF over 3 years was significantly associated with increased 3 years prior time point anti-EBV VCA IgG levels (p<0.001). CONCLUSIONS: The results suggest that the presence of anti-EBV antibodies is associated with MRI markers of GM atrophy in MS and with increased loss of brain volume over 3 years.

Excitatory action of hypocretin/orexin on neurons of the central medial amygdala.

The neurons of the lateral hypothalamus that contain hypocretin/orexin (hcrt/orx) are thought to promote arousal through the excitatory action they exert on the multiple areas to which they project within the CNS. We show here that the hcrt/orx peptides can also exert a strong action on the amygdala, a structure known for its implication in emotional aspects of behavior. Indeed, the hcrt/orx peptides, applied in acute rat brain slices, excite a specific class of "low threshold burst" neurons in the central medial (CeM) nucleus which is considered as a major output of the amygdala. These excitatory effects are postsynaptic, mediated by Hcrt2/OX2 receptors and result from the closure of a potassium conductance. They occur on a class of neurons that are also excited by vasopressin acting through V1a receptors. These results suggest that the hcrt/orx system can act through the amygdala to augment arousal and evoke the autonomic and behavioral responses associated with fear, stress or emotion.

Opposite effects of noradrenaline and acetylcholine upon hypocretin/orexin versus melanin concentrating hormone neurons in rat hypothalamic slices.

Hypocretin/orexin (Hcrt/Orx) and melanin concentrating hormone (MCH) are peptides contained in overlapping cell groups of the lateral hypothalamus and commonly involved in regulating sleep-wake states and energy balance, though likely in different ways. To see if these neurons are similarly or differentially modulated by neurotransmitters of the major brainstem arousal systems, the effects of noradrenaline (NA) and carbachol, a cholinergic agonist, were examined on identified Hcrt/Orx and MCH neurons in rat hypothalamic slices. Whereas both agonists depolarized and excited Hcrt/Orx neurons, they both hyperpolarized MCH neurons by direct postsynaptic actions. According to the activity profiles of the noradrenergic locus coeruleus and cholinergic pontomesencephalic neurons across the sleep-waking cycle, the Hcrt/Orx neurons would be excited by NA and acetylcholine (ACh) and thus active during arousal, whereas the MCH neurons would be inhibited by NA and ACh and thus inactive during arousal while disinhibited and possibly active during slow wave sleep. According to the present pharmacological results, Hcrt/Orx neurons may thus stimulate arousal in tandem with other arousal systems, whereas MCH neurons may function in opposition with other arousal systems and thus potentially dampen arousal to promote sleep.

Early vancomycin-resistant enterococcus (VRE) bacteremia after allogeneic bone marrow transplantation is associated with a rapidly deteriorating clinical course.

Vancomycin-resistant enterococcal (VRE) infection is a growing threat. We studied the incidence, risk factors, and clinical course of early-onset VRE bacteremia in allogeneic hematopoietic stem cell transplant recipients. We carried out a chart review of 281 allogeneic hematopoietic stem cell transplant recipients from 1997-2003, including preparative regimen, diagnosis, status of disease, graft-versus-host disease prophylaxis, antimicrobial therapy, and survival. VRE bacteremia developed in 12/281 (4.3%) recipients; 10 (3.6%) were within 21 days of transplant. Diagnoses were acute leukemia (7), NHL (2), and MDS (1). In all, 70% had refractory/relapsed disease; 30% were in remission. In total, 50% had circulating blasts. Nine of 10 had matched unrelated donors (7/9 with CD8+ T-cell depletion). The average time to positive VRE cultures was 15 days; average WBC was 0.05, and 80% had concomitant infections. Despite treatment, all patients died within 73 days of VRE bacteremia. Intra-abdominal complications were common. Causes of death included bacterial or fungal infection, multiorgan failure, VOD, ARDS, and relapse. A total of 60% of patients engrafted neutrophils, but none engrafted platelets. Early VRE bacteremia after allogeneic bone marrow transplant is associated with a rapidly deteriorating clinical course, although not always directly due to VRE. Early VRE may be a marker for the critical condition of these high-risk patients at the time of transplant.

Orexins/hypocretins excite basal forebrain cholinergic neurones.

The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identified neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998) which are importantly implicated in the control of wakefulness (for reviews see Hungs and Mignot, 2001; van den Pol, 2000; Willie et al., 2001 ). Indeed, alteration in these peptides' precursor, their receptors or the hypothalamic neurones that produce them leads to the sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Thannickal et al., 2000). The mechanisms by which the orexins modulate wakefulness, however, are still unclear. Their presence in fibres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might influence the important sleep and waking neural systems situated there (Jones, 2000). The present study, performed in rat brain slices, demonstrates, however, that the orexins have no effect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory effect on the cholinergic neurones of the contiguous BF. In addition, by comparing the effects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX(2)), which are those lacking in narcoleptic dogs (Lin et al., 1999). These results suggest that the orexins excite cholinergic neurones that release acetylcholine in the cerebral cortex and thereby contribute to the cortical activation associated with wakefulness.

Orexins (hypocretins) directly excite tuberomammillary neurons.

Wakefulness has recently been shown to depend upon the newly identified orexin (or hypocretin) neuropeptides by the findings that alteration in their precursor protein, their receptors or the neurons that produce them leads to the sleep disorder narcolepsy in both animals and humans. The questions of how and where these brain peptides act to maintain wakefulness remain unresolved. The purpose of the present study was to determine whether the orexins could directly affect hypothalamic histaminergic neurons, which are known to contribute to the state of wakefulness by their diffuse projections through the brain. Using brain slices, we recorded in the ventral tuberomammillary nuclei from neurons identified as histaminergic on the basis of their previously described morphological and electrophysiological characteristics and found that they were depolarized and excited by the orexins through a direct postsynaptic action. We then compared the depolarizing effect of orexin A and B and found that they were equally effective upon these cells. This latter finding suggests that the effect of orexins is mediated by orexin type 2 receptors, which are those lacking in narcoleptic dogs. Our results therefore show that the histaminergic neurons of the tuberomammillary nuclei represent an important target for the orexin system in the maintenance of wakefulness.

Identification of sleep-promoting neurons in vitro.

The neurons responsible for the onset of sleep are thought to be located in the preoptic area and more specifically, in the ventrolateral preoptic nucleus (VLPO). Here we identify sleep-promoting neurons in vitro and show that they represent an homogeneous population of cells that must be inhibited by systems of arousal during the waking state. We find that two-thirds of the VLPO neurons are multipolar triangular cells that show a low-threshold spike. This proportion matches that of cells active during sleep in the same region. We then show, using single-cell reverse transcriptase followed by polymerase chain reaction, that these neurons probably contain gamma-aminobutyric acid (GABA). We also show that these neurons are inhibited by noradrenaline and acetylcholine, both of which are transmitters of wakefulness. As most of these cells are also inhibited by serotonin but unaffected by histamine, their overall inhibition by transmitters of wakefulness is in agreement with their relative inactivity during waking with respect to sleep. We propose that the reciprocal inhibitory interaction of such VLPO neurons with the noradrenergic, serotoninergic and cholinergic waking systems to which they project is a key factor for promoting sleep.

Post-lesional plasticity in the central nervous system of the guinea-pig: a "top-down" adaptation process?

Vestibular compensation for the postural and oculomotor deficits following unilateral labyrinthectomy is a model of functional plasticity in the brain of adult vertebrates. The mechanisms involved in this recovery are still controversial. The post-lesional lack of vestibular input might be compensated by changes in the efficacy of the remaining sensory inputs involved in gaze and posture stabilization. However, the compensation process could also rapidly become independent of these external cues, and thus be detectable in vitro in preparations obtained from lesioned animals. In agreement with this hypothesis, we have shown recently that prominent traces of the compensation process appeared three days after the lesion on in vitro isolated brains taken from labyrinthectomized guinea-pigs, where the connectivity of the central vestibular-related networks is preserved. We report here that, one week after the lesion, a slight increase in the intrinsic, spontaneous activity of the deafferented, central vestibular neurons was found in brainstem slices. This increase became stronger in slices taken after one month of compensation, and was associated at this stage with a significant decrease in the intrinsic activity of the vestibular neurons on the contralesional side. Vestibular compensation could thus follow a "top-down" strategy: it would first rely on the external cues given by the intact sensory systems, then on an internal reorganization of the vestibular-related networks, and finally on changes in the intrinsic properties of the vestibular neurons themselves. Similar strategies may be used by the mammalian brain to compensate for other types of deafferentations or environmental changes.

Plastic changes underlying vestibular compensation in the guinea-pig persist in isolated, in vitro whole brain preparations.

Vestibular compensation for the postural and oculomotor deficits induced by unilateral labyrinthectomy is a model of post-lesional plasticity in the central nervous system. Just after the removal of one labyrinth, the deafferented, ipsilateral vestibular nucleus neurons are almost silent, and the discharge of the contralateral vestibular nucleus neurons is increased. The associated static disorders disappear in a few days, as normal activity is restored in both vestibular nuclei. In this study, we searched for traces of vestibular compensation in isolated whole brains taken from adult guinea-pigs. The electrophysiological responses evoked in control brains were compared to those evoked in brains taken from animals that had previously been labyrinthectomized. Guinea-pigs compensated for an initial labyrinthectomy within three days. In vivo, subsequent deafferentation of vestibular nucleus neurons on the intact side triggered "Bechterew's phenomenon": a new postural and oculomotor syndrome appeared, similar to the one induced by the first lesion, but directed to the newly deafferented side. These disturbances would be caused by the new imbalance between the discharges of neurons in the two vestibular nuclei triggered by the second deafferentation. Experiments were designed to search for a similar imbalance in vitro in brains taken from labyrinthectomized animals, where the intact vestibular nerve is cut during the dissection. Isolated whole brains were obtained from young guinea-pigs at various times (one to seven days) following an initial labyrinthectomy. An imbalance between the resting activities of medial vestibular nucleus neurons on both sides of the brainstem was revealed in brains taken more than three days after the lesion: their discharge was higher on the compensated, initially lesioned side than on the newly deafferented side. In some cases, an oscillatory pattern of discharge, reminiscent of the spontaneous nystagmus associated in vivo with Bechterew's syndrome, appeared in both abducens nerves. These data demonstrate that most of the changes underlying vestibular compensation persist, and can thus be investigated in the isolated whole brain preparation. Brains removed only one day after the lesion displayed normal commissural responses and symmetric spinal inputs to vestibular nucleus neurons. However, an unusually large proportion of the neurons recorded on both sides of the preparation had very irregular spontaneous discharge rates. These data suggest that the first stages of vestibular compensation might be associated with transient changes in the membrane properties of vestibular nucleus neurons. Brains taken from compensated animals displayed a significant, bilateral decrease of the inhibitory commissural responses evoked in the medial vestibular nucleus by single-shock stimulation of the contralateral vestibular nerve. The sensitivity of abducens motoneurons on the initially lesioned, compensated side to synaptic activation from the contralesional vestibular nucleus neurons was also decreased. Both changes may explain the long-term, bilateral decrease of vestibular-related reflexes observed following unilateral labyrinthectomy. Spinal inputs to vestibular nucleus neurons became progressively asymmetric: their efficacy was increased on the lesioned side and decreased on the intact one. This last modification may support a functional substitution of the deficient, vestibular-related synergies involved in gaze and posture stabilization by neck-related reflexes.

Neuronal correlates of vestibulo-ocular reflex adaptation in the alert guinea-pig.

The spiking behaviour of 66 second-order vestibular neurons was studied in alert, chronically prepared guinea-pigs during the horizontal vestibulo-ocular reflex (VOR). Among the 66 studied neurons, seven were held long enough (> 1 h) to compare their spiking behaviour before and after a training procedure inducing a decrease in the gain of the VOR. When tested in darkness following adaptation, five of them showed a significant decrease of their sensitivity to head rotation. However, the resting discharge of these five neurons remained unchanged. This suggests that VOR adaptation is mediated not only by changes in synaptic efficacities but also by modifications in the spike generator which transforms synaptic inputs into a pattern of action potentials.

The serotonin inhibition of high-voltage-activated calcium currents is relieved by action potential-like depolarizations in dissociated cholinergic nucleus basalis neurons of the guinea-pig.

The aim of the present study was to investigate whether the voltage-dependent inhibition of calcium currents by serotonin 5-HT1A agonists can be alleviated (facilitated) by action potential-like depolarizations. In dissociated cholinergic basal forebrain neurons using whole-cell recordings, it is shown that a selective serotonin 5-HT1A agonist (8-OH-DPAT) predominantly blocks N-type HVA calcium current, although a minor reduction of P-type current was also observed. The inhibition may principally occur through Gi-Go subtypes of G-proteins because it was prevented by N-ethylmaleimide, a substance known to block specifically pertussis-sensitive G-proteins. The inhibitory effect of 8-OH-DPAT on calcium currents is voltage-dependent because it was alleviated by long-lasting depolarizing prepulses. Interestingly, the inhibition could also be reversed by prepulses made-up of action potential-like depolarizations that were given at a frequency of 200 Hz. This observation may have important implications during periods of high-frequency rhythmic bursts, a firing pattern that is prevalent in cholinergic basal forebrain neurons.

GABAergic input to cholinergic nucleus basalis neurons.

The potential influence of GABAergic input to cholinergic basalis neurons was studied in guinea-pig basal forebrain slices. GABA and its agonists were applied to electrophysiologically-identified cholinergic neurons, of which some were labelled with biocytin and confirmed to be choline acetyltransferase-immunoreactive. Immunohistochemistry for glutamate decarboxylase was also performed in some slices and revealed GABAergic varicosities in the vicinity of the biocytin-filled soma and dendrites of electrophysiologically-identified cholinergic cells. From rest (average - 63 mV), the cholinergic cells were depolarized by GABA. The depolarization was associated with a decrease in membrane resistance and diminution in firing. The effect was mimicked by muscimol, the specific agonist for GABA(A) receptors, and not by baclofen, the specific agonist for GABA(B) receptors, which had no discernible effect. The GABA- and muscimol-evoked depolarization and decrease in resistance were found to be postsynaptic since they persisted in the presence of solutions containing either high Mg2+/low Ca2+ or tetrodotoxin. They were confirmed as being mediated by a GABA(A) receptor, since they were antagonized by bicuculline. The reversal potential for the muscimol effect was estimated to be approximately -45 mV, which was -15 mV above the resting membrane potential. Finally, in some cholinergic cells, spontaneous subthreshold depolarizing synaptic potentials (average 5 mV in amplitude), which were rarely associated with action potentials, were recorded and found to persist in the presence of glutamate receptor antagonists but to be eliminated by bicuculline. These results suggest that GABAergic input may be depolarizing, yet predominantly inhibitory to cholinergic basalis neurons.

Pharmacological characterization and differentiation of non-cholinergic nucleus basalis neurons in vitro.

Using intracellular recordings in guinea pig brain slices, the pharmacology of electrophysiologically identified and immunohistochemically confirmed non-cholinergic nucleus basalis neurons was studied to determine their response to the major neurotransmitters of the subcortical afferents to this region. The cells were differentiated into three types: Type A cells (approximately 44%) were depolarized by noradrenaline (NA) and muscarine, Type B cells (approximately 23%) were depolarized by NA but hyperpolarized by muscarine, and Type C cells (approximately 15%) were hyperpolarized by both agonists. These cell types were also differentially responsive to serotonin (hyperpolarizing B, C) and histamine (depolarizing A, B). Accordingly, the non-cholinergic neurons share certain discharge properties but appear nonetheless to comprise distinct types which respond differentially to the major modulatory neurotransmitters and thus play potentially different roles in cortical modulation across the sleep-wake cycle.