References

Tai S, Leung L (2009) Facilitation of dentate gyrus population spike may involve septohippocampal GABAergic input. Neuroscience 2009 Abstracts 566.18/EE33. Society for Neuroscience, Chicago, IL.

Summary: The medial septum (MS) is known to modulate the neural circuitry in the hippocampus. It have been demonstrated that, in anaesthetized rats, MS stimulation prior to perforant path stimulation facilitated the population spike in the dentate gyrus and such facilitation was unaffected by muscarinic and nicotinic cholinergic antagonists, suggesting that this facilitation may be mediated by septohippocampal GABAergic neurons. To study the effects of septohippocampal neurons in modulating neural circuitry in the dentate gyrus, selective lesion of septohippocampal GABAergic cells was made by infusion of orexin-saporin in the MS. Evoked field potentials were recorded in the dentate gyrus following stimulation of the medial perforant path in urethane-anesthetized rats using multichannel silicon probes and analyzed as current source density. High-frequency stimulation of the pontis oralis (PnO) activated a hippocampal theta rhythm. Theta peak power was attenuated in lesion rats as compared to control (sham lesion) rats. PnO stimulation resulted in a larger average increase in dentate population spike (pSpike) in control rats compared to lesion rats (26.4 ± 8.8 % vs 5.1 ± 8.7 %) but the difference was not significant (P = 0.1082; control n = 9, lesion n = 8). The number of choline acetyltransferase-immunopositive (cholinergic) cells in the MS was not significantly different (P = 0.88, unpaired t-test) between lesion and control rats, while a significant decrease (about 50%) of the number of parvalbumin-immunopositive GABAergic cells in the MS was observed in lesion as compared to control rats (P<0.05, unpaired t-test). The effect of PnO stimulation on the paired-pulse pSpike response versus interpulse interval (30 - 400 ms) was not different between control and lesion rats. No difference between control and lesion rats was detected in the pSpike enhancement after PnO stimulation following scopolamine (5 mg/kg i.p.). Additional studies were needed to demonstrate the facilitation of dentate gyrus population spike by septohippocampal GABAergic neurons.

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Gaykema RP, Thacker GC, Shapiro NJ, Goehler LE (2009) Immunotoxic lesion of hypothalamic noradrenergic/adrenergic input ameliorates the effects of peripheral LPS challenge on sickness behavior and associated brain c-Fos expression. Neuroscience 2009 Abstracts 570.11/EE120. Society for Neuroscience, Chicago, IL.

Summary: Caudal medullary catecholamine neurons that innervate the hypothalamus play a major role in the activation of paraventricular neurons that drive pituitary adrenocorticotropin and adrenal corticosteroid release in response to peripheral pro-inflammatory challenges with interleukin-1 or lipopolysaccharide (LPS). Pro-inflammatory challenges also lead to marked behavioral changes, including fatigue, loss of social interest, anorexia, somnolence, but the precise neuronal mechanisms that underlie sickness behavior remain elusive. We reasoned that the medulla-hypothalamic catecholaminergic pathway may also contribute to the behavioral manifestations in illness. To investigate such possible role, we applied a targeted lesion approach in rats to determine whether or not caudal brainstem catecholaminergic neurons that innervate the hypothalamus are also necessary for the expression of sickness behavior. Anti-dopamine beta hydroxylase antibodies conjugated to saporin (DSAP), when injected into a target region, selectively poisons and destroy noradrenergic/adrenergic neurons that innervate the target. DSAP was micro-injected bilaterally into the hypothalamic paraventricular nucleus (PVN), whereas control rats received unconjugated saporin (SAP controls). Fourteen days later the animals were injected intraperitoneally with either LPS or saline, and 2h later were submitted to the open field to record their exploratory behavior, 1h after which the rats were sacrificed for brain immunohistochemical analyses. LPS-treated SAP control rats showed drastic reduction in exploratory behavior (reduced locomotion distance and velocity). Prior DSAP microinjections largely reversed the LPS-induced reduction in locomotor behavior. The brains of these DSAP rats showed a dramatic loss of noradrenergic innervation of the PVN but also in other parts of the medial, tuberal and tuberomammilary regions of the hypothalamus. The behavioral resilience to LPS coincided with diminished LPS-related c-Fos staining in the PVN, and increased c-Fos staining in the lateral and tuberomammillary regions related to behavior and/or arousal. In summary, our findings support the hypothesis that hypothalamic catecholaminergic projections originating in the lower brainstem play a critical role in the expression of sickness behavior in the context of novelty-induced exploratory activity, but we cannot determine with precision in which part of the hypothalamus the noradrenergic/adrenergic input contributes to the expression of sickness behavior due to extensive collateralization of the ascending projections throughout the hypothalamus.

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Gulino R, Gulisano M (2009) Poster: Expression of cell fate determinants and plastic changes after neurotoxic lesion of adult mice spinal cord by cholera toxin-B saporin. Neuroscience 2009 Abstracts 563.15/DD51. Society for Neuroscience, Chicago, IL.

Summary: Recent studies have attempted to achieve recovery after spinal cord (SC) injury or disease by either increase neurogenesis or stimulate neuroplasticity. Sonic hedgehog (Shh) Notch-1 and Numb are involved in the regulation of stem cell function. Additionally, Notch-1 has a role as modulator of synaptic plasticity. Little is known about the role of these proteins in the adult SC after selective removal of motoneurons. We injected Cholera toxin-B saporin into the gastrocnemius muscle to induce a selective depletion of motoneurons within lumbar mice 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. The neurotoxin lesion determined a motoneuron depletion and a decrease of ChAT and Synapsin-I protein levels in the lumbar SC. ChAT and Synapsin-I appeared correlated each other and with the motor performance, suggesting that the recovery of locomotion could depend from synaptic plasticity. Moreover, we observed a number of proliferating cells within the depleted SC, which were identified as active astrocytes. 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 Therefore, given the role of these proteins in adult neurogenesis, we presume their involvement also in the observed glial reaction. The in vivo manipulation of Shh, Notch-1 and Numb signalling after lesion could be a way to reduce glial reaction and improve functional recovery.

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Huang T-Y, Lin L-S, Chen H-I, Jen C (2009) Chronic treadmill exercise improves cerebellar functions: Alterations in mitochondrial protein expression, rotarod performance, and toxin resistance. Neuroscience 2009 Abstracts 660.18/CC34. Society for Neuroscience, Chicago, IL.

Summary: The effects of exercise on cerebellar functions were studied. Five-week-old male Wistar rats were divided into exercise and sedentary groups. For exercise groups, rats were subjected to 8 weeks of treadmill exercise at moderate intensity. In some groups, rats were administered with OX7-saporin, a cerebellar Purkinje cell toxin, into the lateral ventricle during the 5th week of training. At the end of training period, they were tested for rotarod performance. Brain tissues were obtained for measurement of mitochondria-related protein, including Mfn2, OPA1, Drp1 and CcOx-IV. The morphology of Purkinje cells was also examined by two photon microscopy. Our results showed that exercise training improve rotarod performance, and increased cerebellar protein levels of Mfn2 and OPA1 (mitochondrial fusion proteins) but not Drp1 (mitochondrial fission protein) or CcOx-IV (a mitochondrial complex IV marker). The dendritic field of Purkinje cells was significant modified in exercise groups. OX7-saporin application impaired the rotarod performance and decreased cerebellar Purkinje cell number only in sedentary rats. In summary, chronic exercise enlarged dendritic field of Purkinje cells and improved cerebellar function, including the rotarod performance, the mitochondrial fusion protein expression, and the resistance to toxin insult.

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Jeong D, Hwang Y, Lee D, Chang J (2009) Behavioral and histological characteristics of 192 IgG-saporin injected rats depending on injection site and dose. Neuroscience 2009 Abstracts 526.23/H8. Society for Neuroscience, Chicago, IL.

Summary: Cholinergic neuronal deficits are evident in both Alzheimer’s disease dementia (AD) and vascular dementia (VaD). Forebrain Cholinergic neurons in the nucleus basalis magnocellularis (NBM) project primarily to the neocortex, and those in the medial septum project to the hippocampus and they make an important role in memory function. We used 192 IgG-saporin to mimic deficits of cholinergic neurons at AD and VaD. 192 IgG-saporin is composed with monoclonal antibody had a low affinity to the rat nerve growth factor receptor p75 and ribosomal inactivating protein, called saporin. When injected intracerebroventricularly or directly into the basal forebrain cholinergic complex, 192 IgG-saporin selectively destroys cholinergic neurons. Many experimenters had used 192 IgG-saporin to investigate cholinergic function but it had been used in different doses and sites of lesion. This makes it difficult to compare the degrees of impairment produced by different lesions. Consequently, our aim is observation of behavioral and histological changes depending on injection site and dose of 192 IgG-saporin. We injected 192 IgG-saporin (0.63ug/ul) in medial septum (dose: 0.05ul, 0.1ul, 0.2ul) or lateral ventricle (dose: 6ul, 8ul, 10ul). 192 IgG-saporin injected rats were compared with Dulbecco’s phosphate buffered saline injected rats. Neurological deficit and functional outcome were determined by immuohistochemistry using anti-cholineacetyltransferase antibody and behavioral test, called water maze. In immunohistological study, the extent of the cholinergic lesion was showed in the basal forebrain complex region of 8ul and 10ul of 192 IgG-saporin injected rats. In behavioral study, sham and lesion groups were able to learn the reference aspect of the water maze within 5day of training. In probe test, we observed significant decrease in time in target quadrant, platform and platform crossings, and increase in latency to first crossing at 8ul and 10ul of 192 IgG-sapoin injected rats (p<0.05). Therefore, our study evaluated that 8ul 192 IgG-saporin injections were sufficient to make an AD mimic dementia model.

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Matchynski JJ, Lowrance S, Rossignol J, Puckett N, Derkorver N, Radwan J, Trainor K, Sandstrom M, Dunbar G (2009) Intracerebroventricular injections of mu-P-75 saporin can produce memory deficits without impairing motor deficits in a mouse model of Alzheimer’s disease. Neuroscience 2009 Abstracts 528.1/H34. Society for Neuroscience, Chicago, IL.

Summary: Intracerebroventricular injections of mu-P-75 saporin (Advanced Targeting Systems, San Diego, CA) effectively and efficiently destroys cholinergic neurons and creates memory deficits in mice, mimicking some of the key symptoms of Alzheimer’s disease. Early attempts to use mu-P-75 saporin in mice required a relatively high mean effective dose (ED50) of 3.6 µg in order to create behavioral deficits (Berger-Sweeney et al., 2001, The Journal of Neuroscience, 21: 8164-8173; Hunter et al, 2004, European Journal of Neuroscience, 19: 3305-3316). Recent advances in producing the saporin have lowered the ED50 to doses to 0.4 µg, although the resulting memory deficits are transient, and doses above 0.8 µg can cause motor deficits (Moreau et al., 2008, Hippocampus, 18: 610-622). In an effort to elucidate the behavioral effects of a higher (0.8 µg) dose, we gave bilateral intracerbroventricular injections of mu-P-75 saporin (n=6) or sterile phosphate buffered saline (n=3) into C57/BL6 mice and assessed their cognitive abilities on both a Morris water maze (MWM) and an object-recognition task, while monitoring their motor abilities using a rotarod task. Mice receiving the mu-P-75 saporin performed significantly worse than sham animals on an object recognition task and tended to have longer latencies and swim paths during the seven days of MWM testing. Importantly, no between-group differences were observed for latency to fall on the rotarod task. Collectively, these results suggest that the 0.8 µg dose of saporin is both safe and effective for mimicking AD-like memory deficits, without causing significant motor deficits.

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Laplante FP, Dufresne M, Lappi DA, Sullivan RM (2009) Depletion of cholinergic neurons in the nucleus accumbens impairs dopamine function in the prefrontal cortex in the rat. Neuroscience 2009 Abstracts 341.7/O16. Society for Neuroscience, Chicago, IL.

Summary: Studies of post mortem schizophrenic brains have revealed a selective loss of cholinergic interneurons, most pronounced in the ventral striatal region. We have previously shown in the rat, that a novel saporin immunotoxin coupled with an antibody targeting choline acetyltransferase (ChAT) and microinjected (0.5 _g/_l; 0.5 _l) into the nucleus accumbens (N. Acc) of adult rats, reduces the number of cholinergic neurons in N. Acc. by 40-50 %. Such lesions result in a markedly heightened response to the locomotor activating effects of amphetamine and impair prepulse inhibition of the acoustic startle response. We proposed that this local cholinergic deficit leads to a hyperresponsiveness in subcortical dopamine (DA) systems of relevance to schizophrenic symptomatology. Presently, we hypothesize that the same local cholinergic defect may trigger broader changes in cortical/subcortical networks, specifically prefrontal cortex (PFC) deficits in DA-mediated functions, also proposed in schizophrenia. Young adult male Srpague-Dawley rats were injected bilaterally in the N. Acc. as described above with either the cholinergic immunotoxin or vehicle. Two weeks later, they were trained in a working memory task dependent on PFC function, using the delayed alternation paradigm in the T-maze. Lesioned rats took significantly longer to reach criterion performance during training than controls. During testing, lesioned rats were significantly impaired in the percentage of correct arm choices across delay intervals, but especially with longer (40 sec) delays. The same animals were then implanted with voltammetric recording electrodes in the ventromedial PFC to examine the increases in in vivo extracellular DA release in response to a brief tail pinch stress. Lesioned rats showed a significantly reduced activation of the mesocortical DA system compared to controls. Taken together, the data suggest that reduction in the density of cholinergic neurons in the N. Acc also triggers deficits in prefontally-mediated function known to be under mesocortical DAergic regulation. This raises the possibility that ventral striatal cholinergic deficits may be causally linked to cortical/subcortical functional imbalances proposed to exist in schizophrenia.

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Brink TS, Khasabov SG, Fliss PM, Simone DA (2009) Ablation of NK-1 expressing neurons in the rostral ventromedial medulla attenuates inflammatory hyperalgesia. Neuroscience 2009 Abstracts 361.5/BB31. Society for Neuroscience, Chicago, IL.

Summary: Substance P (SP) is a neuropeptide synthesized by many nociceptive primary sensory neurons and is released into the spinal cord following noxious stimulation where it binds to neurokinin-1 (NK-1) receptors, mostly located on ascending spinal neurons. Spinal NK-1 receptors are involved in the development of hyperalgesia and central sensitization. NK-1 expressing neurons are also present in the rostral ventromedial medulla (RVM), a brainstem area involved in descending modulation of nociceptive transmission in the spinal cord. ON cells in the RVM are involved in facilitation of nociceptive transmission and their activity may be modulated by SP. SP injected into the RVM excites ON cells through NK-1 receptors, and NK-1 receptor antagonists into the RVM attenuate hyperalgesia produced by capsaicin. Here, we studied the role of RVM NK-1 positive neurons in modulating hyperalgesia following acute (intraplantar capsaicin injection) or sustained (complete Freund’s adjuvant (CFA) in the hindpaw) inflammation. We used the ribosomal toxin saporin (SAP) conjugated to a stable agonist of SP (SSP) to selectively ablate RVM cells that possess NK-1 receptors. In male Sprague-Dawley rats, withdrawal responses to noxious heat and mechanical stimuli were obtained using the Hargreaves method and a 15 g von Frey monofilament applied to the plantar hindpaw, respectively. Rats were treated with either the SSP-SAP toxin (0.5 µg/0.5 µl) or blank-SAP, and were tested 10-24 days after injection, when NK-1 expressing RVM neurons are ablated. In control rats, injection of capsaicin (10 µl of 0.1%) produced a 63% decrease in withdrawal latency to heat and an increase in withdrawal response frequency evoked by the monofilament from 16% up to 87%. However, SSP-SAP attenuated capsaicin-evoked hyperalgesia to heat (15% decrease in withdrawal latency) and mechanical (increase to 44% withdrawal frequency) stimuli. Elimination of NK-1 positive neurons in the RVM also attenuated the development of hyperalgesia following CFA. Whereas control rats exhibited a 60% decrease in withdrawal latency to heat and an increase in withdrawal frequency the monofilament from 10% up to 78%, withdrawal latency decreased 27% and withdrawal frequency increased to only 46% in rats treated with SSP-SAP. We conclude that neurons in the RVM that contain NK-1 receptors are pronociceptive and contribute to the hyperalgesia produced by capsaicin or CFA.

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Li A-J, Wang Q, Dinh TT, Ritter S (2009) Leptin-saporin injection into the arcuate nucleus lesions NPY/AGRP and POMC neurons and produces hyperphagia, obesity and changes in diurnal feeding patterns in rats. Neuroscience 2009 Abstracts 374.5/EE116. Society for Neuroscience, Chicago, IL.

Summary: Leptin-saporin (Lep-SAP), a conjugate of leptin with a ribosomal inactivating toxin, saporin (Advanced Targeting Systems), is a novel toxin designed to destroy leptin receptor-expressing cells selectively in vitro. However, its lesioning properties in vivo are currently unknown. Here, we injected Lep-SAP into the arcuate nucleus (Arc), to examine its effects on feeding behavior and on leptin receptor-expressing NPY/AGRP and POMC neurons in this area. Immunohistochemical studies showed unilateral injection of Lep-SAP into the Arc dramatically reduced numbers of NPY-Y1- and α-MSH- positive neurons compared to the contralateral side injected with SAP control. Real-time PCR revealed only 11-21% of Agrp and Pomc expression remaining in the Arc after Lep-SAP injection into this region. Rats injected bilaterally with Lep-SAP were unresponsive to central leptin administration and showed dramatic increases in feeding, body weight and light-phase feeding, compared pre-injection baseline. Two weeks after injection, total daily feeding was increased by 75%, light phase feeding by 359% and dark phase feeding by 33%. Control SAP injections did not produce these changes. Clock gene expression in homogenates of whole hypothalamus and liver were quantified at ZT 5-7. Bmal1 expression in hypothalamus and liver of Lep-SAP rats was decreased, while hepatic Per1 expression was increased compared to control. Results demonstrate that Lep-SAP effectively lesions Arc leptin receptor-expressing NPY/AGRP and POMC neurons in vivo, and that rats with this lesion are hyperphagic and obese, possibly due to enhanced hunger drive, lack of responsiveness to leptin and/or changes in circadian control of feeding behavior.

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Carvalho AF, Reyes AS, Van Bockstaele EJ (2009) The role of limbic norepinephrine in cannabinoid-induced aversion. Neuroscience 2009 Abstracts 449.3/V29. Society for Neuroscience, Chicago, IL.

Summary: The endocannabinoid system has been implicated in diverse physiological mechanisms including modulation of pain and analgesia, learning and memory and feeding, among others. Thus, targeting the cannabinoid system has risen to the forefront in the development of novel treatments for a number of pathophysiological processes. Consistent with this, agonists of the cannabinoid receptor type 1 (CB1R) have been successfully used in the treatment of severe anorexia in patients with AIDS and in alleviating nausea and vomiting in patients undergoing chemotherapy. However, significant side effects have been observed in clinical trials raising concerns regarding the potential clinical utility of cannabinoid-based agents. Disturbances in mood and affect, including paranoia, anxiety and nervousness, have been reported in patients. Understanding the neural circuits and neurochemical substrates impacted by cannabinoids will provide a better means of gauging their actions within the central nervous system that contribute to the expression of unwanted side effects. We have previously shown an increase in anxiety-like behaviors in rats receiving repeated administration of cannabinoid agonists. This increase in anxiety was accompanied by increases in indices of noradrenergic activity. In the present study, we investigated whether norepinephrine in the limbic forebrain of rats is required for cannabinoid-induced aversion using an immunotoxin lesion approach combined with behavioral analysis using a place conditioning paradigm. Male Sprague Dawley rats received bilateral injections of a ribosomal toxin, saporin (SAP) conjugated to an antibody that specifically recognizes the enzyme dopamine-beta-hydroxylase (DSAP), into the limbic forebrain. Control rats received saporin alone. As previously reported, administration of the synthetic cannabinoid receptor agonist, WIN 55,212-2 (3.0mg/kg), induced aversion in a place conditioning paradigm in SAP-only treated rats. The rats’ spatial memory was also evaluated using the Morris Water Maze. Depletion of norepinephrine using DSAP in specific limbic regions impaired cannabinoid-induced aversion to WIN 55,212-2 without affecting learning and memory processes. Taken together, noradrenergic projections to the limbic forebrain may be critical in the manifestation of aversive behaviors associated with cannabinoid agonist exposure.

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