Abstracts from Society for Neuroscience (SFN) November 14-17, 2010 • San Diego, CA

37 entries found for : sfn2010

The effects of a combination of antioxidants and essential fatty acids as treatment for Alzheimer’s disease in the mu-p75 saporin-injected model

Matchynski JJ, Lowrance SA, Rossignol J, Dekorver NW, Puckett ND, Pappas CA, Trainor KJ, Delongchamp JL, Radwan J, Heldt JC, Dey ND, Dunbar GL (2010) The effects of a combination of antioxidants and essential fatty acids as treatment for Alzheimer’s disease in the mu-p75 saporin-injected model. Neuroscience 2010 Abstracts 856.15/I21. Society for Neuroscience, San Diego, CA.

Summary: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is marked by a progressive loss of memory and affects over five million people nationwide (Alzheimer’s Association, 2010). It is characterized by an increase in oxidative stress, amyloid plaques, neurofibrillary tangles, and the loss of cholinergic neurons. Mice injected with the ribosome deactivating protein, mu-p75 saporin, model the deficits in memory, loss of cholinergic neurons, and increased oxidative stress observed in AD. The current study aimed to decrease the deficits observed in the saporin mouse model using a combination of antioxidants from tart cherries and essential fatty acids, Cerise© total body rhythm (TBR). Mice dosed with TBR or methylcellulose were given bilateral ventricular injections of phosphate buffer saline or saporin. Memory and motor functioning were then measured in a series of behavioural tests. Results indicate that TBR decreased the memory deficits observed in object recognition, place recognition, and Morris-water-maze tasks, as well as the inflammatory response and loss of cholinergic neurons in the medial septum. The findings suggest that TBR could provide an effective, adjunctive treatment that may delay the onset or decrease the severity of AD.

Related Products: mu p75-SAP (Cat. #IT-16)

Molsidomine promotes the recovery of cognitive deficit induced by 192 IgG saporin in rats

Hernandez MA, Hernández-Melesio M, Gonzalez-Ezquivel D, Quevedo-Corona L, Jiménez-Cataño M, Santoyo-Pérez M, Rios-Castañeda C, Pérez-Severiano F (2010) Molsidomine promotes the recovery of cognitive deficit induced by 192 IgG saporin in rats. Neuroscience 2010 Abstracts 856.27/I33. Society for Neuroscience, San Diego, CA.

Summary: The NO donor molsidomine (MOLS) has been used as a pharmacological tool in order to antagonize the cognitive deficit associated to cholinergic hypofunction produced by scopolamine. However, the participation of NO in the recovery of cholinergic deficit due to the administration of the cholinergic immunotoxin, 192 IgG saporin (SAP) has not been analyzed. The aim of the present study was to determine the effect of MOLS to counteract the cognitive deficits induced by cholinergic denervation in the object recognition task. Male Wistar rats were divided in the follow experimental groups according to the intraseptal administration of SAP and its vehicle PBS, and the injection (i.p) of the NO donor molsidomine and its vehicle saline: SAP (0.22 µg), PBS (0.1M pH 7.4), molsidomine (4mg/kg), SAP/molsidomine. The single dose of MOLS used in this study antagonized the cognitive failure related to SAP administration and increased the exploration time of novel object. We conclude that MOLS promote the acquisition of recognition memory in the model of cholinergic denervation associated to 192 IgG SAP and further immunohistological studies are being carried out in order to demonstrate that nitric oxide could have an effect over the cholinergic functionality.

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

Leptin-saporin lesions of the arcuate nucleus induce an arrhythmic circadian feeding pattern

Wiater MF, Oostrom M, Barfield R, Dinh TT, Li A-J, Ritter S (2010) Leptin-saporin lesions of the arcuate nucleus induce an arrhythmic circadian feeding pattern. Neuroscience 2010 Abstracts 733.6. Society for Neuroscience, San Diego, CA.

Summary: The endogenous circadian rhythm of feeding is incompletely understood. The leptin sensitive network within the arcuate nucleus (Arc) of the hypothalamus is important for the control of feeding. Genetic deletion of leptin or leptin receptors results in profound obesity, hyperphagia, and the loss of day/night differences in food intake. Because the Arc is critically involved in control of food intake and contains leptin receptors, we hypothesized that the Arc plays an important role in maintenance of feeding rhythms. To examine this hypothesis, we injected a newly developed targeted toxin, leptin conjugated to saporin (LSAP), into the Arc to lesion leptin receptor-expressing neurons in the vicinity of the injection. Controls were injected saporin conjugated to a peptide with no known action or receptor (blank-saporin, BSAP). We expected the Arc LSAP would disrupt the circadian rhythm of food intake, as seen in rats with genetic deletion of leptin or its receptor. Eating rhythms were monitored continuously (each minute) over a 60-day period using BioDAQ (Research Diets) automated meal monitoring equipment. Data were analyzed for circadian rhythm using ClockLab (ActiMetrics) software. Eatograms (food intake in actogram format), showing eating times and durations comparable to actograms used for wheel-running activity, and Chi-square periodograms were generated. Feeding was monitored in light:dark, dark:dark, or light:light conditions. The LSAP injection caused profound hyperphagia, weight gain and disrupted circadian feeding patterns. Although LSAP rats remained sensitive to light and dark under certain circumstances and were capable of an apparent rhythm during light:dark conditions, feeding was arrhythmic by all measures when photic cues were removed (i.e., in dark:dark and light:light conditions). At the end of experimentation, lesions were analyzed using immunohistochemistry to detect agouti gene related protein (AGRP) and α-melanocortin stimulating hormone (α-MSH) neurons, both known to express leptin receptors. Cell bodies positive for these peptides were greatly diminished in the Arc. Results indicate that the Arc contributes importantly to the expression of circadian rhythms of food intake.

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

Krx-0501 reduces cognitive deficits in a saporin mouse model of Alzheimer’s disease

Lowrance SA, Matchynski JJ, Rossignol J, Dekorver N, Fink K, Salibi P, Dunbar GL (2010) Krx-0501 reduces cognitive deficits in a saporin mouse model of Alzheimer’s disease. Neuroscience 2010 Abstracts 856.8/I14. Society for Neuroscience, San Diego, CA.

Summary: Alzheimer’s disease (AD) is a progressive neurodegenerative disease marked by memory loss and dysfunction of cholinergic neurons. Neurotrophic factors, like nerve growth factor (NGF), have shown to improve cognitive function in AD patients. The inability of NGF to cross the blood brain barrier (BBB) and painful side effects have caused serious concerns over its future use as a treatment. Substituted pyrimidines, such as KRX-0501 (KRX; Keryx Biopharmaceuticals, New York, NY) on the other hand, readily cross the BBB and exert beneficial neurotrophic-like effects in vivo. In this study KRX was administered daily to mice that were given intra-cerebroventricular injections of mu p-75 Saporin (SAP; Advanced Targeting Systems, San Diego, CA) or vehicle (phosphate buffered saline). KRX treatment began at seven weeks of age and continued for 32 days. Doses were set at 10, 15, and 20mg/kg respectively. Animals were tested for cognitive impairment using the Morris water maze (MWM) task, object recognition (OR) and place recognition (PR) tasks while motor deficits were tested using MWM swim speed, rotarod (RR) and the open field (OF) tasks. On day 33 of KRX treatment, mice were sacrificed by transcardial perfusion. In a second experiment, mice received SAP or vehicle surgery and were sacrificed for enzyme-linked immunosorbent assay (ELISA) analysis. MWM results revealed significantly lower escape latencies of control animals relative to SAP animals. In addition mice treated with the low and middle doses of KRX displayed decreased escape latency on the MWM. In the OR task, only mice in the highest treatment group performed significantly above chance levels. No between group differences were seen in the PR task, swim speed, latency to fall from the RR, and distance travelled in the OF. Immunohistochemistry (IHC) using the glial fibrillary acidic protein antibody indicated that astrocyte activation took place primarily around the surgical injection sites. IHC labeling against choline-acetyltransferase revealed a significant decrease in cholinergic neurons of the medial septum. Finally, ELISA protein analysis of midbrain sections revealed that the KRX treatments did not increase levels of endogenous NGF. These results show that SAP injections produced a reproducible destruction of cholinergic neurons, accompanied by memory deficits in the MWM, in the absence of motoric deficits. The KRX treatment attenuated memory deficits, despite unabated cholinergic cell loss in the medial septum, and did so without affecting levels of endogenous NGF.

Related Products: mu p75-SAP (Cat. #IT-16)

Defining the role of norepinephrine in cannabinoid-induced aversion and anxiety

Carvalho AF, Van Bockstaele E (2010) Defining the role of norepinephrine in cannabinoid-induced aversion and anxiety. Neuroscience 2010 Abstracts 833.3. Society for Neuroscience, San Diego, CA.

Summary: In the central nervous system, cannabinoids have been shown to regulate neurotransmitter release, control the hypothalamic-pituitary-adrenal axis and impact several physiological systems, such as food intake, pain and emotion perception. Manipulation of the cannabinoid system using exogenous compounds has been explored as a potential therapeutic for several disorders; however some severe side effects have been reported. 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 may contribute to the expression of unwanted side effects. Previous work from our lab had shown that the cannabinoid receptor (CBr) agonist WIN 55,212-2 is able to induce changes in noradrenergic transmission in limbic structures such as prefrontal cortex (PFC) and the nucleus accumbens (Acb). Moreover, we have previously reported that norepinephrine in the nucleus accumbens (Acb) is critical for WIN 55,212-2-induced aversion, as measured by the place conditioning paradigm. In the present study, we further explore the role of norepinephrine in cannabinoid-induced behaviors. More specifically, we investigate whether norepinephrine in the limbic forebrain of rats is important for the anxiety induced by WIN 55,212-2 (3.0 mg/kg, i.p.). Lesion of noradrenergic neurons in the Acb and bed nucleus of the stria terminalis (BNST) was achieved by the intracerebral injection of the toxin saporin conjugated with an antibody that recognizes the enzyme dopamine-beta-hydroxylase (DBH). This toxin yields a specific lesion of noradrenergic neurons. The anxiogenic effects of WIN 55,212-2 were then measured in the elevated zero maze. The results show that depletion of noradrenergic innervation of the Acb and BNST did not reduce the anxiogenic properties of WIN 55,212-2. These results, together with our previous findings, suggest that the anxiogenic and aversive properties of the CBr agonist WIN 55,212-2 are differentially regulated, with the aversive effects being dependent on noradrenergic transmission within the Acb and the anxiogenic effects being regulated by a, yet to be determined, alternative mechanism/circuit.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Role of the septohippocampal GABAergic system in spatial orientation

Koppen JR, Winter SS, Cheatwood JL, Wallace DG (2010) Role of the septohippocampal GABAergic system in spatial orientation. Neuroscience 2010 Abstracts 806.16/KKK21. Society for Neuroscience, San Diego, CA.

Summary: Spatial orientation depends on the integrity of multiple neural systems. For example, during the progression of Alzheimer’s Disease, degeneration of the basal forebrain is associated with cognitive impairments including episodes of wandering. The medial septum projects both cholinergic and GABAergic fibers into the hippocampus. Research and therapies have typically focused on enhancing function of the cholinergic component; however, the GABAergic component has also been shown to contribute to hippocampal function. Previous attempts to characterize the role of the GABAergic system in spatial orientation involved non-selective lesion techniques in combination with the water maze task have failed to characterize the nature of the deficit mediating the impaired performance. Development of GAT1-Saporin immunotoxin provides a novel tool to selectively destroy GABAergic neurons in the medial septum. The current study examined the effects of injecting GAT1-Saporin or saline (sham lesion) into the medial septum on spatial orientation using the food-hoarding paradigm. The food-hoarding paradigm involves training rats to search for food pellets on a large circular table and carrying the food pellet directly to a visible refuge. Three probes dissociate the use of environmental and self-movement cues: 1) Hidden probe involved placing the refuge below the surface of the table, limiting rats to use distal environmental or self-movement cues to locate the refuge; 2) Dark Probe involved using the hidden refuge with the room lights off, limiting rats to use self-movement cues to locate the refuge; 3) New probe involved placing the hidden refuge on the opposite side of table, placing environmental and self-movement cues in conflict. Both sham and GAT1-Saporin rats were accurate in returning to the refuge during the Hidden probe. Only sham rats were accurate in carrying food to the refuge during the Dark probe. During the New probe, both groups initially carried the food pellet to the former refuge location. Although sham rats consistently carried the food pellet to the new refuge location after their initial error, GAT1-Saporin rats continued to perseverate to the former refuge location. The current study demonstrates a role for the septohippocampal GABAergic system in spatial orientation related to processing self-movement cues.

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

Evidence that focal hippocampal interneuron loss disrupts theta-rhythm activity in dorsal CA1

Rossi CA, Lehmkhule MJ, Kesner RP, Dudek FE (2010) Evidence that focal hippocampal interneuron loss disrupts theta-rhythm activity in dorsal CA1. Neuroscience 2010 Abstracts 811.1/LLL64. Society for Neuroscience, San Diego, CA.

Summary: Hippocampal theta activity (6-12 Hz) is an oscillatory local field potential that is thought to play a critical role in the encoding and storage of new information. As a hypothetical mechanism for theta rhythm generation, interneurons have been proposed to appropriately time the GABAergic inhibition of principal cells, as a means of organizing the theta pattern; however, little experimental work has been done to test this hypothesis directly. The current study aims to test in a relatively direct manner the hypothesis that interneurons synchronize the activity of pyramidal cells into theta-band oscillations. In the current study, SSP-saporin (a selective interneuron-targeting neurotoxic lesioning agent) was infused into six sites located in dorsal CA1 in order to create an interneuron-only lesion confined to that area. Animals were also implanted with chronic field potential recording electrodes aimed at areas CA1, CA3, and dentate gyrus. All animals were then monitored, using video and EEG recordings, 24 h per day for the next 7 to 10 days. In addition, EEG was recorded while animals were allowed to explore a novel open field for 30 min in order to create a situation where theta rhythm activity is highly likely to occur. Local field potentials from animals that received SSP-Saporin injections into the dorsal CA1 area revealed attenuation of theta rhythm activity in the lesioned area. Recordings from controls, however, showed a robust peak of activity in the theta frequency band, similar to what has been traditionally described in the hippocampus of naive rats. Together, these results suggest that local elimination of interneurons disrupts local theta rhythm without induction of seizure activity. These experiments provide evidence concerning the possible organizational role of GABAergic interneurons in theta rhythm, an important component of normal hippocampal function.

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

Do GAT1-saporin lesions of the medial septum damage GABAergic afferents to the medial septum?

Gielow M, Roland J, Servatius, RJ Pang KCH (2010) Do GAT1-saporin lesions of the medial septum damage GABAergic afferents to the medial septum?. Neuroscience 2010 Abstracts 811.3/LLL66. Society for Neuroscience, San Diego, CA.

Summary: 192-IgG saporin is an antibody directed to the p75 receptor conjugated to the ribosomal-inactivating compound saporin. 192-IgG saporin has been widely used to selectively ablate cholinergic cells of the basal forebrain. Cholinergic lesions are typically made by injections of 192-IgG saporin at the soma in basal forebrain nuclei. However, 192-IgG saporin is also effective in damaging specific cholinergic projections by administration of the toxin in the axon terminal region. Recently, GAT1-saporin has been developed as a tool to selectively damage cells expressing the GABA transporter GAT1. GAT1-saporin combines an antibody to the GABA transporter GAT1 with saporin. GAT1 transporters are primarily localized to neurons and GAT1-saporin has been shown to selectively damage GABAergic neurons in the BNST and medial septum. Given the similarity to 192-IgG saporin, a major question is whether GAT1-saporin is effective in damaging GABAergic afferents to the area of administration. Our previous studies found that GAT1-saporin administered to the medial septum / diagonal band of Broca (MSDB) damages GABAergic septohippocampal neurons and impaired performance on delayed match to position tasks. While it seems likely that damage of GABAergic MSDB neurons is responsible for these behavioral impairments, one cannot rule out the possibility that destruction of GABAergic afferents to the MSDB may also contribute. Therefore, the present study was undertaken to determine whether GAT1-saporin lesions of the MSDB damage the GABAergic hippocamposeptal projection. Male Sprague Dawley rats received both fluorogold and either GAT1-saporin or vehicle in medial septum. Immunocytochemistry for choline acetyltransferase and parvalbumin confirmed the extent of the lesion. The majority of hippocamposeptal GABAergic neurons contain the neuropeptide somatostatin. Quantification of double-labeled hippocampal fluorogold-positive cell bodies with anti-somatostatin immunofluorescence was performed using unbiased stereology. Preliminary data suggest that GABAergic hippocamposeptal neurons are intact. These results will be important in understanding the damage produced by GAT1-saporin.

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

Effects of basal forebrain cholinergic lesions and estradiol on relative levels of estrogen receptor mRNAs in the rat forebrain

Hammond R, Shinde A, Gibbs RB (2010) Effects of basal forebrain cholinergic lesions and estradiol on relative levels of estrogen receptor mRNAs in the rat forebrain. Neuroscience 2010 Abstracts 611.16/MMM67. Society for Neuroscience, San Diego, CA.

Summary: Beneficial effects of estradiol on cognitive performance are lost in response to cholinergic denervation of the hippocampus and frontal cortex. Effects of estradiol also decline with age and time following the loss of ovarian function, which parallels naturally-occurring declines in basal forebrain cholinergic function. We hypothesize that cholinergic impairment may alter the expression of estrogen receptors in specific regions of the brain, thereby decreasing estradiol effects. In the present study, quantitative RT-PCR was used to evaluate the effects of septal cholinergic lesions ± estradiol treatment on relative levels of three estrogen receptors, ERα, ERß, and GPR30. Young adult ovariectomized (OVX) rats received intraseptal injections of saline or 192 IgG-saporin (a selective cholinergic immunotoxin). One week later, rats received either silastic capsules containing 17ß-estradiol or a blank capsule, implanted s.c. Seven days later, rats were killed and the brains were dissected. Tissues from the hippocampus, frontal cortex, prefrontal cortex, striatum, and septum were collected. RNA was extracted and relative levels of ER mRNA determined. Levels within each sample were normalized to levels of GAPDH. Differences between treatments and controls were calculated using the ΔΔCt method. Preliminary data indicate that septal cholinergic lesions produced significant decreases in relative levels of ERα and ERß mRNA in the hippocampus, and an increase in ERß mRNA in the frontal cortex. Estradiol alone produced decreases in levels of ERα, ERß, and GPR30 mRNA in the frontal cortex, decreased levels of ERα and ERß mRNA in the septum, and increased levels of ERα mRNA in the striatum. In rats with cholinergic lesions that also received estradiol, decreased levels of ERα mRNA were detected in hippocampus and septum, and decreased levels of ERß mRNA also were detected in septum. Data suggest that some of the effects of cholinergic denervation on ER mRNA expression may be mitigated by estradiol treatment. These data show that cholinergic lesions significantly affect ER mRNA expression in the brain, and that effects are region-specific. Such effects could account for the loss of beneficial effects of estradiol on cognitive performance in association with age and time following menopause, as well as in association with specific neurodegenerative diseases such as Alzheimer’s disease.

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

Cholinergic modulation of both visual and olfactory attention with the five-choice serial reaction time test

Ljubojevic V, Luu P, De Rosa E (2010) Cholinergic modulation of both visual and olfactory attention with the five-choice serial reaction time test. Neuroscience 2010 Abstracts 535.8. Society for Neuroscience, San Diego, CA.

Summary: The nucleus basalis magnocellularis (NBM) sends acetylcholine (ACh) to neocortical regions that are involved in attentional cognitive processes. Using the five choice serial reaction time task (5CSRTT), the rodent analog of sustained attention in the human cognitive literature, it has been shown that a loss of cholinergic cells in the NBM causes impaired visual attentional performance in rats (Lehmann et al., 2003; McGaughy et al., 2002). The present research examined the neurochemical modulation of attentional processes using both a visual and an olfactory version of the 5CSRTT. To that purpose, we trained 14 male adult Long-Evans rats to attend and react to the briefly presented visual or odor stimuli until they achieved a stable performance under the baseline task conditions, i.e., low attentional demand with stimulus duration (SD) of 1s. Following the successful acquisition of both versions of the 5CSRTT, the rats were subjected to selective cholinergic lesions of the NBM with the cholinergic immunotoxin 192 IgG-saporin to remove the cholinergic innervation from the neocortical mantle. This allowed an examination of the role of ACh in modulation of visual and olfactory attention. After the two week post-surgical recovery period, we compared the attentional performance of the saporin-lesioned (SAP) group (N=8) to that of the sham-lesioned (SHAM) group (N=6) on the two versions of the 5CSRTT task. We observed the impaired attentional performance of the SAP rats on the visual 5CSRTT under the baseline conditions (SD=1s); shortening the SD = 0.5s increased the extent of their deficits. With the olfactory 5CSRTT, the SAP impairment was only observed under the attentional challenge of SD=0.5s. However, in both modalities the difference between two groups trended toward statistical significance due to the low number of the experimental subjects in each group. We are currently performing further parametric manipulations to further challenge the rats in both modalities. We will then collect data from an additional 14 rats to increase the statistical power of our experiment. After the completion of the behavioral data collection, we will conduct acetylcholinesterase histochemistry and choline acetyltransferase immunohistochemistry in order to determine the extent of the loss of cholinergic afferents in fronto-parietal target cortical areas and the loss of cholinergic cell bodies in the NBM, respectively. In addition, parvalbumin immunohistochemistry will be carried out to quantify GABA-releasing neurons colocalized in NBM to confirm the selectivity of our lesion.

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

The arcuate nucleus of the hypothalamus controls the circadian distribution of sleep and feeding

Wiater MF, Mukherjee S, Dinh TT, Rooney E, Li A-J, Simasko SM, Ritter S (2010) The arcuate nucleus of the hypothalamus controls the circadian distribution of sleep and feeding. Neuroscience 2010 Abstracts 648.16/H17. Society for Neuroscience, San Diego, CA.

Summary: Integration of daily sleep and feeding rhythms is incompletely understood. We examined the role of the hypothalamic arcuate nucleus (Arc) in these processes using Arc microinjections of the targeted toxin, NPY-saporin (NPY-sap), or control blank-saporin (B-sap). NPY-sap targets and destroys NPY receptor-expressing neurons. We monitored 24 hr feeding over a 30-day period beginning 2 wks after the Arc injections, and used EEG recordings to assign vigilance states. Vigilance was divided into rapid-eye movement sleep (REMS), non-REMS (NREMS) and wake. NPY-sap lesioned rats were hyperphagic , consuming up to 225% of pre-injection baseline. They rapidly became obese. While in the sleep-monitoring chambers, their body weight change per week ranged from 56 ± 9 g to 40.5 ± 4.5g, compared to 6 ± 0.4 g/wk for B-sap rats. Their circadian pattern of food intake was severely disrupted, such that intake in light and dark periods were approximately equal (43% of their total intake was consumed in the light period vs. 25% in B-sap controls). Sleep patterns were also significantly disrupted in the NPY-sap animals. The occurrence of rapid eye movement sleep (REMS) was inverted in phase, occurring mainly at night, rather than during the day. NonREMS was distributed equally across day and night, instead of occurring predominantly during the day. However, 24-hr total REMS and NREMS time was normal. B-sap controls had normal sleep patterns, with NREMS and REMS occurring predominantly in the light phase. To determine if the change in sleep pattern was due to the change in feeding patterns, we restricted access to food to the dark period for 4 days. NPY-sap treated animals doubled their food intake in the dark period. However, sleep patterns were not changed compared to the ad libitum feeding period in either NPY-sap or B-sap rats. After 7 days of ad libitum feeding, we restricted food access to the light period for 4 days. Again, NPY-sap animals doubled their intake during the feeding period, this time during the light phase, and sleep patterns were not changed in either group by the restricted feeding. By 100 days post-lesion, the NPY-sap animals were still obese, but the patterning and amount of their food intake were becoming similar to controls. However, when evaluated again, sleep patterns were still altered to the same degree as observed early post-lesion. These results confirm the importance of NPY-receptive Arc neurons in controlling food intake. They also reveal an unexpected role for the Arc in the timing of both NREMS and REMS that appears to be independent of the patterning of food intake.

Related Products: NPY-SAP (Cat. #IT-28), Blank-SAP (Cat. #IT-21)

NK-1 Receptors in the RVM: Involvement in hyperalgesia produced by naloxone but not in morphine analgesia

Khasabov SG, Fliss PM, Rao AS, Simone DA (2010) NK-1 Receptors in the RVM: Involvement in hyperalgesia produced by naloxone but not in morphine analgesia. Neuroscience 2010 Abstracts 678.15/QQ2. Society for Neuroscience, San Diego, CA.

Summary: The rostral ventromedial medulla (RVM) is a crucial supraspinal site for opioid analgesia. Descending modulation of nociceptive transmission by the RVM can be antinociceptive, which is associated with increased activity of OFF cells, or pronociceptive, which is related to activation of ON cells. Analgesia produced by opioids at the RVM level is due to direct inhibition of ON cells and the indirect increase in discharge of OFF cells. A subpopulation of neurons in the RVM (approximately 7%) express neurokinin-1 receptors (NK-1R), which are receptors for substance P (SP). We have shown that NK-1R in the RVM are located primarily on ON cells and contribute to descending facilitation of nociception. We suggest that elimination of NK-1R expressing neurons by the specific saporin toxin conjugate SSP-SAP, will reduce the number of ON cells and thereby decrease descending facilitation without affecting antinociception associated with activity of OFF cells. We therefore determined the contribution of NK-1R expressing neurons in the RVM to changes in nocifensive behaviors produced by morphine or the opioid receptor antagonist naloxone by eliminating NK-1R expressing neurons. Adult male Sprague Dawley rats were pretreated with injection of SSP-SAP (1 µM/0.5 µl) or inactive toxin into the RVM. Ablation of NK-1R possessing neurons was determined histologically and did not alter tale flick or paw withdrawal latencies to heat for up to 4 weeks following treatment, indicating that these neurons do not modulate acute nociception. Morphine (30 µg/0.5 µl) injected into the RVM of control rats or rats pretreated with SSP-SAP increased tail flick latencies approximately 133.5 ± 20.8% and 140.4 ± 8.3%, respectively. The increase in paw withdrawal latency following morphine was also similar between groups. However, injection of naloxone (50 µg/0.5 µl) in control rats decreased tail flick latencies for 90 min with a maximal reduction of 32.2 ± 4.1%, whereas in rats treated with SSP-SAP latencies decreased by 17.8 ± 4.9% and for only 30 min. A similar pattern of effects was found on paw withdrawal latencies to heat. These data support the notion that ON cells possess NK-1Rs and contribute to facilitation of nociceptive transmission.

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

Morphine-induced pain hypersensitivity, but not opioid tolerance, depends on microglia-mediated alteration of Cl- homeostasis in spinal dorsal horn

Ferrini F, Mattioli TAM, Lorenzo L-E, Godin A, Wiseman PW, Ribeiro-Da-Silva A, Cahill CM, Milne B, De Koninck Y (2010) Morphine-induced pain hypersensitivity, but not opioid tolerance, depends on microglia-mediated alteration of Cl- homeostasis in spinal dorsal horn. Neuroscience 2010 Abstracts 678.9/PP14. Society for Neuroscience, San Diego, CA.

Summary: Prolonged morphine exposure leads to a reduction of the antinociceptive effect (opioid tolerance) and to an increase in pain sensitivity. Recent evidences suggest that these side effects share similar mechanisms with those underlying neuropathic pain. We have shown that the release of BDNF by activated microglia following peripheral nerve injury causes a decrease in KCC2 activity in the spinal dorsal horn (DH) and weakens Cl−-mediated inhibition through GABAA and glycine receptors. Here, we tested the hypothesis that a similar cascade of events underlies morphine-induced pain hypersensitivity. Adult rats, receiving either morphine (10mg/Kg s.c. twice a day) or saline, were tested for nociceptive thresholds prior to and 1 h after morphine injections each day. Morphine induced tolerance within 2 days and hyperalgesia within 5 days. The hyperalgesia, but not the tolerance, was reversed by intrathecal (i.t.) administration of the anti-mac1 saporin-conjugated antibody (an immunotoxin targeted against microglia) or a TrkB blocking antibody, confirming involvement of both microglia and BDNF in the morphine-dependent hyperalgesia. Microglial activation was confirmed by an increased OX-42 staining after chronic morphine and was blocked by i.t. (-)-naloxone, as well as by (+)-naloxone. Interestingly, (+)-naloxone, while prevented microglia activation, had little effect on morphine tolerance. After 7 days of treatment, rats were sacrificed and DH lamina I-II neurons were recorded by imposing a Cl- load (29 mM). A depolarizing shift in EGABA was observed in lamina I neurons from morphine-treated rats (-42 ± 1 mV, n=6) compared to controls (-50 ± 2 mV, n=5, P<0.05) indicating a weaker Cl- extrusion capacity in these cells. A similar effect was also observed following 3h in vitro incubation of spinal cord slices with morphine (1 μM). No change in EGABA was observed either in the presence of opioid receptor antagonists or the TrkB blocking antibody, confirming the involvement of BDNF in the morphine-signalling pathway. Interestingly, morphine did not produce any change in EGABA in lamina II neurons. To confirm the participation of altered Cl- homeostasis on morphine-induced hyperalgesia in vivo, we administered the carbonic anhydrase inhibitor acetazolamide (i.t.) to minimize the bicarbonate-mediated component of GABAA/glycine currents. Acetazolamide was sufficient to restore inhibition in spinal DH neurons and to reverse the morphine-dependent hyperalgesia. Our data suggest that microglial activation and BDNF release following chronic morphine treatment may alter Cl- extrusion capacity of spinal lamina I neurons and increase pain hypersensitivity.

Related Products: Mac-1-SAP rat (Cat. #IT-33)

Patterning of somatosympathetic reflexes: Identification of distinct bulbospinal sympathoexcitatory RVLM projections by conduction velocity and catecholamine phenotype

Burke PG, Neale J, Korim WS, Mcmullan S, Pilowsky PM, Goodchild AK (2010) Patterning of somatosympathetic reflexes: Identification of distinct bulbospinal sympathoexcitatory RVLM projections by conduction velocity and catecholamine phenotype. Neuroscience 2010 Abstracts 694.11/HHH34. Society for Neuroscience, San Diego, CA.

Summary: The aim of this study was to examine the somatosympathetic reflex (SSR) response of different sympathetic nerves to identify distinct projections of presympathetic vasomotor RVLM neurons by axonal conduction and catecholamine phenotype. All experiments were conducted in urethane-anaesthetised (1.3 g/kg ip), paralysed, vagotomised and artificially ventilated Sprague Dawley rats (n = 44). First, we determined the simultaneous activity of dorsal root potentials and the splanchnic SSR to single shock sciatic nerve (SN) stimulation (single 0.2 ms pulse, 50 sweeps at 0.5-1 Hz, 1-80 V, n=4). Second, we simultaneously recorded the sympathoexcitatory response of multiple, sympathetic nerves (cervical, renal, splanchnic and lumbar) to low (A-fibre afferent; 4-10 V) and high (A- and C-fibre afferents; +40 V) SN stimulation (n=19). Third, we examined the cervical or splanchnic SSR to low intensity SN stimulation in rats following RVLM microinjection of somatostatin (SST) or muscimol (n=8). Fourth, we examined the splanchnic SSR in rats pretreated with intraspinal anti-dopamine-beta-hydroxylase-saporin (anti-DβH-SAP; 24 ng/side, n=8), a neurotoxin that depleted ~70% of catecholamine (C1) neurons in the RVLM compared to IgG-saporin control (n=5). Low intensity SN stimulation evoked biphasic responses in the renal, splanchnic and lumbar nerves but a single peak in the cervical nerve. High intensity SN stimulation evoked triphasic responses in the renal, splanchnic and lumbar nerves and a biphasic cervical response. RVLM injections of SST abolished the early peak of the cervical and splanchnic SSR. Intraspinal pretreatment with anti-DβH-SAP eliminated the late peak of the splanchnic SSR and attenuated the first peak. It is concluded that the mono- or bi-phasic SSR responses are generated by A-fibre afferent inputs driving two classes of bulbospinal sympathoexcitatory RVLM neurons with myelinated or unmyelinated axonal conduction. Secondly, unmyelinated RVLM presympathetic neurons, presumed to be all C1, innervate splanchnic, renal and lumbar SPN, whereas myelinated C1 and non-C1 neurons innervate all sympathetic outflow examined. These findings extend prior evidence that the RVLM expresses several types of phenotypically distinct descending sympathoexcitatory pathways.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Mouse IgG-SAP (Cat. #IT-18)

Donepezil enhances effects of estradiol on DMP acquisition in rats with partial loss of septal cholinergic neurons

Gibbs RB, Chipman AM, Nelson D (2010) Donepezil enhances effects of estradiol on DMP acquisition in rats with partial loss of septal cholinergic neurons. Neuroscience 2010 Abstracts 710.23/NNN23. Society for Neuroscience, San Diego, CA.

Summary: We hypothesize that effects of estradiol on cognitive performance decrease in association with decreased basal forebrain cholinergic function, and that this accounts for the loss of estradiol effect with age and time post-menopause. In the present study, we tested the feasibility of using donepezil, a cholinesterase inhibitor commonly used to treat Alzheimer’s disease, to enhance beneficial effects of estradiol on cognitive performance in rats with septal cholinergic lesions. Young adult, ovariectomized rats received intraseptal injections of 192IgG-saporin or vehicle. Two weeks later, rats started receiving daily injections of donepezil (Don, 5 mg/Kg/day, i.p.) or vehicle. A week later, rats received either silastic capsules containing 17ß-estradiol (E) or empty capsules, implanted s.c. Rats were then trained on a delayed matching-to-position (DMP) T-maze task. Upon completion, brains were collected and sections through the medial septum were processed for detection of choline acetyltransferase (ChAT). The severity of the cholinergic lesions was ranked on a 5-point scale by estimating the loss of ChAT-positive cells in the septum. Eighty-one rats completed the study. Lesions produced a decline in performance that correlated with the severity of cholinergic cell loss (F[4,76]=10.0, p<0.0001). In rats with >50% loss of septal cholinergic neurons, treating with E and/or Don had no significant effect on the rate of DMP acquisition (F[3,36]=0.90, p=0.45). In rats with <50% loss of septal cholinergic neurons, treating with the combination of Don+E significantly increased the rate of acquisition relative to controls (p<0.05 by Tukey test), and reduced deficits associated with increasing the intertrial delay (F[9, 111]=2.30, p=0.02 for Delay x Tx interaction). Treating with Don or E alone had no significant effect, although E alone produced a strong trend toward improvement. Cholinergic lesions also increased the likelihood that rats would adopt a persistent turn χ2=13.3, p=0.0003), and treatment with Don+E reduced this effect (χ2=8.8, p=0.03). These findings demonstrate that cholinergic dysfunction produces a learning impairment as well as a loss of estrogen effect on cognitive performance which can be attenuated by treating with a cholinesterase inhibitor. The findings also indicate a critical threshold for cholinergic function below which combined therapy is not effective. We propose that combining donepezil with estrogen therapy in postmenopausal women may offer significant cognitive benefits, particularly in relatively healthy older women showing early signs of cognitive impairment or Alzheimer’s disease.

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

Targeting inhibitory neurons in the superficial dorsal horn: Somatostatin-saporin

Chatterjee K, Lemons LL, Wiley RG (2010) Targeting inhibitory neurons in the superficial dorsal horn: Somatostatin-saporin. Neuroscience 2010 Abstracts 585.1/XX15. Society for Neuroscience, San Diego, CA.

Summary: Intrathecal injection of somatostatin (SST), or the long-acting congener, octreotide, have been reported analgesic in humans with intractable pain. The principal SST receptor, sst2a, is expressed by GABAergic neurons in the superficial dorsal horn of the spinal cord. In the present study, we sought to determine the nocifensive behavioral effects of selectively destroying sst2a-expressing dorsal horn neurons using intrathecal injection of the targeted toxin, SST-saporin. SST-sap (500-625 ng) was injected intrathecally into rats followed by thermal plate and thermal preference shuttle box testing for up to three weeks. One of three rats injected with 625 ng of SST-sap developed severe persistent scratching of its lower body. Compared to vehicle controls and rats injected with 500 ng of corticotrophin releasing factor (CRF)-saporin, the SST-sap rats showed initially prolonged latencies and decreased nocifensive reflex responses on the 44 C hotplate that persisted for up to 17 days before returning to control levels. SST-sap rats also showed decreased reflex responses on the 0.3 C cold plate. Lastly, SST-sap rats showed no change in thermal preference in a shuttle box with floor temperatures of 15 C vs 45 C. CRF-sap rats showed delayed onset (after 8 days) of decreased hotplate responding and increased hot side occupancy in the thermal preference shuttle box. These results suggest, at the doses used, that SST-sap produced incomplete depletion of target neurons followed by compensatory plasticity, whereas, CRF-sap produced no primary effect but induced secondary plasticity resulting in long term decrease in responses to aversive heat. Higher dose studies and anatomic analysis of lesions produced by these agents are planned.

Related Products: CRF-SAP (Cat. #IT-13)

Targeting inhibitory neurons in the superficial dorsal horn: Neurotensin-saporin (NTS-sap) and neurotensin-cholera toxin A subunit (NTS-CTA)

Wiley RG, Lemons LL, Chatterjee K (2010) Targeting inhibitory neurons in the superficial dorsal horn: Neurotensin-saporin (NTS-sap) and neurotensin-cholera toxin A subunit (NTS-CTA). Neuroscience 2010 Abstracts 585.2/XX16. Society for Neuroscience, San Diego, CA.

Summary: Neurotensin (NTS) and high affinity neurotensin receptors (NTSR-1) are found in the superficial dorsal horn, primarily lamina II. Intrathecal NTS has been reported to be anti-nociceptive, naloxone does not block the anti-nociceptive effects of intrathecal NTS and NTS acting at the NTSR-1 is excitatory. Based on these facts, we hypothesized that intrathecal neurotensin produces anti-nociception by exciting inhibitory interneurons in the superficial dorsal horn. In the present study, we sought to determine the effects, on modified thermal plate responses, of lumbar intrathecal injections of NTS-saporin, that is expected to selectively kill NTSR-1-expressing dorsal horn neurons, and NTS-Cholera toxin A subunit (NTS-CTA), that is expected to excite the same neurons. NTS-sap (200-625 ng) produced sustained, remarkable, vigorous scratching of hindquarters, often to the exclusion of any other activity. 12-15 ng of NTS-sap produced no scratching and increased lick/guard responding on the 44 C hotplate. Lumbar intrathecal injections of NTS-CTA (500 ng) produced profound decrease in lick/guard responding on the 44.5 C hotplate that lasted for 100-150 hours. This unique pattern of effects is consistent with the hypothesis that NTSR-1-expressing lamina II dorsal horn neurons are both inhibitory and anti-nociceptive. These results also are consistent with the intrathecal injections of NTS-CTA producing sustained excitation of these inhibitory interneurons resulting in inhibition of nociceptive projection neurons. This strategy of exciting NTSR-1-expressing inhibitory interneurons of the superficial dorsal horn is a novel approach to achieve non-opioid-mediated analgesia which may prove valuable in treating refractory chronic pain.

Related Products: Neurotensin-SAP (Cat. #IT-56), Neurotensin-CTA (Cat. #IT-60)

Neuropeptide receptor co-expression in superficial dorsal horn: Effects of galanin-saporin, neuropeptide y-saporin and dermorphin-saporin

Lemons LL, Chatterjee K, Wiley RG (2010) Neuropeptide receptor co-expression in superficial dorsal horn: Effects of galanin-saporin, neuropeptide y-saporin and dermorphin-saporin. Neuroscience 2010 Abstracts 585.5/XX19. Society for Neuroscience, San Diego, CA.

Summary: We have previously shown that the role of specific neurons in behavioral processes can be fruitfully studied using targeted toxins. Toxins composed of a targeting neuropeptide coupled to the ribosomal-inactivating toxin, saporin, are used to selectively destroy superficial dorsal horn neurons expressing the cognate peptide receptors followed by assessment of changes in pain behavior. In the present study, we sought to compare the anatomic effects of three closely related targeted toxins, each with different nocifensive behavioral effects. Rats were given single lumbar intrathecal injections of either galanin-saporin (Gal-sap), neuropeptide Y-saporin (NPY-sap), or dermorphin-saporin (Derm-sap). Lumbar spinal cord sections from each rat were stained for each of the three receptors, GalR-1, Y1R and MOR (mu opiate) using standard immunoperoxidase technique. Each toxin produced a significant decrease in staining for its cognate receptor. Gal-sap animals showed no change in either MOR or Y1R staining. NPY-sap rats showed decreased staining for both GalR1 and MOR, and Derm-sap rats were assessed for changes in expression of GalR1 and Y1R. These findings suggest overlaps between the populations of neurons that express the GalR1, Y1R, and MOR. Specifically, Y1R-expressing neurons also express GalR1 and MOR, probably by separate subpopulations of Y1R neurons. The results also suggest either that Gal-sap only kills neurons that do not express either of the other two receptors, or some of the observed loss of receptors after NPY-sap is due to secondary (transsynaptic) effects. Double- and triple-label fluorescent immunohistochemistry will be used to directly visualize receptor co-expression patterns and targeted toxin effects. These results will be valuable in interpreting the unique nocifensive behavioral effects of each of these targeted toxins.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12), Galanin-SAP (Cat. #IT-34), NPY-SAP (Cat. #IT-28)

Damage of GABAergic neurons in the medial septum-diagonal band (MSDB) reduces behaviorally-activated hippocampal acetylcholine efflux and impairs spatial working memory

Roland JJ, Janke KL, Savage LM, Servatius RJ, Pang KCH (2010) Damage of GABAergic neurons in the medial septum-diagonal band (MSDB) reduces behaviorally-activated hippocampal acetylcholine efflux and impairs spatial working memory. Neuroscience 2010 Abstracts 611.13/MMM64. Society for Neuroscience, San Diego, CA.

Summary: The septohippocampal pathway is mostly composed of cholinergic and GABAergic projections and has an established role in learning, memory and disorders of cognition. Most studies have focused on the role of the cholinergic system in learning, memory and disorders of cognition. Although MSDB cholinergic lesions do not result in learning impairments, changes in hippocampal acetylcholine (ACh) levels have been tied to memory functions where deficits or enhancements in memory were correlated with hippocampal ACh decreases or increases, respectively. The activity of MSDB cholinergic neurons is greatly influenced by GABAergic afferents, including those from GABAergic neurons within the MSDB. Recently, we’ve demonstrated that toxins that preferentially damage MSDB GABAergic neurons impair delayed match to position tasks, but not spatial reference memory. Interpretation of these results needs to take into account the fact that a MSDB GABAergic lesion would influence both septohippocampal cholinergic and GABAergic transmission. The current study examined the effect of MSDB GABAergic lesions on spontaneous alternation (Experiment 1) and a non-matching to position task (NMTP; Experiment 2) while concurrently using in vivo microdialysis to measure hippocampal ACh efflux. Adult male Sprague-Dawley rats received vehicle (PBS) or GABAergic (GAT-1 saporin) MSDB lesion and a hippocampal microdialysis cannula. In Experiment 1, treatment groups did not differ in terms of activity, alternation rates, or baseline and maze-activated ACh efflux. In Experiment 2, hippocampal ACh efflux was measured at two time points (early and late) across the acquisition of a delayed NMTP task. Overall, GAT1-saporin treated rats had lower accuracy scores across 10 days of maze training compared to the vehicle treated rats. Basal ACh release in the hippocampus was similar in vehicle and GAT1-saporin rats. During the two microdialysis sampling points, both groups of rats displayed significant increases in ACh efflux while performing the task. However, behaviorally activated ACh efflux was reduced in GABA-lesioned animals compared to vehicle treated rats. The results demonstrate that MSDB GABAergic lesions do not alter basal hippocampal ACh efflux, but can reduce ACh efflux when challenged cognitively. Future studies will attempt to determine whether reduced ACh efflux is due to damage of MSDB GABAergic neurons or a result of impaired working memory performance.

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

Functional cholinergic neurons from human embryonic stem cells

Liu Y, Krencik R, Liu H, Ma L, Zhang X, Zhang S-C (2010) Functional cholinergic neurons from human embryonic stem cells. Neuroscience 2010 Abstracts 331.5/B19. Society for Neuroscience, San Diego, CA.

Summary: Basal forebrain cholinergic neurons play a critical role in regulating memory and cognition. Degeneration or dysfunction of these neurons is associated with neurological conditions including Alzheimer’s disease and dementia. In this study, we aimed at generating cholinergic neurons from human embryonic stem cells (hESCs) for therapeutic development. hESCs were first differentiated to primitive neuroepithelial cells in a chemically defined medium. In the presence of sonic hedgehog, over 97% of the differentiated cells became Nkx2.1-expressing ventral forebrain progenitors. These ventral progenitors further differentiated to cholinergic neurons with basal forebrain characteristics by expressing ChAT, VAChAT, FoxG1, Nkx2.1, Islet1, ßIII-tubulin, MAP2, P75, Synapsin but not GABA, Glutamate, or Mash2. The hESC-generated cholinergic neurons were electrophysiologically active in vitro. Following transplantation into the hippocampus of mice, in which cholinergic neurons in the medial septum were destroyed by IgG-P75-saporin, the grafted human cells produced large cholinergic neurons. The animals transplanted with cholinergic neurons demonstrated an improvement in learning and memory deficit. These results indicate that the human stem cell-generated cholinergic neurons are functional, thus providing a new source for drug discovery and cell therapy for neurological disorders that affect cholinergic neurons.

Related Products: mu p75-SAP (Cat. #IT-16)

ATS Poster of the Year Winner

The effects of neonatal cholinergic lesion on age-related changes in behaviour, neurogenesis and CA1 pyramidal cell morphology

Rennie KE, Frechette M, Pappas BA (2010) The effects of neonatal cholinergic lesion on age-related changes in behaviour, neurogenesis and CA1 pyramidal cell morphology. Neuroscience 2010 Abstracts 349.8/J12. Society for Neuroscience, San Diego, CA.

Summary: Age-related cognitive decline is associated with dysfunction of the basal forebrain cholinergic (BFC) system, and cortico-hippocampal cholinergic denervation is a hallmark neurochemical feature of the Alzheimer’s-afflicted brain. It has been suggested that cognitive deficits that emerge with age may be rooted in early dysfunction of the BFC system and that impaired cholinergic transmission might interact with ageing-associated factors to produce cognitive decline. The purpose of this study was to examine the effects of neonatal cholinergic lesion on age-related changes in spatial working memory, neurogenesis and hippocampal CA1 pyramidal cell morphology. We have previously reported that neonatal cholinergic lesion results in only minor behavioural deficits, but impairs the birth and/or survival of new neurons and reduces CA1 dendritic complexity in the young adult rat. We hypothesized that memory impairments would become apparent in lesioned rats as they age, and that this impairment would be accompanied by more drastic reductions in neurogenesis and cytoarchitectural alterations than those that have been documented in the young adult animal after neonatal cholinergic lesion. Seven-day-old male Sprague-Dawley rats were subjected to basal forebrain cholinergic lesion by infusion of the cholinotoxin 192-IgG-Saporin into the lateral ventricles. At the age of 12 or 21 months, the rats were tested on a working memory version of the Morris water maze. While aging had only a slight effect on the memory performance of control rats, lesioned rats showed pronounced memory impairments with age. This occurred without CA1 cell loss or astrogliosis in 21-month-old lesioned rats when compared to age-matched controls. However, golgi analysis revealed that while cholinergic lesion did not alter the total dendritic length, branching, number of spines, or spine density of CA1 pyramidal cells in 21-month-old rats, the distribution of these parameters across branch orders was shifted. The lesion caused a slight reduction in apical branch length and spine density, and basal branch number, length and number of spines at low/middle branch orders, but increased these parameters at upper branch orders. Thus, perinatal cholinergic lesion precipitates spatial memory dysfunction during old age, and this seems to be associated with cytoarchitectural changes to neurons rather than neuronal loss.

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

Hindbrain catecholamine neurons are required for rapid switching of metabolic substrate utilization during glucoprivation

Li A-J, Wang Q, Dinh TT, Ritter S (2010) Hindbrain catecholamine neurons are required for rapid switching of metabolic substrate utilization during glucoprivation. Neuroscience 2010 Abstracts 392.14/III1. Society for Neuroscience, San Diego, CA.

Summary: Glucoprivation is a metabolic emergency in which a rapid and effective system-wide switch to fat metabolism must occur to conserve any available glucose for use by the brain. Glucoprivation stimulates secretion of corticosterone, which is known to play an important role in promoting fat utilization. In previously published work, we showed that injections of the retrogradely transported catecholamine neuron immunotoxin, anti-dopamine beta-hydroxylase conjugated to saporin (DSAP) into the paraventricular nucleus of the hypothalamus (PVH) eliminate the corticosterone response to glucoprivation without impairing the response to a nonmetabolic stressor (swim stress), without altering the circadian rhythm of corticosterone secretion and without damaging the PVH CRF-secreting neurons. Here we microinjected DSAP into the PVH to selectively lesion hindbrain catecholamine neurons innervating this site, thus impairing the glucoprivation-induced corticosterone response. Using indirect calorimetry, we examined metabolic fuel utilization and other metabolic parameters in these lesioned rats under basal and glucoprivic conditions. Under basal conditions, energy expenditure and locomotor activity did not differ between DSAP rats and controls injected with unconjugated saporin (SAP). However, DSAP rats had a higher respiratory exchange ratio (RER) than SAPs, indicating their greater dependence on carbohydrate utilization. Glucoprivation induced by 2-deoxy-D-glucose (2DG, 250 mg/kg) reduced energy expenditure equally in SAP and DSAP rats. However, 2DG rapidly decreased RER to 0.8 (a value indicating ongoing fat metabolism) in the SAP group, but not in the DSAP group. Responses to 2DG persisted for about 6 hours. Adrenal dennervation, which eliminates the adrenal medullary response to glucoprivation, did not alter these responses to 2DG in either SAP or DSAP rats. Results indicate that in the absence of hypothalamically-projecting hindbrain catecholamine neurons, rats cannot efficiently switch their fuel utilization from carbohydrate to fat during glucoprivation, presumably due to a deficient corticosterone response. Results also suggest a previously unrecognized role for these catecholamine neurons in control of basal substrate utilization.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Leptin-saporin lesion of hypothalamic arcuate neurons impairs circadian feeding rhythms

Li A-J, Dinh TT, Wang Q, Wiater MF, Ritter S (2010) Leptin-saporin lesion of hypothalamic arcuate neurons impairs circadian feeding rhythms. Neuroscience 2010 Abstracts 498.6/III29. Society for Neuroscience, San Diego, CA.

Summary: To examine the role of leptin receptor-expressing neurons in the arcuate nucleus (Arc) in circadian control of spontaneous feeding and energy expenditure, we injected a novel targeted toxin, leptin conjugated to saporin (Lep-SAP) into the Arc in rats. Lep-SAP effectively lesioned Arc neurons in a leptin-receptor dependent manner, indicated by an 80% reduction of agouti gene-related protein- or melanocyte-stimulating hormone-immunoreactive neurons in Sprague Dawley rats, but not in leptin receptor deficient Zucker fa/fa rats. Food intake and metabolism were monitored 3-5 weeks after Arc Lep-SAP and control blank-saporin (B-SAP) injections using an Oxymax system. Lep-SAP rats consumed 49% of their total daily intake during the day, compared to 34% in B-SAP rats. Eatograms (feeding actograms), cosinar analysis and Chi-square periodograms of continuous feeding records failed to detect a circadian oscillation in the feeding patterns of Lep-SAP rats, but did detect significant circadian rhythms in B-SAP controls. Unlike feeding, metabolic rate, respiratory exchange ratio and locomotor activity continued to exhibit significant circadian periodicity in both groups, though dampened in amplitude in Lep-SAPs, suggesting that rhythms of feeding and metabolism may be controlled by separate mechanisms. Expression of clock-related genes (Per1 and Bmal1) in hypothalamus, liver and white fat tissue was asynchronous in Lep-SAP rats. These results suggest that leptin-receptive neurons in the Arc exert a critical influence on the circadian patterning of food intake.

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

Mesolimbic-basal forebrain circuitry mediating the motivational activation of attention

St Peters MM, Bruno JP, Sarter M (2010) Mesolimbic-basal forebrain circuitry mediating the motivational activation of attention. Neuroscience 2010 Abstracts 506.12/LLL52. Society for Neuroscience, San Diego, CA.

Summary: Prefrontal circuitry mediating cue detection is modulated by the tonic component of cholinergic activity. Performance-associated increases in tonic cholinergic activity are augmented by demands on the cognitive control of attention. Highest levels of tonic cholinergic activity are observed while animals perform below baseline as a result of, for example, a distractor, but while they remain motivated to stabilize and recover attentional performance. Cortico-mesolimbic-basal forebrain circuitry is thought to mediate such motivated activation of attentional performance. We previously observed that stimulation of ionotropic glutamate receptors in the shell of the nucleus accumbens (NAs) stimulates tonic cholinergic activity in the prefrontal cortex. Here we test the hypothesis that such stimulation benefits attentional performance while distractors evoke cognitive control. Rats were trained in an operant sustained attention task (SAT) before undergoing surgery for implantation of a bilateral guide cannula targeting the NAs or, in separate animals, the core of the NA (NAc). NMDA (0.01-0.15 µg/0.5 µL/hemisphere) or vehicle (0.9% saline) was infused bilaterally into task-performing animals during SAT and the more challenging distractor version (dSAT). For the dSAT, the operant chamber ceiling lights flashed on/off at 0.5 Hz during the middle block of three blocks of trials that constituted a session. NMDA infusions in the NAs, but not into the NAc, significantly improved the animals’ attentional performance in the presence of the distractor. These findings are consistent with the hypothesis that activation of the NAs mediates attentional performance under conditions that require top-down control. The next set of experiments determined whether the effects of NAs activation require the cortical cholinergic system. We infused the immunotoxin 192 IgG saporin into prefrontal or parietal regions, in addition to implantation of guide cannula targeting the NAs. Replicating the initial finding, NAs NMDA infusions enhanced dSAT performance. Both PFC and PPC cholinergic deafferentation prevented this effect of NMDA. These findings suggest that the motivated activation of the cholinergic attention system during demands on top-down control modulates fronto-parietal attention networks to optimize attentional performance.

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

Novel object recognition and social interaction in rats lacking cortical cholinergic innervation; comparing manual and digital video tracking systems

Savage ST, Olson L, Mattsson A (2010) Novel object recognition and social interaction in rats lacking cortical cholinergic innervation; comparing manual and digital video tracking systems. Neuroscience 2010 Abstracts 506.9/LLL49. Society for Neuroscience, San Diego, CA.

Summary: Alterations in cholinergic signaling in the brain have been implicated as a contributing factor in the pathogenesis of schizophrenia. We have shown that cholinergic denervation of cortex cerebri by stereotaxic infusion of the immunotoxin 192 IgG-saporin into nucleus basalis magnocellularis in adult rats leads to an enhanced locomotor sensitivity to amphetamine, as well as, a potentiated dopamine release in nucleus accumbens. We have also shown that this cortical cholinergic denervation leads to an increased locomotor response to the NMDA receptor antagonist phencyclidine (PCP), suggesting that disruption of cortical cholinergic activity can lead to disturbances of glutamatergic transmission. We hypothesize that this loss of cortical cholinergic input alters the activity of cortical glutamatergic neurons and in turn, their regulation of subcortical dopamine neurons. In current studies we are investigating memory functions using the novel object recognition task (NOR) and social interaction in adult male Lister hooded rats with cholinergic denervation of neocortex. The behavioral tasks are being conducted under normal conditions and with a PCP-challenge. The data are analyzed both manually by a trained observer, and with a nose point digital video tracking system (Clever Sys Inc.). Manually scoring behavioral data requires extensive observer training, is subject to inter-observer variability, and is time consuming. An automated tracking system could potentially improve upon these issues, however is prone to other problems, including the difficulty of accurately tracking multiple body points. Furthermore, the Lister hooded fur has two different colors which proves difficult for computerized systems to accurately determine the body points. A comparison of the manual scoring and the computerized tracking system is being conducted to determine the most reliable method for each behavioral task. Preliminary results indicate that the cholinergically denervated rats performed the NOR task under normal conditions as well as the controls, however failed to show a preference for the novel object under PCP-challenge. These results were obtained through analysis with both the manual and automated system. Despite fur color difficulties, the video tracking system was able to analyze the NOR task and accurately calculate the distance traveled, which is not easily obtained through manual scoring. These initial results indicate that cortical cholinergic deficits, in addition to a potentiation of the locomotor response to PCP, can also lead to an enhanced sensitivity to PCP-induced cognitive impairments.

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

Decrease of Arc protein expression and delay of memory acquisition by immunolesion

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

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

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

Time- and dose-response of 6-hydroxydopamine on locus coeruleus noradrenegric neurons in c57bl/6 mice

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

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

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Decreasing abnormal nocifensive responses in the bilateral chronic constriction injury (bCCI) model of neuropathic pain: Effects of lumbar intrathecal CCK-saporin

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

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

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Gastrin-releasing peptide receptor in the spinal cord mediates mechanical allodynia following nerve injury

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

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

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The recovery of locomotion after lumbar spinal cord motoneuron depletion is affected by the modulation of Sonic Hedgehog and Notch-1 pathways

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

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

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Lesioning of the ventrolateral preoptic nucleus alters isoflurane-induced hypnosis in a time-dependent fashion

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

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

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Subplate neurons promote the formation of barrels within rat primary somatosensory cortex

Sheikh A, Kanold PO (2010) Subplate neurons promote the formation of barrels within rat primary somatosensory cortex. Neuroscience 2010 Abstracts 33.1/E1. Society for Neuroscience, San Diego, CA.

Summary: Subplate neurons are a transient neuronal population present in the neonatal cortex. Subplate neurons receive thalamic afferents and project into the developing cortical plate. Selective removal of subplate neurons in cat visual cortex prevents the normal development of ocular dominance columns and the functional maturation of thalamocortical connections (Ghosh & Shatz 1992, Kanold et al. 2003) . A role of subplate neurons in the development of other sensory cortices is unknown. In rodents, thalamocortical afferents representing the whiskers segregate into barrels in the primary somatosensory cortex (S1). This segregation occurs postnatally and can be disrupted by manipulations of neuronal activity. We previously showed that subplate removal disrupts the development of patterned cortical activity in S1 (Tolner, Yukin, Kaila, Kanold, Abstr. SFN 2009). Thus we hypothesized that disruption of patterned activity in S1 alters the development of barrels. Thus here we investigated if subplate neurons play a role in the development of barrels in rat S1. Subplate neurons were ablated in the somatosensory cortex of rat pups at postnatal day (P) 0 by immunotoxin injections. 10-14 days later we investigated the pattern of barrels in S1 via cytochrome oxidase staining. After subplate ablation there was a disturbance in the barrel patterning when compared to the un-manipulated or control-toxin injected hemispheres. Therefore, subplate neurons are involved in the formation of barrels in S1.

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

Anxiety-like behavior in the elevated plus maze (EPMZ) depends on noradrenergic (NA) inputs to the anterolateral bed nucleus of the stria terminalis (alBST) in rats

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

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

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The role of GABA-ergic interneurons in CA1 and dentate gyrus for sequence learning

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

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

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Cholinergic innervation of the hippocampus is not neccesary for episodic memory, but is required for context-place learning in rats

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

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

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Carageenan evoked P-Akt in deep dorsal horn neurons is prevented by loss of neurokinin1 positive neurons in superficial dorsal horn

Sorkin LS, Choi J-I, Koehrn FJ (2010) Carageenan evoked P-Akt in deep dorsal horn neurons is prevented by loss of neurokinin1 positive neurons in superficial dorsal horn. Neuroscience 2010 Abstracts 81.21/VV19. Society for Neuroscience, San Diego, CA.

Summary: P-Akt expression increases in dorsal horn after prolonged noxious stimulation and participates in synaptic strengthening. Recent work from our lab, showed separate p-Akt peaks in superficial (45 min) and deep dorsal horn (DH) neurons (2 hrs) after paw carrageenan. It has been suggested that NK1 receptor-expressing projection neurons (NK1+) in superficial DH are necessary for deep DH neuronal sensitization, possibly via a spino-bulbo-spinal loop. In this study, we examined whether the pattern of p-Akt expression was modulated by elimination of superficial DH NK1(+) neurons. Male Holtzman rats (250-275 g) were injected over the lumbar enlargement with substance P-saporin conjugate (SSP-SAP ([Sar9Met(O2)11] 100 ng/µl, n=8), SAP (n=8), or BSA vehicle (n=3). Catheters were removed 20 min post-injection. Two weeks later, carrageenan (2%, 100 µl) was injected into the hindpaw. Animals were perfused 45 min or 2 hrs after carrageenan (or sham) injection. Immunohistochemistry was performed on frozen sections (20mm). After correction for background density, NK-1 immunoreactivity was measured as number of bright pixels (intensity value >50 of 256)/total pixels within user-defined boxes in laminae I-III and IV-V. Neuronal p-Akt was measured using double labeling with rabbit anti-p-Akt ser 473 and mouse anti-NeuN; cells immunopositive for both were counted separately for lamina I-III and IV-V. For both measures, values from four random sections taken from segments L4 and 5 were averaged for each animal. The histologist was blinded as to spinal treatment. Density of NK-1 immunoreactivity was markedly reduced in laminae I-III in rats treated with SSP-SAP compared to SAP or BSA (24.7± 5.6% vs 78.3 ± 3.2%, 76.9 ± 2.0% respectively; p<0.01) with no differences in lamina IV-V (72.2 ± 3.4% vs 75.9 ± 1.7%, & 76.5 ± 3.4%. Counts of p-Akt neurons did not differ at any time point among animals with no pretreatment, BSA or SAP prior to carrageenan. However, the carrageenan-evoked increases in p-Akt neurons seen in laminae I-III at 45 min (9.3 ± 1.0 SAP and 2.4 ± 1.6 SSP-SAP at 45 min) and in laminae IV-V at 2 hour (11.3±1.0 SAP and 3.5± 0.9 SSP-SAP) were totally blocked by loss of NK1 neurons. Selective ablation of NK1+ neurons in superficial DH blocked peripheral inflammation-induced increase of p-Akt expression in both superficial and deep DH neurons. While some p-Akt reduction in laminae I-III was probably due to neuronal loss, i.e. NK1 receptor bearing neurons become p-AKT positive, we propose that reduction in deeper laminae was due to elimination of the first leg of a facilitatory spino-bulbo-spinal loop although loss of local NK+ interneurons could also have contributed.

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Early post-natal cholinergic lesion impairs normal development and maturation of the motor cortex in rats

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

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

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