References

Van Der List DA, Chapman B (2011) Targeted ablation of intrinsically photosensitive melanopsin expressing retinal ganglion cells early in development alters retinal morphology within the inner plexiform layer of mice. Neuroscience 2011 Abstracts 232.12. Society for Neuroscience, Washington, DC.

Summary: It has been demonstrated in adult mice, that eliminating a small subset of retinal ganglion cells expressing the photopigment melanopsin (ip-RGCs) with an immunotoxin alters the effects of light on circadian rhythms. The immunotoxin was made by conjugating the melanopsin antibody with ribosome-inactivating protein, saporin. It has also been observed that the ablation of ip-RGCs in adult mice did not alter retinal morphology. Specifically, it was found that dendrites arising from starburst amacrine cells retained their position within the inner plexiform layer (IPL) suggesting no reorganization within this synaptic layer (Goz et al. 2008). In this study, we used the same melanopsin immunotoxin (Mx) (Advanced Targeting Systems) to perform intravitreal injections into mice at postnatal day one. The animals were sacrificed at P26 and the retina fixed in 4%PFA, frozen transverse sections were then immunostained with antibodies against melanopsin, choline acetyl transferase (ChAT), calreinin, calbindin, PKC and Kv4.2. In control retinae, melanosin antibody stained ip-RGC cell bodies and dendrites stratifying in both On and Off layers of the IPL, whereas retinae treated with Mx shows a loss of melanopsin-containing cell bodies and dendrites. In control retinae, ChAT stains starburst amacrine cells with cell bodies in the RGC and INL layers and two distinct bands in the IPL. In Mx treated retinae, most starburst amacrine cells appear to be eliminated along with melanopsin RGCs. Interestingly, if there is a hint of residual melanopsin expressing dendrites remaining, there is also a ChAT expressing cell body and a hint of dendrites in the synaptic layer. In control retinae, calretinin and calbindin antibodies stain a subset of RGCs and amacrine cells and show a characteristic three-layered pattern of dendrites in the IPL. In Mx treated retinae, the calretinin and calbindin layers within the IPL are altered showing an absent or more diffuse labeling pattern in the ON and OFF bands. Antibodies against PKC (staining rod bipolar cells) and Kv4.2 (stains a subset of retinal ganglion cells) do not show an altered staining pattern. These findings suggest that the initial stratification and structural development of synaptic layers in the IPL are altered by Mx treatment.

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Spuz CA, Paolone G, Briscoe S, Bradshaw M, Albin RL, Sarter MF (2011) Deficits in attentional control of balance, mobility, and complex movements in a rat model of early state, multisystem Parkinson disease. Neuroscience 2011 Abstracts 244.02. Society for Neuroscience, Washington, DC.

Summary: In Parkinson disease (PD), basal forebrain cholinergic loss coincides with midbrain dopaminergic neuron loss and contributes to attentional deficits in PD. We hypothesize that these attentional deficits contribute to L-DOPA-insensitive impairments of mobility and postural control in PD. To assess complex movement control, we developed a novel Complex Motor Control Test (CMCT) for rats. The CMCT consists of several 2 m long beams (plank, 13.34 cm width; round rod, 3.81 cm diameter; square rod, 2.54 cm side length), which can be placed at zero, 22.5° or 45° angles in the vertical plane. Rods can rotate at 1 rpm or 10 rpm. A separate ladder apparatus (100 cm long, 7 cm wide, 2 cm between rungs, 5 mm rung diameter) can be placed at zero, 22.5° or 45° angles in the vertical plane and tilted laterally at 15° or 30° angles. Four high-resolution cameras and mirror system record animals’ performances. Rats are habituated by learning that plank traversal allows entry of home compartments containing individual bedding and palatable food. To separately assess attentional performance, we employed our Sustained Attention Task (SAT), including a distractor condition (dSAT). Our initial experiments determined CMCT and SAT performance in three groups: (1) animals with limited (40-60%) loss of cortical cholinergic afferents following immunotoxin 192-IgG saporin basal forebrain lesions (SAP); (2) animals with dopaminergic deafferentation following 6-OHDA dorsal striatal lesions (6-OHDA); (3) animals with both types of deafferentation (DUAL). SAT performance was dramatically impaired in SAP and DUAL animals. Control animals rapidly traversed angled and rotating rods and angled and tilted ladders. Deafferented animals were able to traverse the plank at all angles as effectively as control animals. Cholinergic lesions robustly impaired animals’ ability to maintain balance on the rods, to re-adjust posture on and traverse rotating rods, and had falls (into a net) or dismounts more frequently than control animals. These data reveal unexpectedly striking impairments in complex gait and movement control resulting from loss of corticopetal cholinergic neurons. These results support the hypothesis that basal forebrain cholinergic cell loss in PD contributes to complex posture and movement control deficits.

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Rossi CA, Lehmkuhle MJ, Dudek FE (2011) Evidence that focal interneuron lesions in the hippocampus may lead to a model of epileptogenesis in the mouse. Neuroscience 2011 Abstracts 249.09. Society for Neuroscience, Washington, DC.

Summary: A selective loss of part of the overall population of GABAergic interneurons is a seminal component of many forms of human epilepsy, and is manifest in many animal models of acquired epilepsy, including those based on chemoconvulsant-induced status epilepticus. The current study specifically tests the hypothesis that partial interneuron loss in the dorsal CA1 area of the hippocampus induces epileptiform activity, and the subsequent hypothesis that interictal-like spikes and seizures progressively worsen during the following weeks and months. Focal interneuron lesions were made by intra-hippocampal injection of SSP-Saporin into dorsal CA1 in the hippocampus of GAD67-GFP transgenic mice. Chronic recording electrodes were implanted at the injection site, and local field potentials (LFPs) were monitored continuously during video recording for several weeks. LFP recordings were analyzed for the occurrence of inter-ictal-like paroxysmal events (hippocampal sharp waves of 50-100 msec), and frank seizures. Although interneuron lesions alone were seen to generate inter-ictal-like activity within several days following surgery, full-blown seizure activity was not observed until several weeks later. The current data suggest that disruption of the local GABAergic interneuron population may be a key event that triggers alteration of neural networks in the hippocampus, leading to paroxysmal events and ultimately seizures. The delay in onset suggests other factors besides interneuron loss play a role in the generation of seizures and the development of epilepsy. Thus, loss of local inhibition may be a necessary, but not sufficient condition for epileptogenesis.

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Ljubojevic V, De Rosa E, Luu P (2011) Cholinergic modulation of cross-modal attentional orienting. Neuroscience 2011 Abstracts 294.07. Society for Neuroscience, Washington, DC.

Summary: We modified the classic cued target detection paradigm, using odor cues to predict a visual target, to examine cholinergic modulation of attentional control signals. It has been proposed that without ACh top-down processing will inappropriately dominate in the presence of a low validity cue, i.e., the cue will still drive attention. Thus, we reduced central cholinergic influences in rats after they acquired high validity cues to examine whether top-down processing would dominate even when these same cues changed to a lower validity. The validity effect (VE = invalid cue RT – valid cue RT) is thought to measure the ability to reorient attention, i.e., it reflects the time a subject needs to disengage from an invalidly cued location and shift attention to the actual target location. We trained 8 male Long-Evans rats until they reached the stable performance under baseline conditions: cue validity (CV) = 100%, target duration = 1s. Then we simultaneously manipulated the rats’ cholinergic system and cue validity within a testing session in a 3×3 repeated measures design. The three drug conditions were: muscarinic antagonist scopolamine (0.2mg/kg), muscarinic antagonist methylscopolamine as a peripheral nervous system control (0.2mg/kg), and saline. CV in each session was set to 100%, 75%, or 50%. In sessions with the lower cue validity of 75% and 50%, rats with scopolamine showed the predicted higher validity effect when compared to their performance with the control drugs. Based on the increased VE in scopolamine condition, we conclude that ACh plays a role in attentional orienting when cue and target are presented in a different sensory modality. We hypothesize that scopolamine may have exacerbated the top-down expectations from the cue and increased the validity effect. Thus, we are collecting data from rats that had selective cholinergic lesions of the nucleus basalis magnocellularis, which provides ACh input into the neocortex, with the cholinergic immunotoxin 192 IgG-saporin to support this pharmacological effect. We expect that NBM-ACh-lesioned rats will also have an increased validity effect as the CV decreases relative to the sham-lesioned rats. Also, we will collect the data from 8 additional rats to increase the statistical power of the experiment.

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Bates AT, Duys AN, Miller CE, Miller R, Mcgaughy J (2011) The effects of noradrenergic lesions to the orbitofrontal cortex on reversal learning. Neuroscience 2011 Abstracts 296.03. Society for Neuroscience, Washington, DC.

Summary: Poor impulse control is one of the major symptoms of disorders such as attention deficit disorder and is hypothesized to result from dysfunction in the prefrontal cortex. Specifically the ability to inhibit responding to a previously reinforced stimulus as required in tests of reversal rely on the functional integrity of the orbitofrontal cortex. Previous work from our lab and others have shown that norepinephrine in the prelimbic cortex is necessary to perform attentional set shifting. Lesions to this region result in attentional set shifting impairments that can be remediated by the administration of a selective noradrenergic reuptake blocker. Though many studies have shown monoamine levels in the orbitofrontal cortex are critical to reversal learning, few studies have directly addressed the impact of norepinephrine depletion in the orbitofrontal cortex on reversal learning. In the present study, we assess the effects of noradrenergic deafferentation of the orbitofrontal cortex in the intra-dimensional/extra-dimensional set shifting task using adult male, Long-Evans hooded rats. Preliminary data support the hypothesis that norepinephrine in orbitofrontal cortex is critical to successful reversal learning as the lesioned animals required more trials to reach criterion performance on reversals than sham-lesioned rats. Performance on the ID and ED portions of the task were not impacted by lesion. After behavioral testing was completed, brains were processed to elucidate norepinephrine transporters (NET). Fiber density of NET positive fibers was assessed in the regions of the orbitofrontal, prelimbic, and cingulate cortices for all subjects. These findings point to the function of the noradrenergic system within the orbitofrontal cortex on mediating impulse control while leaving attentional set shifting performance intact.

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Pacheco-Herrero M, Thyssen D, Ramos-Rodriguez J, Berrocoso E, Bacskai B, Garcia-Alloza M (2011) Rapid beta-amyloid deposition and behavioural impairment after cholinergic denervation in APPswe/PS1dE9. Neuroscience 2011 Abstracts 47.02. Society for Neuroscience, Washington, DC.

Summary: Alzheimer’s disease (AD) is the most common cause of dementia. Although the ultimate neurotoxic mechanisms are not known, extensive evidence supports the role of amyloid-beta (Aβ) deposition as senile plaques (SP) in the disease. On the other hand, neuronal loss is the pathological feature that best correlates with the duration and severity of the illness and specifically, cholinergic denervation of the basal forebrain seems to be a good predictor of clinical dementia in AD. A close relationship has been documented between Aβ deposition and neurodegeneration, however, whether specific neurodegeneration may lead to senile plaque deposition remains unclear. We addressed this by inducing selective cholinergic lesions in APPswe/PS1dE9 mice with murine p-75 saporin, an inmunotoxin that selectively removes cholinergic innervation. We performed intracerebroventricular murine p-75 lesions in animals with an incipient (~3 months) and robust (~7 months of age) Aβ deposition and removed ~50% of basal forebrain cholinergic innervation to cortex and hippocampus. Immediately after injections, cranial windows were implanted and Aβ deposition was monitored in vivo and in real time in the cortex using methoxy-XO4 and multiphoton microscopy. We observed increased SP deposition as soon as 1 week after the lesion. We further corroborated our in vivo data post-mortem, using anti- Aβ and anti-fibrils antibodies as well as thioflavin S staining, both in the cortex and the hippocampus. 7 days after the surgery, when the lesion is established, animals were tested in the new object discrimination and Morris water maze tests. We observed an early memory impairment in young lesioned mice (~3 months) and this effect worsened with age (~7 months of age), when Aβ deposition is more robust. Altogether, our data suggest that cholinergic denervation may contribute to the deposition of Aβ and synergistically contribute to the cognitive impairment observed in AD.

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Holschbach MA, Lonstein JS (2011) Diminished norepinephrine release in the BSTv decreases anxiety but does not promote maternal behavior in nulliparous female rats. Neuroscience 2011 Abstracts 86.06. Society for Neuroscience, Washington, DC.

Summary: Postpartum caregiving heavily depends on both increased motivation to interact with offspring and decreased emotional reactivity. The early postpartum period is associated with reduced anxiety in mammals, which may promote contact with potentially anxiogenic young. The ventral bed nucleus of the stria terminalis (BSTv) is associated with both anxiety and maternal behaviors in laboratory rats and may be a site of integration for mediating tradeoffs between mothering and emotional reactivity. Our laboratory has previously shown that increasing norepinphrine (NE) release in the BSTv of postpartum rats via infusion of an autoreceptor antagonist increases dams’ anxiety behaviors to levels seen in untreated virgin rats. Interestingly, this treatment also disrupts maternal retrieval of pups (Smith and Lonstein, SFN 2009). Unlike postpartum rats, nulliparous females are not spontaneously maternal, and we hypothesized that if NE release in the BSTv disrupts maternal behaviors even in highly motivated postpartum rats, it may greatly hinder expression of maternal behaviors in virgins. To investigate whether depleting NE input to the BSTv is sufficient to reduce anxiety and promote maternal behavior in virgin female rats we injected an antiserum- based neurotoxin selective for noradrenergic fibers and cells (anti-dopamine beta-hydroxylase-saporin; anti-DBH-SAP; 50 mg/side), into the BSTv of ovariectomized virgin female rats. Two weeks later, we examined females’ anxiety behavior in an elevated plus maze and the next day began a maternal sensitization procedure. We placed three recently fed pups into each animal’s homecage and observed behavior for the following fifteen minutes each day until rats exhibited full maternal behavior (i.e. retrieved all three pups to a common nest site and hovered over them) during three consecutive tests. Histological analysis of the brains confirmed that anti-DBH-SAP greatly reduced NE fiber content in the BSTv. Compared to control animals injected with artificial CSF, animals injected with anti-DBH-SAP showed reduced anxiety in an elevated plus maze. Anti-DBH-SAP did not, however, reduce the latency to show full maternal behavior. Thus, although reduced anxiety permits or promotes expression of maternal behaviors in already motivated postpartum rats, reducing BSTv-mediated anxiety is not sufficient to facilitate maternal responsiveness without otherwise activating maternal motivational systems.

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Ritter S, Li A-J, Wang Q, Dinh TT (2011) Basal metabolic substrate utilization is altered by lesion of hindbrain catecholamine neurons that innervate the medial hypothalamus and substrate selection during glucoprivation is impaired. Neuroscience 2011 Abstracts 88.05. Society for Neuroscience, Washington, DC.

Summary: Central injection of the targeted immunotoxin, anti-dopamine beta hydroxylase (DBH)-saporin (DSAP), retrogradely and selectively lesions norepinephrine (NE) and epinephrine (E) neurons with projections to the injection site. Previous work has shown that DSAP injections targeting the hypothalamic paraventricular nucleus eliminate key counterregulatory responses to acute glucose deficit, including feeding and corticosterone secretion. To examine the role of these NE an E neurons in metabolic control under basal conditions, we injected rats in the PVH with DSAP or control unconjugated saporin (SAP) and analyzed their metabolic profiles using metabolic chambers (Columbus Instruments). Rats were maintained on a standard pelleted rodent diet. We found that the respiratory exchange ratio (RER) was consistently elevated in DSAP rats across the entire circadian cycle under basal conditions, compared to the RER of SAP controls, indicating increased dependence on carbohydrate utilization. Metabolic rate and activity did not differ between groups. This result suggests a chronic enhancement of glucose mobilization or an impairment of the ability to mobilize fatty acids in the DSAP rats. We also found that when challenged by 2-deoxy-D-glucose induced glucoprivation, SAP controls exhibited a rapid decrease in RER, indicating a switch to fat metabolism, whereas DSAP rats did not exhibit this response. Together these results favor the possibility that a central mechanism for fat mobilization is impaired in DSAP rats and that this impairment is reflected under both basal and glucoprivic conditions. The previously reported observation that PVH DSAP-injected rats exhibit a slowly-developing obesity also supports this possibility. Additional findings suggest that this impairment may be due to the loss of NE/E control of corticosterone secretion in the DSAP rats.

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Cyr, M Maclaren DA, Bédard M-A, Clark SD, Mechawar N, Rochford J, Winn P (2011) Highly selective lesion of the cholinergic pedunculopontine neurons using a minimally-invasive angular stereotaxic surgery with the Diphteria-Urotensin-II neurotoxin in rat. Neuroscience 2011 Abstracts 37.06. Society for Neuroscience, Washington, DC.

Summary: Highly selective cholinergic lesions of the basal forebrain can be achieved with the immunotoxin 192-IgG saporin. This toxin has no effect however on the cholinergic neurons of the pedunculopontine tegmental nucleus (PPTg). For many years, most studies have used excitotoxins such as ibotenate, quisqualate, kainate, or N-methyl-D-aspartate, with a relative efficacy in targeting the PPTg cholinergic neurons, however these toxins also destroy the interdigitated glutamatergic and GABAergic neurons. More recently, selective cholinergic lesions were obtained with the Dtx-UII neurotoxin in both rats (Clark et al., 2007) and monkeys (Karachi et al., 2010). This toxin binds at the Urotensin-II receptor predominantly expressed in the pedunculopontine and the laterodorsal, but not the basal forebrain cholinergic nuclei. Because of the scattered distribution of the cholinergic neurons in the rat PPTg, infusion of the Dtx-UII requires multiple skull holes and needle lowering through areas containing critical blood vessels, increasing therefore surgery time, incidence of bleeding and mortality rate. Here, we report that these disadvantages can be avoided by doing a single Dtx-UII infusion, through an angular stereotaxic pathway. Results were contrasted with those obtained from the classical flat skull stereotaxic surgery used by Clark et al. (2007). Long Evans rats (males 250g – 300g) were operated according to three different methods. In group one, 3μl of Dtx-UII (3% concentration) was infused evenly in three unilateral stereotaxic coordinates along the PPTg (pars oralis, centralis, caudalis), using a flat skull position. In groups two and three, 2μl and 3μl of Dtx-UII were infused respectively using the angular stereotaxic method described by Wishaw et al. (1977). Incisor bar was elevated such that there was an 8º29’ angle (.147) between the latter and the interaural line. Following rat sacrifices, ChAT and NeuN immunohistochemistry were conducted in order to determine the cholinergic specificity and magnitude of the lesions. Results revealed similar PPTg cholinergic lesions between the three groups, reaching > 80% on the side of the lesions. Group 1 showed the greatest non specific lesions outside the PPTg, attributable to the needle pathways. This group of rats also showed the greatest number of surgical complications. We conclude that the cholinergic PPTg neurons can be optimally lesioned by using an angular surgical approach with the Dtx-UII toxin. Clark S.D., et al. (2007). J Neurochem., 102, 112-120. Karashi C., et al. (2010). J Clinical Investigation, 120, 2745-2754. Wishaw et al. (1977). Physiol. Behav., 19, 719-722.

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Parent M, Rosa-Neto P, Aliaga A, Soucy J-P, Bedard M-A (2011) [18F] fluoroethoxybenzovesamicol (FEOBV): A reliable PET radiocompound for the in-vivo assessment of cholinergic terminals. Neuroscience 2011 Abstracts 37.13. Society for Neuroscience, Washington, DC.

Summary: The vesicular acetylcholine transporter (VAChT) can be used as a surrogate target for PET imaging of brain cholinergic terminals. [18F]fluoroethoxybenzovesamicol (FEOBV) appears as a promising VAChT radioligand for PET imaging (Mulholand et al., 1998). Its pharmacokinetics, metabolism, and brain distribution have been well described in rodent and in primates (Kilbourn et al., 2010; Landry-St-Pierre et al., 2006; Soucy et al., 2010). The current study aims to assess the availability of VAChT binding sites in animals with presynaptic deficits induced by age or experimental lesions. We predict declines of FEOBV binding in brain regions innervated by cholinergic fibers. Twenty-one male Long-Evans rats were evenly divided in three groups: 1) Young rats (one month old); 2) Older rats (18 months old); 3) Rats with unilateral cholinergic lesions. In the latter group, 192-IgG-saporin (0.5 μg/μl) was infused under stereotaxic control into the nucleus basalis magnocellularis (NBM). A three weeks recovery period followed the surgery. FEOBV PET was conducted with a microPET (Siemens R4) on anesthetized animals. FEOBV (~11MBq) was injected and radioactivity measured in 27 sequential time frames of increasing duration, from 30 s to 5 min, for a total duration of 60 min. Images were reconstructed using a Maximum A Posteriori (MAP) algorithm, coregistered to a typical rat MRI template, and binding potential (BP) was calculated using the cerebellar cortex as reference tissue. Student t-tests were carried out at the voxel level: 1) Between lesioned (n=7) and non-lesioned (n=14) rats; 2) Between young (n=7) and old (n=7) non-lesioned rats. In lesioned rats, maximal BP reduction was observed in the ventral frontal cortex on the side of the lesion (t=6.5, p<0.0005, µ=41.88 mm3). Aged rats show significant clusters of BP reduction in both hippocampi (t=7.5 p<0.0005 24.61 mm3). We conclude that FEOBV PET allows quantification of cholinergic denervation following both normal aging and surgically induced cholinergic lesions.

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