References

Jeong D, Lee D, Chang J (2010) Decrease of Arc protein expression and delay of memory acquisition by immunolesion. Neuroscience 2010 Abstracts 145.5/H6. Society for Neuroscience, San Diego, CA.

Summary: Cholinergic neuronal deficit is one of the common characteristics in both Alzheimer’s disease dementia (AD) and vascular dementia (VaD). Forebrain Cholinergic neurons in the basal forebrain project to the neocortex and the hippocampus which make an important role in memory function. We used 192 IgG-saporin to produce selective lesion of cholinergic basal forebrain neurons including the medial septum (MS) and the nucleus basalis magnocellularis (NBM). We intracerebroventricularly injected 192 IgG-saporin (0.63 µg/µl dose, 6 µl, 8 µl and 10 µl) or phosphate buffered saline (8 µl). Morris water maze and tissue perforation for immunohistochemistry and western blotting were sequentially performed 2 weeks after injection of 192 IgG-saporin. In the acquisition phase of Morris water maze, latency of 6ul group (2nd day), 8 µl group (2nd day) and 10 µl group (3rd day) was significantly delayed but it was recovered within 1week. Time in platform and the number of crossing were significantly different between 8 µl LV injection group and sham group in probe test. In immunohistological study, the extent of the cholinergic lesion was showed in the basal forebrain complex region of all 192 IgG-saporin injected rats. Expression of Arc protein is significantly decreased in the frontal cortex (8 µl and 10 µl groups) but hippocampus. Decrease of parvalbumin in the frontal cortex (8ul and 10 ul groups) and the hippocampus (10 µl) means nonselective lesion because of high dose of immunotoxin. We observed recovery after memory acquisition delay and decrease of synaptic activity in the frontal cortex except in the hippocampus. High dose of immunotoxin injured not only cholinergic neuron but also GABAergic neuron in the frontal cortex and the hippocampus. Hippocampal GABAergic cell synapse on to glutamatergic pyramidal cells. Deficit of the hippocampal inhibitory cell may facilitate hippocampal synaptic plasticity and the recovery.

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Szot P, Franklin A, White S, Raskind M (2010) Time- and dose-response of 6-hydroxydopamine on locus coeruleus noradrenegric neurons in c57bl/6 mice. Neuroscience 2010 Abstracts 157.20/R1. Society for Neuroscience, San Diego, CA.

Summary: Locus coeruleus (LC) noradrenergic neurons are severely reduced in Alzheimer’s and Parkinson’s disease. However, it is unclear why these neurons are lost and the consequence of this loss on the progression and symptoms of these neurodegenerative disorders. Therefore, establishing an animal model of LC noradrenergic neuronal loss is critical in determining how the LC contributes to these disorders. The purpose of this study was to determine the dose- and time-response of noradrenergic neurotoxicity of 6-hydroydopamine (6OHDA) in adult male C57BL/6 mice. Our laboratory recently demonstrated that DSP4 does not result in a loss of LC noradrenergic neurons. Neurotoxicity of 6OHDA on LC noradrenergic neurons was determined by measuring tyrosine hydroxylase (TH) mRNA expression and TH-immunoreactivity (IR) in LC noradrenergic neurons. TH mRNA was quantitated using MCID (OD), while TH-IR was used to determine if protein levels reflected what was observed with mRNA. 6OHDA (20 µg/µl bilaterally) and dopamine beta-hydroxylase-saporin (DBH-saporin; 1 µg/µl bilaterally) were initially administered into the lateral ventricles (icv) and sacrificed 2 weeks later. 6OHDA reduced TH mRNA and -IR in both the dopaminergic neurons of the substantia nigra (SNpc) and ventral tegmental nucleus (VTA), and LC by -46%, -65% and -63%, respectively. DBH-saporin icv injection did not affect dopaminergic or noradrenergic neurons. Injection of DBH-saporin into the LC (0.1 µg/µl unilaterally) also did not affect LC noradrenergic neurons 2 weeks later. As a time-course 6OHDA (7 µg/µl) was injected unilaterally into the LC (vehicle was administered in the alternate LC) and sacrificed 3 days, 2 and 3 weeks later. A loss of LC noradrenergic neurons was observed only 3 weeks later (-81.4%). 6OHDA was then injected unilaterally into the LC at 7, 10, and 14 ug/ul (vehicle was administered in the alternate LC) and sacrificed 2 weeks later. The 7 µg/µl dose of 6OHDA did not affect TH mRNA in the LC as compared to control side (-19%), 10 ug/ul 6OHDA significantly reduced TH mRNA in the LC by ~55%, and 14 ug/ul 6OHDA dramatically reduced TH mRNA in the LC by ~90%. TH-IR in the LC of the three different 6OHDA doses reflected closely the TH mRNA data. 6OHDA at the dose of 14 µg/µl, which resulted in a near complete loss of LC noradrenergic neurons, did not affect dopaminergic neurons in the SN (-9%) and VTA (+17%). These data indicate that DBH-saporin, at the parameters studied, did not affect mouse LC noradrenergic neurons. 6OHDA demonstrated a time- and dose-response reduction of mouse LC noradrenergic neurons. The consequence of this LC neuronal loss on forebrain noradrenergic markers will also be presented.

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Datta S, Chatterjee K, Wiley R (2010) Decreasing abnormal nocifensive responses in the bilateral chronic constriction injury (bCCI) model of neuropathic pain: Effects of lumbar intrathecal CCK-saporin. Neuroscience 2010 Abstracts 175.22/MM12. Society for Neuroscience, San Diego, CA.

Summary: The bCCI model produces long lasting -cold hyperalgesia (at least 100 days) along with decreases in staining for cholecystokinin (CCK) in the dorsal horn (DH). Spinal cholecystokinin (CCK) has anti-opiate activity, and selective destruction of DH neurons expressing CCK receptors by injection of intrathecal CCK-saporin, in naïve rats decreases thermal nocifensive reflex responses and is additive with morphine in decreasing nocifensive responses to heat. In the present study, we sought to determine the effects of intrathecal CCK-sap in the bCCI model of neuropathic pain in Long Evans female rats. bCCI rats underwent bilateral ligation of the sciatic nerves with chromic gut sutures. Controls underwent sham surgery with no ligation. Rats were tested on 0.3 C cold plate, thermal preference task (TPT) (shuttle box with floor temperatures of 15 C vs 45 C) and mechanical stimulation (von Frey). bCCI produced increased responses on the cold plate. 21 days after the bCCI surgery, the rats were injected with 1500 ng CCK-sap into the lumber CSF. Then, thermal and mechanical testing was repeated at intervals. Intrathecal CCK-sap injections decreased abnormal nocifensive responding of bCCI rats on the cold plate. CCK-sap reduced withdrawal responses to mechanical stimulation in bCCI rats. In TPT testing, the bCCI animals were hyperalgesic to cold (reduced cold side occupancy). After intrathecal CCK-sap injections, thermal preference was reversed (increased cold side occupancy). We interpret these results as showing that CCK-sap reverses abnormal nocifensive responses of bCCI in rats to aversive cold and mechanical stimuli. These results suggest that silencing CCK receptor-expressing superficial DH neurons is a potential strategy for development of new treatments for chronic neuropathic pain.

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Li C, Back S, Lee J, Baek SK, Na H (2010) Gastrin-releasing peptide receptor in the spinal cord mediates mechanical allodynia following nerve injury. Neuroscience 2010 Abstracts 176.2/OO4. Society for Neuroscience, San Diego, CA.

Summary: Gastrin-releasing peptide receptor (GRPR) has been suggested as an itch-specific gene in the spinal cord (Sun et al., Nature, 2009). They described that selective ablation of GRPR-expressing lamina I neurons led to deficits in itch-related scratching behaviors without any effects on pain behaviors including nerve injury-induced mechanical allodynia. It has been known that two types of mechanical allodynia, such as static and dynamic allodynia, can be detectable in neuropathic patients, and may be mediated by distinct mechanisms. In the present study, we investigated the role of spinal GRPR in each of static and dynamic allodynia using both rat- and mouse-tail models of neuropathic pain. Bombesin-saporin (bombesin-sap) was administered intrathecally to ablate spinal GRPR-expressing neurons. Scratching behaviors evoked by pruritogenic agents, such as serotonin and chloroquine, and physiological pain behaviors were analyzed before nerve injury. Static or dynamic allodynia was assessed by the application of von Frey filaments to the tail or brushing the tail with a filament, respectively. RC3095, a GRPR antagonist, was given intrathecally to see its effects on static and dynamic allodynia in neuropathic rats. Bombesin-sap treatment resulted in reduction of GRPR-immunoreactive cells in lamina I of spinal dorsal horn and scratching deficits. Physiological pain behaviors of these animals were not different from those of control animals. Following the partial injury of tail-innervating nerves, animals treated with bombesin-sap exhibited comparable dynamic allodynia to control one. However, they failed to manifest static allodynia during the entire experimental period. In addition, RC3095 relieved static, but not dynamic, allodynia. These findings suggest that spinal GRPR mediates nerve injury-induced static mechanical allodynia as well as itching sensation in normal state.

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Gulino R, Gulisano M (2010) The recovery of locomotion after lumbar spinal cord motoneuron depletion is affected by the modulation of Sonic Hedgehog and Notch-1 pathways. Neuroscience 2010 Abstracts 259.19/W12. Society for Neuroscience, San Diego, CA.

Summary: Sonic hedgehog (Shh) and Notch-1 are involved in the regulation of stem cell function. Additionally, Notch-1 has a role as modulator of synaptic plasticity. In our previous work, we injected Cholera toxin-B saporin (CTB-sap) into the gastrocnemius muscle to induce a selective depletion of motoneurons within lumbar mice spinal cord (SC) and analysed the expression levels of Shh, Notch-1, Numb, Choline acetyltransferase (ChAT) and Synapsin-I proteins. The functional outcome of the lesion was monitored by grid walk test and rotarod. Shh and Notch-1 appeared reduced in the lesioned tissue and correlated with ChAT and Synapsin-I levels, suggesting a role in modulating synaptic plasticity. Numb expression was also reduced after lesion and appeared correlated with motor performance but not with synaptic plasticity. We sought to determine if the pharmacological manipulation of the expression of Shh and Notch-1 could affect functional recovery by a mechanism involving synaptic plasticity. Therefore, we used CTB-sap to induce lesion as above, and injected Cyclopamine, recombinant Shh or recombinant DLL4 chronically into the intrathecal space by means of osmotic minipumps for two weeks. The functional recovery was monitored for one month after lesion by means of grid walk test, two times a week, whereas the modifications of Shh, Notch-1, ChAT and Synapsin-I protein expression levels were measured by western blot and immunohistochemistry. Here, we show that the modulation of Shh or Notch-1 pathways could affect the recovery of locomotion. Moreover, the molecular mechanisms underlying this process is discussed.

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Moore JT, Mccarren HS, Beck SG, Kelz MB (2010) Lesioning of the ventrolateral preoptic nucleus alters isoflurane-induced hypnosis in a time-dependent fashion. Neuroscience 2010 Abstracts 300.28/KKK36. Society for Neuroscience, San Diego, CA.

Summary: Despite 160 years of clinical use, the neural mechanisms through which general anesthetics act remain unknown. One possibility is that anesthetics exert their hypnotic effects by acting on the endogenous arousal neural circuitry, including the wake-promoting orexinergic neurons of the hypothalamus and the sleep-promoting GABAergic and galaninergic neurons of the ventrolateral preoptic nucleus (VLPO). We have previously demonstrated that orexinergic neurons play an essential role during emergence from general anesthesia but not during anesthetic induction (Kelz et al., 2008). Here, we present evidence that the VLPO exerted a modulatory role in the induction of anesthetic hypnosis. We used c-Fos immunohistochemistry to analyze the activity of VLPO neurons in brain slices of mice sacrificed after two hours of anesthetic exposure. Whereas anesthetic exposure produced a decrease in the number of c-Fos-positive nuclei in most brain areas, this was not true for the VLPO: exposure to the volatile anesthetics isoflurane or halothane produced a rapid, dose-dependent increase in the number of c-Fos-positive nuclei in the VLPO, implying that hypnotic doses of volatile anesthetics increased the firing rates of VLPO neurons. To determine whether activation of the VLPO was necessary for anesthetic-induced hypnosis, galanin-saporin was used to produce targeted lesions of VLPO neurons. Six days following surgery, the bilaterally lesioned mice were more resistant to induction with isoflurane than control animals in a loss of righting reflex assay. However, 24 days following surgery the lesioned animals were more sensitive to isoflurane than controls. This time-dependent effect was likely due to the build-up of sleep debt–which is known to reduce the anesthetic dose needed to induce hypnosis–as a result of the insomnia-producing VLPO lesions (Lu et al., 2000). These findings are consistent with the VLPO playing a key role in the induction of volatile anesthetic-induced hypnosis, though formal proof will require acute manipulations of VLPO activity that do not produce a sleep debt confound.

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Zheng H, Rinaman LM (2010) Anxiety-like behavior in the elevated plus maze (EPMZ) depends on noradrenergic (NA) inputs to the anterolateral bed nucleus of the stria terminalis (alBST) in rats. Neuroscience 2010 Abstracts 90.7/FFF13. Society for Neuroscience, San Diego, CA.

Summary: The a2 adrenoceptor antagonist yohimbine (YO) increases transmitter release from NA neurons, activates the HPA stress axis, and increases anxiety-like behavior in rats. YO-induced HPA axis activation depends on collateralized projections from caudal brainstem NA neurons that innervate both the alBST and the medial parvocellular paraventricular nucleus of the hypothalamus (mpPVN) [Banihashemi & Rinaman, J. Neurosci., 2006]. The current study examined whether the same NA projections underlie the anxiogenic behavioral effects of YO. Adult male Sprague-Dawley rats were tested for baseline anxiety-like behavior in the EPMZ. Subsequently, anesthetized rats received bilateral microinjections of saporin toxin conjugated to an antibody against dopamine beta hydroxylase (DSAP) into the alBST to remove sources of NA input; sham control rats were similarly microinjected with vehicle. Two weeks after surgery, rats were re-tested in the EPMZ on three different days, with the first test conducted 30 min after i.p. YO (1.0 mg/kg BW), the second test conducted 30 min after i.p. saline, and the final test conducted without injection. As expected, the number of “anxiogenic” open arm entries was significantly reduced in sham control rats after YO compared to pre-surgery baseline behavior (P<0.01). Conversely, open arm entries were unaffected by YO in DSAP rats, and the YO-induced reduction of time spent in the open arms was significantly attenuated in DSAP rats vs. sham controls (P<0.01). Interestingly, in the final EPMZ test with no i.p. injections, sham control rats but not DSAP rats displayed increased anxiety-like behavior compared to their pre-surgery baseline (sham controls P<0.01; DSAP rats P=0.3). These findings support the view that caudal brainstem NA neurons projecting to the alBST are important for anxious behavior in the EPMZ, consistent with an earlier study utilizing pharmacological blockade of BST NA receptors [Cecchi et al., Neurosci., 2002]. Our ongoing studies are examining whether similar DSAP lesions attenuate conditioned and unconditioned fear and anxiety responses in rats under more ethologically relevant experimental conditions.

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Weeden CS, Morris AM, Rossi CA, Roberts JM, Kesner RP (2010) The role of GABA-ergic interneurons in CA1 and dentate gyrus for sequence learning. Neuroscience 2010 Abstracts 99.26/KKK12. Society for Neuroscience, San Diego, CA.

Summary: The hippocampus (HPP) plays an important role in temporal and spatial memory. Lesion investigations of the CA1 region of HPP indicate the region’s importance in temporal processing and lesions of the dentate gyrus (DG) demonstrate an important role in spatial processing. It has been suggested that a subset of GABAergic interneurons that express Substance P mediate the inhibition of pyramidal and granule cells, which further affects the pattern of their output. This synchronizing action may directly affect information processing of CA1 pyramidal and DG granule cells. A form of temporal processing involves learning specific sequences of events for spatial locations, which incorporates both temporal and spatial qualities attributed to CA1 and DG, respectively. In order to investigate whether interneurons mediate CA1 and DG processing of newly learned locations of sequential patterns, Long-Evans male rats were randomly assigned to the following surgical groups: CA1 pyramidal cell (ibotenic acid), CA1 interneuron (peptidase-resistant substance P analog conjugated to the neurotoxin saporin) (SSP-Saporin), DG granule cell (colchicine), DG interneuron (SSP-Saporin) lesions and controls (PBS). Following recovery from surgery, rats were tested on a sequential learning task for spatial locations using an eight-arm radial maze. Six arm locations were pseudo-randomly assigned to a sequence; each of the arms was baited with a food reward. Doors remained closed until the rat oriented in front of the correct door in the sequence, at which time the door was opened and the rat was allowed access to the reward; the choice was scored as a correct response. However, if the rat oriented to an incorrect door in the sequence, the choice was scored as incorrect and the animal was allowed to reorient to the correct door. The same sequence was repeated ten times per day for a total of ten consecutive days. The percentage of correct choices per day was compared across all ten days. The results indicate that subjects with CA1 pyramidal cell, CA1 interneuron, and DG interneuron, but not DG granule cell lesions, had difficulty acquiring the sequential task when compared to controls. These results suggest an important role for CA1 pyramidal cells, and for interneurons in both CA1 and DG subregions of the HPP in temporal processing of spatial locations.

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Easton A, Phil D, Fitchett A, Eacott MJ, Baxter MG (2010) Cholinergic innervation of the hippocampus is not neccesary for episodic memory, but is required for context-place learning in rats. Neuroscience 2010 Abstracts 99.27/KKK13. Society for Neuroscience, San Diego, CA.

Summary: Loss of cholinergic cortical input is associated with diseases in which episodic memory impairment is a prominent feature, but the degree to which this neurochemical lesion can account for memory impairment in humans with neurodegenerative diseases remains unclear. Removal of cholinergic input to hippocampus impairs some of its functions in memory, perhaps by reducing the plasticity of information representation within the hippocampus, but the role of cholinergic hippocampal input in episodic-like memories has not been investigated. To address this question we tested rats with selective lesions of basal forebrain neurons in the medial septum and vertical limb of the diagonal band (MS/VDB), which contains hippocampal-projecting cholinergic neurons, on a task of integrated memory for objects, places, and contexts (“what-where-which” memory). This task serves as a rodent model of human episodic memory (episodic-like memory) and is sensitive to damage to the hippocampal system. Rats with lesions of cholinergic MS/VDB neurons performed as well on the what-where-which task as controls, but were impaired in a task that simply required them to associate places with contexts (“where-which” memory). Thus, episodic-like memories that rely on the hippocampus do not require cholinergic neuromodulation to be formed. Nevertheless, some more specific aspects of where-which memory, which may be more dependent on the plasticity of hippocampal spatial representations, require acetylcholine. These results suggest that cholinergic projections to hippocampus are not necessary for episodic memory, and furthermore, that hippocampal spatial representations may be to some extent dissociable from episodic memory function.

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Ramanathan D, Conner JM, Anilkumar AA, Tuszynski MH (2010) Early post-natal cholinergic lesion impairs normal development and maturation of the motor cortex in rats. Neuroscience 2010 Abstracts 32.14/D20. Society for Neuroscience, San Diego, CA.

Summary: Prior studies have indicated that sensory and motor representations develop over a defined postnatal period and are dependent upon behavioral experience to achieve appropriate adult patterns. In adult animals, behaviorally driven forms of cortical map plasticity are critically dependent upon the basal forebrain cholinergic system. Based on the critical role cholinergic mechanisms play in mediating experience-dependent plasticity in adulthood, we postulated that cholinergic mechanisms may also play a critical role in shaping initial cortical map formation during development. In this study, using 25 male Fisher rats between the ages of 15 days and 60 days, we first characterized the normal motor map development in the rat. We found that motor maps underwent a significant change in overall size and refinement over time, with more mature animals having larger overall maps (p < 0.001) and an increase in the size of distal forelimb representations (p < 0.01). Following the initial characterization of normal motor map development in the rat, we used 192-IgG-saporin (SAP) to create selective cholinergic lesions early in map development (PND 24), in 5 animals (with 6 animals receiving ACSF as controls). This early cholinergic depletion impaired the normal maturation and refinement of cortical motor representations: the total caudal forelimb area (comprising elbow and wrist) was decreased by 33% in cholinergically depleted animals, from 5.1 ± 0.3 mm2 to 3.4 ± 0.3 mm2 (t-test p < 0.01). This decrease in caudal forelimb area in cholinergically-depleted animals was primarily driven by a significant 37% reduction in the size of the distal forelimb (wrist) representation, from 3.1 ± 0.1 mm2 to 2.0 ± 0.1 mm2 (p < 0.001). In a follow-up experiment with 12 additional animals (6 with cholingeric lesions and 6 controls), we found that early (PND 24) cholinergic depletions resulted in long-term impairments in skilled motor learning, with significant differences in daily motor performance beginning at day 3 of training (repeated measures ANOVA < 0.05). These results suggest a novel role for the basal forebrain cholinergic system in establishing normal cortical map formation during development.

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