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Alterations in gene expression following cortical cholinergic denervation in rats
Savage ST, Olson L, Mattsson A (2011) Alterations in gene expression following cortical cholinergic denervation in rats. Neuroscience 2011 Abstracts 790.01. Society for Neuroscience, Washington, DC.
Summary: Alterations in cholinergic signaling in the brain have been implicated as a contributing factor in the pathogenesis of schizophrenia. Altered function and expression of both nicotinic and muscarinic acetylcholine receptors have been reported in cortical and subcortical regions in post-mortem schizophrenic brains. Pharmacologically, dopamine-releasing compounds, such as amphetamine, can induce the psychotic symptoms in healthy volunteers and exacerbate the symptoms in schizophrenics. Furthermore, the NMDA receptor antagonist phencyclidine (PCP) induces both negative symptoms (such as social withdrawal) and cognitive deficits similar to those exhibited in schizophrenics. We have previously shown that cholinergic denervation of cortex cerebri by stereotaxic infusion of the immunotoxin 192 IgG-saporin in the nucleus basalis magnocellularis (nbm) in adult rats leads to an enhanced sensitivity to both amphetamine and PCP. The enhanced sensitivity to amphetamine, shown by a potentiated dopamine release in nucleus accumbens, along with a marked increase in locomotor activity in response to both amphetamine and PCP, suggested that the disruption of cortical cholinergic activity can lead to disturbances of glutamatergic and dopaminergic transmission. Furthermore, bilateral lesioning of nbm led to a decrease in active social interaction, as well as, impairment after an acute PCP challenge in a cognitive task (novel object recognition). To further evaluate the consequences of cortical cholinergic denervation, we are analyzing the possible changes in mRNA expression levels of selected genes in rats with unilateral removal of the cortical cholinergic innervation by 192 IgG-saporin injections into nbm following acute PCP administration. Our data indicate that the induction of c-fos mRNA expression in cortex in response to PCP administration is markedly reduced in cholinergically denervated animals as compared to controls. Other genes are under investigation to elucidate the interplay between the cholinergic, dopaminergic, and glutamatergic systems.
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Decrease of GABAergic markers and Arc protein expression in the frontal cortex by injection of intraventricular 192 IgG-saporin
Jeong D, Chang W, Lee D, Chang J (2011) Decrease of GABAergic markers and Arc protein expression in the frontal cortex by injection of intraventricular 192 IgG-saporin. Neuroscience 2011 Abstracts 878.08. Society for Neuroscience, Washington, DC.
Summary: Previous studies used 192 IgG-saporin to study cholinergic function because of its facility for selective lesioning; however, results varied due to differences in the methods of administration and behavioral tests used. We investigated whether intraventricular injections of 192 IgG-saporin were suitable to make a dementia animal model for the evaluation of therapeutic drugs or electrical stimulation techniques. We examined the effects of 192 IgG-saporin using the Morris water maze, immunochemistry, and western blotting. Animals were examined 2 weeks after intraventricular injection of 192 IgG-saporin (0.63 µg/µl, 6 µl, 8 µl, and 10 µl) or phosphate buffered saline (8 µl). In the acquisition phase of the Morris water maze, the latencies of the injection groups were significantly delayed, but recovered within 1 week. In the probe test, two of four indices (time in the platform zone and the number of crossings) were significantly different between the control group and the group injected with 8 µl of 192 IgG-saporin. Immunohistochemistry revealed the extent of cholinergic destruction that was apparent in the basal forebrain of all 192 IgG-saporin injected rats. We found significantly decreased activity-regulated cytoskeleton associated protein (Arc) and glutamate decarboxylase (GAD) expression in the frontal cortex (8 µl and 10 µl groups), but not in the hippocampus, using western blotting. Further, spatial memory impairment was associated with cholinergic basal forebrain injury as well as fronto-cortical GABAergic hypofunction and synaptic plasticity deceleration. We conclude that intraventricular injection of 192 IgG-saporin is a suitable method for making a rat model of dementia.
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P300-like event related potentials in IgG192-saporin induced rat model of Alzheimer´s disease
Clausen B, Klipec W, Bastlund J, Collins M (2011) P300-like event related potentials in IgG192-saporin induced rat model of Alzheimer´s disease. Neuroscience 2011 Abstracts 550.01. Society for Neuroscience, Washington, DC.
Summary: The P300 event-related potential (ERP) is a time-locked response to rare, response-relevant stimuli. Decreased ERP amplitude is correlated with decreased memory function. Not surprisingly, alterations in P300 ERP amplitude are commonly associated with the progressive disruption of cognitive function in human Alzheimer’s disease. Here, a rat model of Alzheimer’s disease was created by injecting the antibody-linked toxin, IgG192-saporin, into the basal forebrain, producing a progressive degeneration of cholinergic cells to mimic the cholinergic degeneration that is part of Alzheimer’s disease. The goal of this experiment was to investigate the rat model by examining the relationship between the expected degenerative deficits and possible changes in the EEG patterns. Following preliminary training that has produced reliable P300-like ERPs in prior experiments in our lab, half of the rats were injected with IgG192-saporin (lesioned), while the other half were injected with saline (controls). Recording electrodes were surgically implanted on the surface of the brain and in the prefrontal cortex (PFC) and ventral hippocampus (vHipp). Following recovery, P300-like ERP data was recorded for three weeks, after which a ChAT analysis of choline acetyltransferase activity confirmed the extent of cholinergic damage in PFC and Hipp. While no systematic increases in latency were found, surprisingly, significant increases in P300-like ERP amplitude occurred in PFC and vHipp in the lesioned compared to the control rats. The implication of these findings for a rat model of Alzheimer’s disease will be discussed.
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Cholinergic reinforcement and temporal learning in rodent visual cortex
Roach EB, Hussain Shuler MG (2011) Cholinergic reinforcement and temporal learning in rodent visual cortex. Neuroscience 2011 Abstracts 608.16. Society for Neuroscience, Washington, DC.
Summary: The idea that neuromodulators act as reinforcement signals has an intricate scientific history, including the well-characterized analogue of prediction error relayed by midbrain dopamine neurons. Neuromodulators released from discrete nuclei are poised to broadcast to many brain regions at once, and so it is an appealing concept to investigate other neuromodulatory systems within a reinforcement learning framework. Reward timing activity, a neural reflection of operantly learned stimulus-reward intervals in the primary visual cortex (V1), offers a tractable in vivo model to examine the role of candidate neuromodulators in temporal reward learning. Reward timing was first characterized in rats trained to lick a delivery tube to receive water rewards, where stimulation to one eye indicated reward availability after x licks, while stimulation to the other eye required y licks. Simultaneously recorded activity in V1 indicated that single unit responses evolve from reporting only visual characteristics to showing persistent increased/decreased firing or peak activity corresponding to the time of anticipated reward. Individual neurons report one interval or the other, even those with binocular peri-stimulus responses, arguing that reward timing is learned locally within V1. Theoretical work suggests that the local expression of reward associated intervals requires an interaction between the visually-evoked network response and a reinforcement signal conveying the time of reward. Based on anatomical and neurophysiological evidence, we hypothesized that cholinergic input from the basal forebrain (BF) could provide such a reward signal to V1. To test its necessity, BF cholinergic innervation in V1 was lesioned — using the selective neurotoxin 192 IgG-saporin — prior to changing the experimental policy between cues and associated reward delays. This allowed an examination of two potential roles for BF cholinergic input: in expressing previously learned intervals and in acquiring information about new intervals. We found that neurons from saline-infused controls, but not lesioned animals, shifted as a population to report the new, behaviorally relevant intervals (Kolmogorov-Smirnov, p < 0.05). Importantly, neurons from lesioned animals continued to report the previously learned intervals, suggesting that BF cholinergic input is required to learn, but not express, reward timing. These results support the notion that acetylcholine released from BF afferents acts as a reinforcement signal that guides cortical network plasticity.
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Cognitive performance-associated increases in cholinergic neurotransmission also serve as a circadian signal to sustain performance-induced diurnal activity patterns.
Paolone G, Lamy D, Sarter M, Lee T (2011) Cognitive performance-associated increases in cholinergic neurotransmission also serve as a circadian signal to sustain performance-induced diurnal activity patterns. Neuroscience 2011 Abstracts 610.12. Society for Neuroscience, Washington, DC.
Summary: Daily practice of a sustained attention task (SAT) during the light phase of the light/dark cycle causes a stable, entrained, diurnal behavioral activity pattern (Gritton et al. 2009). As SAT performance is mediated by increases in cortical cholinergic neurotransmission, this experiment assessed levels of acetylcholine (ACh) release across the light and dark cycle of animals that previously performed the SAT at a fixed time. Circadian behavioral activity was recorded, and prefrontal ACh release was measured, using microdialysis, beginning on the third day following the last SAT session. SAT practice took place in either the light phase [ZT4], the dark phase [ZT16], or in a constant light condition [LL]. A control group practiced a daily fixed interval [FI-9] schedule of reinforcement at ZT4. A second control group was handled at randomly selected times but was neither water-deprived nor performed a task [NP]. Dialysates were collected, in a new environment, for 180 min total, beginning 90 min before the onset of prior task practice and again during the equivalent time period twelve hours later. For all animals, ACh release levels were higher during the dark phase. In SAT-performing animals, ACh levels increased for 45 min at ZT4 and ZT16. In addition, the ZT4 animals’ behavioral activity was robustly increased during this interval. Animals trained at ZT 4 reversed back to a nocturnal activity pattern 8-10 days after cessation of SAT practice, coinciding with the loss of the task time-synchronized cholinergic activity. In order to determine the necessity of these prior task period-synchronized release events for maintaining diurnal activity patterns, basal forerbain cholinergic neruons were lesioned by intra-basalis infusion of 192 IgG-saporin. As was expected, this lesion impaired SAT performance. Furthermore, following cessation of daily SAT practice, prior performance-period synchronized cholinergic release events were abolished in lesioned animals. Moreover, the lesion triggered a rapid post-performance return to a nocturnal acitvity pattern. Collectively, these results indicate that SAT performance-associated increases in prefrontal cholinergic activity not only support SAT performance but also contribute to cognition-induced diurnality. Furthermore, circadian control of cholinergic activation optimizes task performance as well as the generation of a cholinergic zeitgeber signal. In conclusions, the brain’s clocks and increases in cortical cholinergic neurotransmission interact bidirectionally to sustain cognitive performance and performance-evoked diurnal activity patterns.
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Cholinergic neurons of the basal forebrain and nitric oxide-mediated regulation of sleep homeostasis.
Kalinchuk AV, Porkka-Heiskanen T, Mccarley RW, Basheer R (2011) Cholinergic neurons of the basal forebrain and nitric oxide-mediated regulation of sleep homeostasis. Neuroscience 2011 Abstracts 397.15. Society for Neuroscience, Washington, DC.
Summary: The levels of adenosine (AD) and inducible nitric oxide (NO) synthase (iNOS)-mediated NO increase during sleep deprivation (SD) in the basal forebrain (BF), and, with prolongation of SD, in the frontal cortex (FC). NO donor (DETA NONOate) infusion increases AD and sleep, while iNOS/NO inhibition prevents SD-induced AD increase, suggesting that iNOS/NO stimulates AD increase (Kalinchuk et al., 2006). iNOS induction during SD occurs in wake-active neurons in the BF and FC (Kalinchuk et al., 2010, 2011), however, neurotransmitter specificity of these cells has not described. The lesion of BF cholinergic cells attenuates both SD-induced AD increase and recovery sleep response (Kalinchuk et al., 2008). Hence in this study, we tested the role of cholinergic versus non-cholinergic neurons in iNOS/NO release in BF and FC and homeostatic sleep response. Methods. We performed two types of experiments. Experiment #1. Immunohistochemical detection of neurotransmitter specificity of cells inducing iNOS during SD. The brains of SD animals and their non-SD time-of-day matched controls were subjected to double-labeling with specific markers for iNOS, acetylcholinetransferase (ChAT), vesicular glutamate transporters (VGlut) and glutamate decarboxylase (GAD67). Experiment #2. The effects of SD on iNOS/NO production and the effect of NO-donor, DETA NONOate infusion on sleep were investigated before and after destruction of BF cholinergic neurons using 192 IgG-saporin. In both experiments male rats were implanted for electrographic recording and Experiment # 2 used guide cannula for microdialysis probes targeting BF and FC. In Experiment #2, recording of sleep-waking cycle, SD for 3h and infusion of DETA NONOate for 3h were performed on the same animals before and 2 weeks after targeted saporin injections. Results. Experiment #1. SD led to significant increases in number of iNOS+ cells in the BF and FC. Preliminary data showed that in the BF, in SD group, 96% of ChAT+ cells were also iNOS+, while in the non-SD group only 4% of ChAT+ neurons had weak iNOS+ staining. Numbers of iNOS+/ChAT+ cells positively correlated with SD-induced increase in theta power. Experiment #2. Before saporin injection, both SD and infusion of DETA NONOate induced significant increases in subsequent NREM sleep/NREM delta power (by 35/47% and 39/41%, respectively). After saporin injection, both recovery NREM sleep and DETA NONOate-induced sleep were significantly attenuated (8 and 4% increase as compared with baseline) and increases in delta power were totally blocked. Conclusions. We conclude that cholinergic neurons of the BF are important for iNOS/NO-mediated homeostatic sleep control.
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Role of the medial septal-diagonal band nucleus in working memory: Effects of cholinergic or GABAergic lesions on memory demand and interference
Stewart AL, Roland JJ, Servatius RJ, Pang K (2011) Role of the medial septal-diagonal band nucleus in working memory: Effects of cholinergic or GABAergic lesions on memory demand and interference. Neuroscience 2011 Abstracts 513.09. Society for Neuroscience, Washington, DC.
Summary: The medial septum and diagonal band of Broca (MSDB), a major source of afferents to the hippocampal formation, is critical for learning and memory. The primary cells comprising the septohippocampal systems are cholinergic, GABAergic and glutamatergic. Selective damage of cholinergic MSDB neurons results in mild to no impairment of spatial working memory tasks, suggesting that non-cholinergic MSDB projections are important in learning and memory. Recently, we demonstrated that GABAergic MSDB lesions impair a delayed match to position task (DNMTP) with errors suggesting enhanced proactive interference. The current study assesses the effect of manipulating the intertrial interval (ITI) and retention interval (RI) on DNMTP performance in normal rats and those with cholinergic or GABAergic MSDB damage. In addition, activation of MSDB neurons on the last day of training will be assessed. Male Sprague Dawley rats receive sham, 192-IgG saporin (192-Sap) or GAT1-saporin (GAT1-Sap) administration into the MSDB before training on a DNMTP task using a T-maze. On the sample phase of each trial, rats are forced to one arm for reinforcement. Following an RI, a choice phase allows the rats to choose from both arms. Rats are reinforced for choosing the arm not entered during the sample phase. Following the choice phase, an ITI occurs before the sample phase of the next trial. Rats are trained one session per day, 12 trials per session, and 10 sessions. In a 2 x 2 experimental design, each rat is trained on either a 0 or 60s RI and a 0 or 60s ITI. Conditions with a long RI (60 s) are designed to tax working memory, whereas conditions with a similar RI and ITI are designed to increase interference. Immunocytochemistry for c-Fos is used to assess activation of cholinergic or GABAergic MSDB neurons following the last training session. As expected, our preliminary results show that sham rats performed better on 0 s than 60 s RI (0 s = 78% correct vs 60 s = 57%, both ITI’s pooled). Analysis of the 0 s RI demonstrates that performance in conditions with different RI and ITI was better than when RI and ITI were similar (0 s RI/60 s ITI = 74% vs 0 s RI/0 s ITI = 67%). Further analysis of the 60 s RI was difficult due to the near chance performance. Rats treated with either intraseptal 192-Sap or GAT1-Sap were impaired on the 0 s RI/60s ITI condition (Sham: 85%; 192-Sap: 65%; GAT1-Sap: 72%). However, only the 192-Sap rats were impaired in the 0 s RI/0 s ITI condition (Sham: 71%; 192-Sap: 62%; GAT1-Sap: 69%). Anatomical studies are currently underway. The results of this study will further elucidate the role of MSDB neurons in two aspects of working memory: memory demand and interference.
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Deficits in attentional control of balance, mobility, and complex movements in a rat model of early state, multisystem Parkinson disease
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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Cholinergic modulation of cross-modal attentional orienting
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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Highly selective lesion of the cholinergic pedunculopontine neurons using a minimally-invasive angular stereotaxic surgery with the Diphteria-Urotensin-II neurotoxin in rat
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.
Related Products: 192-IgG-SAP (Cat. #IT-01)