sfn2011

Wiater MF, Jansen H, Oostrom M, Li A-J, Dinh T, Ritter S (2011) Lesions targeting leptin-sensitive neurons in the mediobasal hypothalamus dissociate activity and temperature circadian rhythms. Neuroscience 2011 Abstracts 396.11. Society for Neuroscience, Washington, DC.

Summary: Previously we investigated the role of NPY and leptin sensitive networks in the mediobasal hypothalamus in sleep and feeding and found profound regulatory and circadian deficits. We propose that the MBH, particularly the arcuate nuclei (Arc), is required for the integration of homeostatic circadian systems including temperature and activity. We tested this hypothesis with the use of the saporin toxin conjugated to leptin (Lep-SAP) or a blank molecule with no known biological function or receptor (B-SAP) directed to the Arc. Lep-SAP binds to, is internalized by and destroys leptin receptor expressing neurons at the injection site. Lep-SAP rats became obese and hyperphagic and progressed through a dynamic phase to a static phase of growth similar to a ventromedial lesioned rat. Activity and temperature data were collected using intraperitoneal PDT-4000 Emitters with Vital View Data Acquisition Software (Mini Mitter, Philips Respironics, Bend, OR). Circadian rhythms were examined over 49 days during the static phase of obesity in B-SAP (n=10) and Lep-SAP (n=12) rats. Rats were maintained on a 12:12 light:dark (LD) schedule for 13 days and thereafter maintained in continuous dark (DD). After the first thirteen days of DD, food was restricted to four hours per day from 9AM until 1PM for ten days. Immediately thereafter, rats were fasted for three days to evaluate persistence of food-entrained rhythms. Using ClockLab software (Natick, MA) actograms and tempograms were generated as double raster plots. Lomb-Scargle periodograms were used to assess rhythms and their robustness. We found that Lep-SAP rats were arrhythmic for activity in DD, but that food anticipatory activity was nevertheless entrainable to the restricted feeding schedule and the entrained rhythm persisted during the subsequent 3-day fast. Thus, for activity, the light-entrainable oscillator, but not the food entrainable oscillator, was disabled by the MBH lesion. In contrast, temperature remained rhythmic in DD in the Lep-SAP rats, but did not entrain to restricted feeding. We conclude that the leptin-sensitive network of the Arc and MBH is required for entrainment of activity by photic cues and for entrainment of temperature by food and for the integration of these rhythms.

Related Products: Leptin-SAP (Cat. #IT-47), Blank-SAP (Cat. #IT-21)

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.

Related Products: 192-IgG-SAP (Cat. #IT-01)

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.

Related Products: 192-IgG-SAP (Cat. #IT-01), GAT1-SAP (Cat. #IT-32)

Donahue RR, Corder GF, Mcnamara KC, Wiley RG, Taylor BK (2011) Transmission of neuropathic pain by spinal neurons expressing the NPY Y1 receptor. Neuroscience 2011 Abstracts 179.16. Society for Neuroscience, Washington, DC.

Summary: Endogenous neuropeptide Y (NPY) acts at Y receptors in the dorsal spinal cord to exert a tonic inhibitory control of chronic allodynia (Solway et al, PNAS 108:7224-9, 2011). In this and the adjacent presentation, we tested the hypothesis that NPY does this by inhibiting Y1 receptors on pain transmission neurons or on central terminals of primary afferent neurons. We selectively lesioned cells expressing the NPY receptors in the dorsal horn with intrathecal administration of the NPY-conjugated ribosomal toxin, NPY-saporin. NPY-saporin significantly reduced the population of Y1 receptors in the lumbar dorsal horn by over 50%. Neither NK1 receptors in the dorsal horn, nor neuronal counts in the DRG were affected, suggesting a specific effect on Y1+, NK1- neurons in the dorsal horn, while sparing Y1+ central presynaptic terminals. Fourteen days later, we ligated the tibial and common peroneal branches of the sciatic nerve (spared nerve injury, SNI), and evaluated the development of allodynia and hyperalgesia on post-SNI days 1, 3, 5, 7, 14, 21, 28, 35, and 42. When compared to saporin controls, NPY-saporin (1000 ng) decreased mechanical allodynia (von Frey threshold), cold allodynia (paw withdrawal response to application of a drop of acetone) and mechanical hyperalgesia (paw response to blunt pin). This effect began three days after SNI and lasted until forty two days after SNI. When injected in uninjured rats, NPY-saporin did not disrupt motor coordination (accelerating rotarod), baseline heat or mechanical thresholds, or animal activity levels. We conclude that Y1-expressing cells in the dorsal horn exert a tonic facilitatory control of neuropathic pain, and partially mediate the inhibitory actions of NPY.

Related Products: NPY-SAP (Cat. #IT-28)

Corder GF, Donahue R, Winter MK, Chen W, Mccarson KE, Marvizon J, Taylor B (2011) Activation of postsynaptic NPY Y1 and presynaptic Y2 receptors reduce spinal nociceptive transmission. Neuroscience 2011 Abstracts 179.17. Society for Neuroscience, Washington, DC.

Summary: Exogenous (Intondi et al, Neuroscience, 2008) and endogenous (Solway et al, PNAS 108:7224-9, 2011) neuropeptide Y (NPY) acts at Y1 and Y2 receptors in the dorsal horn (DH) to inhibit hypersensitivity to mechanical and thermal stimuli. The adjacent poster (Donahue, et al, SFN 2011) describes our use of a targeted NPY-saporin neurotoxin approach to selectively remove spinal cord (SC) neurons expressing the Y1 receptor — the data implicate a contribution of Y1-expressing, pain transmission neurons to behavioral signs of persistent pain. To determine whether persistent noxious input is associated with a compensatory increase in NPY-mediated inhibitory signaling (presumably at Y1-expressing DH neurons), we performed GTPγS binding assays in SC slices taken from animals following the intraplantar (i.pl) injection of complete Freund’s adjuvant (CFA). CFA significantly reduced the EC50 of Y1 agonist (Leu31,Pro34-NPY)-induced [35S]GTPγS binding in ipsilateral DH to 0.24 ± 0.17 μM, as compared to sham (1.38 ± 0.51 μM). This support the hypothesis that injury increases in the efficiency of coupling between Y1-receptors and G-proteins. To determine whether compensatory NPY inhibition occurs at presynaptic sites, we studied the activity of presynaptic Y2 receptor in NPY-saporin-treated rats. Intrathecal injection of the Y2 receptor antagonist BIIE0246 reduced von Frey thresholds (saporin group from 1.3±0.4 to 0.6 ±0.1g; 750 ng NPY-saporin group from 5.4±1.0 to 1.2±0.2g, p<0.05), suggesting that presynaptic Y2 receptors contribute to a tonic endogenous inhibition of inflammatory pain. In support of this hypothesis, BIIE0246-induced hyperalgesia (21 days after CFA) significantly increased the Emax of Y2 agonist (PYY3-36)-induced [35S]GTPγS binding. We next determined whether NPY acts at presynaptic terminals of primary afferent neurons to reduce the release of substance P (SP). First, in both the i.pl carrageenan and CFA models of inflammatory pain, intrathecal administration of NPY reduced in vivo neurokin-1 (NK1) receptor internalization (an indirect measure of functional SP release). Second, application of either (Leu31,Pro34)-NPY) or PYY3-36 to spinal cord slices concentration-dependently reduced NK1 internalization in the ipsilateral dorsal horn evoked by electrical stimulation of the dorsal root (1000 pulses of 20 V, 0.4 ms at 100 Hz); these effects were reversed by the Y1 antagonist BIBO3304. We conclude that injury up-regulates post-synaptic Y1 and pre-synaptic Y2 spinal inhibitory mechanisms to reduce behavioral signs of persistent pain.

Related Products: NPY-SAP (Cat. #IT-28)

Sinha SP, Roland JJ, Servatius RJ, Pang KCH (2011) The role of medial septal/diagonal band GABAergic neurons in proactive interference: Effects of selective immunotoxic lesions in latent inhibition. Neuroscience 2011 Abstracts 199.22. Society for Neuroscience, Washington, DC.

Summary: The medial septum/diagonal band (MSDB) is a critical structure for learning and memory, yet the functional contributions of its individual neuronal populations (including cholinergic, GABAergic, glutamatergic and peptidergic cells) are still being characterized. Recent studies have implicated a contributing role for the GABAergic MSDB neuronal population, as selective immunotoxic GABAergic lesions of the MSDB (with GAT1-saporin) produce behavioral impairments in spatial and instrumental tasks. Compared to intact controls, rats with GABAergic MSDB lesions are impaired in learning new spatial locations in a delayed match to position procedure and also exhibit a slower rate of extinguishing a previously acquired avoidance response – behaviors that are consistent with an exacerbation of proactive interference. To further establish the role of these neurons in proactive interference, this study examined the effects of selective GABAergic MSDB lesions in latent inhibition (LI) of the classically conditioned eyeblink response. LI in delay eyeblink conditioning is a phenomenon in which pre-exposure to the conditioned stimulus (CS) interferes with the subjects’ ability to subsequently associate the CS with an unconditioned stimulus (US), resulting in slower acquisition of the conditioned response (CR). We hypothesized that if damage of GABAergic MSDB neurons increases proactive interference, then rats with selective lesions of these neurons would show facilitated LI. Male Sprague-Dawley rats (n=18) were administered either phosphate-buffered saline or GAT1-saporin via intracranial injection into the MSDB. After 7-10 days of recovery, electrodes were implanted into the upper eyelids of the rats for delivery of US and EMG recording. Conditioning began after another 5-7 days of recovery, with Day 1 consisting of 30 minutes of acclimation to the conditioning context. Day 2 began with either 30 presentations of the CS (82dB, 500ms white noise, 25 – 35s ITI) or context pre-exposure of equal duration, followed immediately by 100 paired CS-US trials (82 dB, 500ms white noise co-terminating with a 10V, 10ms square-wave stimulus). In preliminary results, intraseptal GAT1-saporin did not alter CR acquisition in context pre-exposed rats. Rats with GABAergic MSDB lesions continued to exhibit latent inhibition. These preliminary results do not support the idea that damage of GABAergic MSDB neurons increase proactive interference of the classically conditioned eyeblink response. Future studies will examine whether manipulations of the number of CS pre-exposures would facilitate LI in rats with GABAergic MSDB lesions.

Related Products: GAT1-SAP (Cat. #IT-32)

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.

Related Products: Melanopsin-SAP (Cat. #IT-44)

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.

Related Products: 192-IgG-SAP (Cat. #IT-01)

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.

Related Products: SSP-SAP (Cat. #IT-11)

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.

Related Products: 192-IgG-SAP (Cat. #IT-01)