References

Khasabov SG, Fliss PM, Rao AS, Simone DA (2010) NK-1 Receptors in the RVM: Involvement in hyperalgesia produced by naloxone but not in morphine analgesia. Neuroscience 2010 Abstracts 678.15/QQ2. Society for Neuroscience, San Diego, CA.

Summary: The rostral ventromedial medulla (RVM) is a crucial supraspinal site for opioid analgesia. Descending modulation of nociceptive transmission by the RVM can be antinociceptive, which is associated with increased activity of OFF cells, or pronociceptive, which is related to activation of ON cells. Analgesia produced by opioids at the RVM level is due to direct inhibition of ON cells and the indirect increase in discharge of OFF cells. A subpopulation of neurons in the RVM (approximately 7%) express neurokinin-1 receptors (NK-1R), which are receptors for substance P (SP). We have shown that NK-1R in the RVM are located primarily on ON cells and contribute to descending facilitation of nociception. We suggest that elimination of NK-1R expressing neurons by the specific saporin toxin conjugate SSP-SAP, will reduce the number of ON cells and thereby decrease descending facilitation without affecting antinociception associated with activity of OFF cells. We therefore determined the contribution of NK-1R expressing neurons in the RVM to changes in nocifensive behaviors produced by morphine or the opioid receptor antagonist naloxone by eliminating NK-1R expressing neurons. Adult male Sprague Dawley rats were pretreated with injection of SSP-SAP (1 µM/0.5 µl) or inactive toxin into the RVM. Ablation of NK-1R possessing neurons was determined histologically and did not alter tale flick or paw withdrawal latencies to heat for up to 4 weeks following treatment, indicating that these neurons do not modulate acute nociception. Morphine (30 µg/0.5 µl) injected into the RVM of control rats or rats pretreated with SSP-SAP increased tail flick latencies approximately 133.5 ± 20.8% and 140.4 ± 8.3%, respectively. The increase in paw withdrawal latency following morphine was also similar between groups. However, injection of naloxone (50 µg/0.5 µl) in control rats decreased tail flick latencies for 90 min with a maximal reduction of 32.2 ± 4.1%, whereas in rats treated with SSP-SAP latencies decreased by 17.8 ± 4.9% and for only 30 min. A similar pattern of effects was found on paw withdrawal latencies to heat. These data support the notion that ON cells possess NK-1Rs and contribute to facilitation of nociceptive transmission.

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Ferrini F, Mattioli TAM, Lorenzo L-E, Godin A, Wiseman PW, Ribeiro-Da-Silva A, Cahill CM, Milne B, De Koninck Y (2010) Morphine-induced pain hypersensitivity, but not opioid tolerance, depends on microglia-mediated alteration of Cl- homeostasis in spinal dorsal horn. Neuroscience 2010 Abstracts 678.9/PP14. Society for Neuroscience, San Diego, CA.

Summary: Prolonged morphine exposure leads to a reduction of the antinociceptive effect (opioid tolerance) and to an increase in pain sensitivity. Recent evidences suggest that these side effects share similar mechanisms with those underlying neuropathic pain. We have shown that the release of BDNF by activated microglia following peripheral nerve injury causes a decrease in KCC2 activity in the spinal dorsal horn (DH) and weakens Cl−-mediated inhibition through GABAA and glycine receptors. Here, we tested the hypothesis that a similar cascade of events underlies morphine-induced pain hypersensitivity. Adult rats, receiving either morphine (10mg/Kg s.c. twice a day) or saline, were tested for nociceptive thresholds prior to and 1 h after morphine injections each day. Morphine induced tolerance within 2 days and hyperalgesia within 5 days. The hyperalgesia, but not the tolerance, was reversed by intrathecal (i.t.) administration of the anti-mac1 saporin-conjugated antibody (an immunotoxin targeted against microglia) or a TrkB blocking antibody, confirming involvement of both microglia and BDNF in the morphine-dependent hyperalgesia. Microglial activation was confirmed by an increased OX-42 staining after chronic morphine and was blocked by i.t. (-)-naloxone, as well as by (+)-naloxone. Interestingly, (+)-naloxone, while prevented microglia activation, had little effect on morphine tolerance. After 7 days of treatment, rats were sacrificed and DH lamina I-II neurons were recorded by imposing a Cl- load (29 mM). A depolarizing shift in EGABA was observed in lamina I neurons from morphine-treated rats (-42 ± 1 mV, n=6) compared to controls (-50 ± 2 mV, n=5, P<0.05) indicating a weaker Cl- extrusion capacity in these cells. A similar effect was also observed following 3h in vitro incubation of spinal cord slices with morphine (1 μM). No change in EGABA was observed either in the presence of opioid receptor antagonists or the TrkB blocking antibody, confirming the involvement of BDNF in the morphine-signalling pathway. Interestingly, morphine did not produce any change in EGABA in lamina II neurons. To confirm the participation of altered Cl- homeostasis on morphine-induced hyperalgesia in vivo, we administered the carbonic anhydrase inhibitor acetazolamide (i.t.) to minimize the bicarbonate-mediated component of GABAA/glycine currents. Acetazolamide was sufficient to restore inhibition in spinal DH neurons and to reverse the morphine-dependent hyperalgesia. Our data suggest that microglial activation and BDNF release following chronic morphine treatment may alter Cl- extrusion capacity of spinal lamina I neurons and increase pain hypersensitivity.

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Burke PG, Neale J, Korim WS, Mcmullan S, Pilowsky PM, Goodchild AK (2010) Patterning of somatosympathetic reflexes: Identification of distinct bulbospinal sympathoexcitatory RVLM projections by conduction velocity and catecholamine phenotype. Neuroscience 2010 Abstracts 694.11/HHH34. Society for Neuroscience, San Diego, CA.

Summary: The aim of this study was to examine the somatosympathetic reflex (SSR) response of different sympathetic nerves to identify distinct projections of presympathetic vasomotor RVLM neurons by axonal conduction and catecholamine phenotype. All experiments were conducted in urethane-anaesthetised (1.3 g/kg ip), paralysed, vagotomised and artificially ventilated Sprague Dawley rats (n = 44). First, we determined the simultaneous activity of dorsal root potentials and the splanchnic SSR to single shock sciatic nerve (SN) stimulation (single 0.2 ms pulse, 50 sweeps at 0.5-1 Hz, 1-80 V, n=4). Second, we simultaneously recorded the sympathoexcitatory response of multiple, sympathetic nerves (cervical, renal, splanchnic and lumbar) to low (A-fibre afferent; 4-10 V) and high (A- and C-fibre afferents; +40 V) SN stimulation (n=19). Third, we examined the cervical or splanchnic SSR to low intensity SN stimulation in rats following RVLM microinjection of somatostatin (SST) or muscimol (n=8). Fourth, we examined the splanchnic SSR in rats pretreated with intraspinal anti-dopamine-beta-hydroxylase-saporin (anti-DβH-SAP; 24 ng/side, n=8), a neurotoxin that depleted ~70% of catecholamine (C1) neurons in the RVLM compared to IgG-saporin control (n=5). Low intensity SN stimulation evoked biphasic responses in the renal, splanchnic and lumbar nerves but a single peak in the cervical nerve. High intensity SN stimulation evoked triphasic responses in the renal, splanchnic and lumbar nerves and a biphasic cervical response. RVLM injections of SST abolished the early peak of the cervical and splanchnic SSR. Intraspinal pretreatment with anti-DβH-SAP eliminated the late peak of the splanchnic SSR and attenuated the first peak. It is concluded that the mono- or bi-phasic SSR responses are generated by A-fibre afferent inputs driving two classes of bulbospinal sympathoexcitatory RVLM neurons with myelinated or unmyelinated axonal conduction. Secondly, unmyelinated RVLM presympathetic neurons, presumed to be all C1, innervate splanchnic, renal and lumbar SPN, whereas myelinated C1 and non-C1 neurons innervate all sympathetic outflow examined. These findings extend prior evidence that the RVLM expresses several types of phenotypically distinct descending sympathoexcitatory pathways.

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Gibbs RB, Chipman AM, Nelson D (2010) Donepezil enhances effects of estradiol on DMP acquisition in rats with partial loss of septal cholinergic neurons. Neuroscience 2010 Abstracts 710.23/NNN23. Society for Neuroscience, San Diego, CA.

Summary: We hypothesize that effects of estradiol on cognitive performance decrease in association with decreased basal forebrain cholinergic function, and that this accounts for the loss of estradiol effect with age and time post-menopause. In the present study, we tested the feasibility of using donepezil, a cholinesterase inhibitor commonly used to treat Alzheimer’s disease, to enhance beneficial effects of estradiol on cognitive performance in rats with septal cholinergic lesions. Young adult, ovariectomized rats received intraseptal injections of 192IgG-saporin or vehicle. Two weeks later, rats started receiving daily injections of donepezil (Don, 5 mg/Kg/day, i.p.) or vehicle. A week later, rats received either silastic capsules containing 17ß-estradiol (E) or empty capsules, implanted s.c. Rats were then trained on a delayed matching-to-position (DMP) T-maze task. Upon completion, brains were collected and sections through the medial septum were processed for detection of choline acetyltransferase (ChAT). The severity of the cholinergic lesions was ranked on a 5-point scale by estimating the loss of ChAT-positive cells in the septum. Eighty-one rats completed the study. Lesions produced a decline in performance that correlated with the severity of cholinergic cell loss (F[4,76]=10.0, p<0.0001). In rats with >50% loss of septal cholinergic neurons, treating with E and/or Don had no significant effect on the rate of DMP acquisition (F[3,36]=0.90, p=0.45). In rats with <50% loss of septal cholinergic neurons, treating with the combination of Don+E significantly increased the rate of acquisition relative to controls (p<0.05 by Tukey test), and reduced deficits associated with increasing the intertrial delay (F[9, 111]=2.30, p=0.02 for Delay x Tx interaction). Treating with Don or E alone had no significant effect, although E alone produced a strong trend toward improvement. Cholinergic lesions also increased the likelihood that rats would adopt a persistent turn χ2=13.3, p=0.0003), and treatment with Don+E reduced this effect (χ2=8.8, p=0.03). These findings demonstrate that cholinergic dysfunction produces a learning impairment as well as a loss of estrogen effect on cognitive performance which can be attenuated by treating with a cholinesterase inhibitor. The findings also indicate a critical threshold for cholinergic function below which combined therapy is not effective. We propose that combining donepezil with estrogen therapy in postmenopausal women may offer significant cognitive benefits, particularly in relatively healthy older women showing early signs of cognitive impairment or Alzheimer’s disease.

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Li A-J, Wang Q, Dinh TT, Ritter S (2010) Hindbrain catecholamine neurons are required for rapid switching of metabolic substrate utilization during glucoprivation. Neuroscience 2010 Abstracts 392.14/III1. Society for Neuroscience, San Diego, CA.

Summary: Glucoprivation is a metabolic emergency in which a rapid and effective system-wide switch to fat metabolism must occur to conserve any available glucose for use by the brain. Glucoprivation stimulates secretion of corticosterone, which is known to play an important role in promoting fat utilization. In previously published work, we showed that injections of the retrogradely transported catecholamine neuron immunotoxin, anti-dopamine beta-hydroxylase conjugated to saporin (DSAP) into the paraventricular nucleus of the hypothalamus (PVH) eliminate the corticosterone response to glucoprivation without impairing the response to a nonmetabolic stressor (swim stress), without altering the circadian rhythm of corticosterone secretion and without damaging the PVH CRF-secreting neurons. Here we microinjected DSAP into the PVH to selectively lesion hindbrain catecholamine neurons innervating this site, thus impairing the glucoprivation-induced corticosterone response. Using indirect calorimetry, we examined metabolic fuel utilization and other metabolic parameters in these lesioned rats under basal and glucoprivic conditions. Under basal conditions, energy expenditure and locomotor activity did not differ between DSAP rats and controls injected with unconjugated saporin (SAP). However, DSAP rats had a higher respiratory exchange ratio (RER) than SAPs, indicating their greater dependence on carbohydrate utilization. Glucoprivation induced by 2-deoxy-D-glucose (2DG, 250 mg/kg) reduced energy expenditure equally in SAP and DSAP rats. However, 2DG rapidly decreased RER to 0.8 (a value indicating ongoing fat metabolism) in the SAP group, but not in the DSAP group. Responses to 2DG persisted for about 6 hours. Adrenal dennervation, which eliminates the adrenal medullary response to glucoprivation, did not alter these responses to 2DG in either SAP or DSAP rats. Results indicate that in the absence of hypothalamically-projecting hindbrain catecholamine neurons, rats cannot efficiently switch their fuel utilization from carbohydrate to fat during glucoprivation, presumably due to a deficient corticosterone response. Results also suggest a previously unrecognized role for these catecholamine neurons in control of basal substrate utilization.

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Li A-J, Dinh TT, Wang Q, Wiater MF, Ritter S (2010) Leptin-saporin lesion of hypothalamic arcuate neurons impairs circadian feeding rhythms. Neuroscience 2010 Abstracts 498.6/III29. Society for Neuroscience, San Diego, CA.

Summary: To examine the role of leptin receptor-expressing neurons in the arcuate nucleus (Arc) in circadian control of spontaneous feeding and energy expenditure, we injected a novel targeted toxin, leptin conjugated to saporin (Lep-SAP) into the Arc in rats. Lep-SAP effectively lesioned Arc neurons in a leptin-receptor dependent manner, indicated by an 80% reduction of agouti gene-related protein- or melanocyte-stimulating hormone-immunoreactive neurons in Sprague Dawley rats, but not in leptin receptor deficient Zucker fa/fa rats. Food intake and metabolism were monitored 3-5 weeks after Arc Lep-SAP and control blank-saporin (B-SAP) injections using an Oxymax system. Lep-SAP rats consumed 49% of their total daily intake during the day, compared to 34% in B-SAP rats. Eatograms (feeding actograms), cosinar analysis and Chi-square periodograms of continuous feeding records failed to detect a circadian oscillation in the feeding patterns of Lep-SAP rats, but did detect significant circadian rhythms in B-SAP controls. Unlike feeding, metabolic rate, respiratory exchange ratio and locomotor activity continued to exhibit significant circadian periodicity in both groups, though dampened in amplitude in Lep-SAPs, suggesting that rhythms of feeding and metabolism may be controlled by separate mechanisms. Expression of clock-related genes (Per1 and Bmal1) in hypothalamus, liver and white fat tissue was asynchronous in Lep-SAP rats. These results suggest that leptin-receptive neurons in the Arc exert a critical influence on the circadian patterning of food intake.

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St Peters MM, Bruno JP, Sarter M (2010) Mesolimbic-basal forebrain circuitry mediating the motivational activation of attention. Neuroscience 2010 Abstracts 506.12/LLL52. Society for Neuroscience, San Diego, CA.

Summary: Prefrontal circuitry mediating cue detection is modulated by the tonic component of cholinergic activity. Performance-associated increases in tonic cholinergic activity are augmented by demands on the cognitive control of attention. Highest levels of tonic cholinergic activity are observed while animals perform below baseline as a result of, for example, a distractor, but while they remain motivated to stabilize and recover attentional performance. Cortico-mesolimbic-basal forebrain circuitry is thought to mediate such motivated activation of attentional performance. We previously observed that stimulation of ionotropic glutamate receptors in the shell of the nucleus accumbens (NAs) stimulates tonic cholinergic activity in the prefrontal cortex. Here we test the hypothesis that such stimulation benefits attentional performance while distractors evoke cognitive control. Rats were trained in an operant sustained attention task (SAT) before undergoing surgery for implantation of a bilateral guide cannula targeting the NAs or, in separate animals, the core of the NA (NAc). NMDA (0.01-0.15 µg/0.5 µL/hemisphere) or vehicle (0.9% saline) was infused bilaterally into task-performing animals during SAT and the more challenging distractor version (dSAT). For the dSAT, the operant chamber ceiling lights flashed on/off at 0.5 Hz during the middle block of three blocks of trials that constituted a session. NMDA infusions in the NAs, but not into the NAc, significantly improved the animals’ attentional performance in the presence of the distractor. These findings are consistent with the hypothesis that activation of the NAs mediates attentional performance under conditions that require top-down control. The next set of experiments determined whether the effects of NAs activation require the cortical cholinergic system. We infused the immunotoxin 192 IgG saporin into prefrontal or parietal regions, in addition to implantation of guide cannula targeting the NAs. Replicating the initial finding, NAs NMDA infusions enhanced dSAT performance. Both PFC and PPC cholinergic deafferentation prevented this effect of NMDA. These findings suggest that the motivated activation of the cholinergic attention system during demands on top-down control modulates fronto-parietal attention networks to optimize attentional performance.

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Savage ST, Olson L, Mattsson A (2010) Novel object recognition and social interaction in rats lacking cortical cholinergic innervation; comparing manual and digital video tracking systems. Neuroscience 2010 Abstracts 506.9/LLL49. Society for Neuroscience, San Diego, CA.

Summary: Alterations in cholinergic signaling in the brain have been implicated as a contributing factor in the pathogenesis of schizophrenia. We have shown that cholinergic denervation of cortex cerebri by stereotaxic infusion of the immunotoxin 192 IgG-saporin into nucleus basalis magnocellularis in adult rats leads to an enhanced locomotor sensitivity to amphetamine, as well as, a potentiated dopamine release in nucleus accumbens. We have also shown that this cortical cholinergic denervation leads to an increased locomotor response to the NMDA receptor antagonist phencyclidine (PCP), suggesting that disruption of cortical cholinergic activity can lead to disturbances of glutamatergic transmission. We hypothesize that this loss of cortical cholinergic input alters the activity of cortical glutamatergic neurons and in turn, their regulation of subcortical dopamine neurons. In current studies we are investigating memory functions using the novel object recognition task (NOR) and social interaction in adult male Lister hooded rats with cholinergic denervation of neocortex. The behavioral tasks are being conducted under normal conditions and with a PCP-challenge. The data are analyzed both manually by a trained observer, and with a nose point digital video tracking system (Clever Sys Inc.). Manually scoring behavioral data requires extensive observer training, is subject to inter-observer variability, and is time consuming. An automated tracking system could potentially improve upon these issues, however is prone to other problems, including the difficulty of accurately tracking multiple body points. Furthermore, the Lister hooded fur has two different colors which proves difficult for computerized systems to accurately determine the body points. A comparison of the manual scoring and the computerized tracking system is being conducted to determine the most reliable method for each behavioral task. Preliminary results indicate that the cholinergically denervated rats performed the NOR task under normal conditions as well as the controls, however failed to show a preference for the novel object under PCP-challenge. These results were obtained through analysis with both the manual and automated system. Despite fur color difficulties, the video tracking system was able to analyze the NOR task and accurately calculate the distance traveled, which is not easily obtained through manual scoring. These initial results indicate that cortical cholinergic deficits, in addition to a potentiation of the locomotor response to PCP, can also lead to an enhanced sensitivity to PCP-induced cognitive impairments.

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Liu Y, Krencik R, Liu H, Ma L, Zhang X, Zhang S-C (2010) Functional cholinergic neurons from human embryonic stem cells. Neuroscience 2010 Abstracts 331.5/B19. Society for Neuroscience, San Diego, CA.

Summary: Basal forebrain cholinergic neurons play a critical role in regulating memory and cognition. Degeneration or dysfunction of these neurons is associated with neurological conditions including Alzheimer’s disease and dementia. In this study, we aimed at generating cholinergic neurons from human embryonic stem cells (hESCs) for therapeutic development. hESCs were first differentiated to primitive neuroepithelial cells in a chemically defined medium. In the presence of sonic hedgehog, over 97% of the differentiated cells became Nkx2.1-expressing ventral forebrain progenitors. These ventral progenitors further differentiated to cholinergic neurons with basal forebrain characteristics by expressing ChAT, VAChAT, FoxG1, Nkx2.1, Islet1, ßIII-tubulin, MAP2, P75, Synapsin but not GABA, Glutamate, or Mash2. The hESC-generated cholinergic neurons were electrophysiologically active in vitro. Following transplantation into the hippocampus of mice, in which cholinergic neurons in the medial septum were destroyed by IgG-P75-saporin, the grafted human cells produced large cholinergic neurons. The animals transplanted with cholinergic neurons demonstrated an improvement in learning and memory deficit. These results indicate that the human stem cell-generated cholinergic neurons are functional, thus providing a new source for drug discovery and cell therapy for neurological disorders that affect cholinergic neurons.

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ATS Poster of the Year Winner

Rennie KE, Frechette M, Pappas BA (2010) The effects of neonatal cholinergic lesion on age-related changes in behaviour, neurogenesis and CA1 pyramidal cell morphology. Neuroscience 2010 Abstracts 349.8/J12. Society for Neuroscience, San Diego, CA.

Summary: Age-related cognitive decline is associated with dysfunction of the basal forebrain cholinergic (BFC) system, and cortico-hippocampal cholinergic denervation is a hallmark neurochemical feature of the Alzheimer’s-afflicted brain. It has been suggested that cognitive deficits that emerge with age may be rooted in early dysfunction of the BFC system and that impaired cholinergic transmission might interact with ageing-associated factors to produce cognitive decline. The purpose of this study was to examine the effects of neonatal cholinergic lesion on age-related changes in spatial working memory, neurogenesis and hippocampal CA1 pyramidal cell morphology. We have previously reported that neonatal cholinergic lesion results in only minor behavioural deficits, but impairs the birth and/or survival of new neurons and reduces CA1 dendritic complexity in the young adult rat. We hypothesized that memory impairments would become apparent in lesioned rats as they age, and that this impairment would be accompanied by more drastic reductions in neurogenesis and cytoarchitectural alterations than those that have been documented in the young adult animal after neonatal cholinergic lesion. Seven-day-old male Sprague-Dawley rats were subjected to basal forebrain cholinergic lesion by infusion of the cholinotoxin 192-IgG-Saporin into the lateral ventricles. At the age of 12 or 21 months, the rats were tested on a working memory version of the Morris water maze. While aging had only a slight effect on the memory performance of control rats, lesioned rats showed pronounced memory impairments with age. This occurred without CA1 cell loss or astrogliosis in 21-month-old lesioned rats when compared to age-matched controls. However, golgi analysis revealed that while cholinergic lesion did not alter the total dendritic length, branching, number of spines, or spine density of CA1 pyramidal cells in 21-month-old rats, the distribution of these parameters across branch orders was shifted. The lesion caused a slight reduction in apical branch length and spine density, and basal branch number, length and number of spines at low/middle branch orders, but increased these parameters at upper branch orders. Thus, perinatal cholinergic lesion precipitates spatial memory dysfunction during old age, and this seems to be associated with cytoarchitectural changes to neurons rather than neuronal loss.

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