References

Jeong D, Chang W, Lee D, Chang J (2011) Decrease of GABAergic markers and Arc protein expression in the frontal cortex by injection of intraventricular 192 IgG-saporin. Neuroscience 2011 Abstracts 878.08. Society for Neuroscience, Washington, DC.

Summary: Previous studies used 192 IgG-saporin to study cholinergic function because of its facility for selective lesioning; however, results varied due to differences in the methods of administration and behavioral tests used. We investigated whether intraventricular injections of 192 IgG-saporin were suitable to make a dementia animal model for the evaluation of therapeutic drugs or electrical stimulation techniques. We examined the effects of 192 IgG-saporin using the Morris water maze, immunochemistry, and western blotting. Animals were examined 2 weeks after intraventricular injection of 192 IgG-saporin (0.63 µg/µl, 6 µl, 8 µl, and 10 µl) or phosphate buffered saline (8 µl). In the acquisition phase of the Morris water maze, the latencies of the injection groups were significantly delayed, but recovered within 1 week. In the probe test, two of four indices (time in the platform zone and the number of crossings) were significantly different between the control group and the group injected with 8 µl of 192 IgG-saporin. Immunohistochemistry revealed the extent of cholinergic destruction that was apparent in the basal forebrain of all 192 IgG-saporin injected rats. We found significantly decreased activity-regulated cytoskeleton associated protein (Arc) and glutamate decarboxylase (GAD) expression in the frontal cortex (8 µl and 10 µl groups), but not in the hippocampus, using western blotting. Further, spatial memory impairment was associated with cholinergic basal forebrain injury as well as fronto-cortical GABAergic hypofunction and synaptic plasticity deceleration. We conclude that intraventricular injection of 192 IgG-saporin is a suitable method for making a rat model of dementia.

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

Lee J, Jeong D, Chang J (2011) The effects of basal forebrain cholinergic neuron on novel object recognition. Neuroscience 2011 Abstracts 878.10. Society for Neuroscience, Washington, DC.

Summary: Medial septum and basal nucleus areas of the basal forebrain project cholinergic neurons to the frontal cortex and the Hippocampus.And degeneration of the cholinergic basal forebrain neurons is a common feature of Alzheimer’s disease (AD) has been correlated with cognitive decline. This research was studied to verify the effects of cholinergic neuron in basal forebrain to the role of the hippocampus and the frontal cortex on recognition through recognition test and immunohistochemistry after damaging cholinergic neuron of the basal forebrain by intraventricular injection of 192 IgG-saporin. 192 IgG-saporin of 8ul (0.63ug/ul) was injected to the bilateral lateral ventricle of rats. After 2 weeks, novel object recognition (NOR) test was conducted to elucidate damage of cholinergic neuron. In the NOR test, rats are exposed to two identical objects for 15 minutes in empty plastic box (60cmx60cmx30cm). After 3 hours, they are reintroduced to the same object and a new novel object for 10 minutes. This procedure was repeated for 4 days After completing the behavioral experiment, the ChAT of cholinergic neuron in the basal forebrain was ascertained to confirm with immunohistochemistry if cholinergic neuron was damaged. In NOR test, the lesion group with 192 IgG-saporin showed 10% lower novel object preference than normal group. However, this rate is not that significant value enough to elucidate behavioral difference between normal group and lesion group. In immunohistochemistry, the number of cholinergic neuron was remarkably decreased in basal forebrain. According to both of the NOR test and Immunohistochemistry in the condition under lesion, Cholingergic input to hippocampus and frontal cortex from basal forebrain affects recognition somewhat, however the effect is not so essential.

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

Clausen B, Klipec W, Bastlund J, Collins M (2011) P300-like event related potentials in IgG192-saporin induced rat model of Alzheimer´s disease. Neuroscience 2011 Abstracts 550.01. Society for Neuroscience, Washington, DC.

Summary: The P300 event-related potential (ERP) is a time-locked response to rare, response-relevant stimuli. Decreased ERP amplitude is correlated with decreased memory function. Not surprisingly, alterations in P300 ERP amplitude are commonly associated with the progressive disruption of cognitive function in human Alzheimer’s disease. Here, a rat model of Alzheimer’s disease was created by injecting the antibody-linked toxin, IgG192-saporin, into the basal forebrain, producing a progressive degeneration of cholinergic cells to mimic the cholinergic degeneration that is part of Alzheimer’s disease. The goal of this experiment was to investigate the rat model by examining the relationship between the expected degenerative deficits and possible changes in the EEG patterns. Following preliminary training that has produced reliable P300-like ERPs in prior experiments in our lab, half of the rats were injected with IgG192-saporin (lesioned), while the other half were injected with saline (controls). Recording electrodes were surgically implanted on the surface of the brain and in the prefrontal cortex (PFC) and ventral hippocampus (vHipp). Following recovery, P300-like ERP data was recorded for three weeks, after which a ChAT analysis of choline acetyltransferase activity confirmed the extent of cholinergic damage in PFC and Hipp. While no systematic increases in latency were found, surprisingly, significant increases in P300-like ERP amplitude occurred in PFC and vHipp in the lesioned compared to the control rats. The implication of these findings for a rat model of Alzheimer’s disease will be discussed.

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

Dinh TT, Smith BR, Wiater MF, Jansen H, Li A-J, Ritter S (2011) Lesions targeting leptin-sensitive neurons in the ventromedial and suprachiasmatic nuclei differentiate sites for circadian control of feeding. Neuroscience 2011 Abstracts 600.13. Society for Neuroscience, Washington, DC.

Summary: We have previously investigated the role of leptin sensitive networks in the mediobasal hypothalamus (MBH) for feeding using the saporin toxin conjugated to leptin (Lep-SAP) and a control conjugate, blank-saporin (B-SAP). Lep-SAP binds to, is internalized by and destroys leptin receptor expressing neurons at the injection site. We found that injections of Lep-SAP directed at the arcuate nuclei (Arc) caused profound regulatory and circadian deficits including arrhythmia for feeding. We have proposed that leptin-sensitive circuitry within the MBH, particularly the arcuate nuclei (Arc), is required for the maintenance of feeding rhythms. Here we tested this hypothesis further by examining effects of Lep-SAP injections into two additional hypothalamic nuclei in the vicinity of the Arc, the ventromedial nucleus (VMN) and the suprachiasmatic nucleus (SCN). In an additional group, the SCN was lesioned electrolytically. Feeding data were collected using BioDAQ computerized meal monitors (Research Diets, Inc) and analyzed using ClockLab software to generate double raster eatogram plots. Lomb-Scargle periodograms were used to assess rhythms and their robustness. Feeding was monitored under light:dark (LD) and dark:dark (DD) conditions in all groups except SCN Lep-SAPs. Unlike Arc-directed injections of Lep-SAP, VMN injections did not alter the diurnal distribution of feeding in either LD or DD and lesioned rats did not become obese or hyperphagic. Lomb-Scargle analysis and eatograms indicated that VMN rats have intact circadian rhythms for feeding. Both Lep-SAP and electrolytic lesions of the SCN caused a slight reduction body weight, compared to controls. Total 24h food intake was unchanged, but light-period food intake was increased. Rats with electrolytic lesions of the SCN were arrhythmic for feeding under both LD and DD conditions. Together with results from Arc Lep-SAP injections, these findings strongly implicate leptin-sensitive circuitry in the MBH in control of circadian feeding rhythms. In addition, they point to the particular significance of the Arc and its connections with the SCN in this circuitry. Leptin-sensitive neurons in the VMN appear to be of less importance in this role.

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

Li AJ, Wang Q, Ritter S (2011) Fourth ventricular glucosamine-induced feeding is catecholamine-dependent. Neuroscience 2011 Abstracts 600.17. Society for Neuroscience, Washington, DC.

Summary: Glucokinase has been identified as a glucose-sensor for detecting glucose changes both in the brain and periphery. Previous reports have shown that lateral ventricular injection of a glucokinase inhibitor, glucosamine, stimulates glucoprivic feeding in rats. Other work has demonstrated involvement of hindbrain glucokinase in glucoregulation. Here we compared the effects of lateral (LV) and fourth ventricular (4V) injections of glucosamine on food intake in rats. We found that glucosamine injected into 4V (0, 0.2, 0.6, and 1.0 mg/rat) enhanced food intake in a dose-dependent manner and that LV and 4V injections were of similar potency. Glucosamine did not elevate blood glucose under the conditions of our test. We also found that enhancement of feeding by 4V glucosamine was abolished by medial hypothalamic injections of anti-dopamine beta hydroxylase saporin, a retrogradely transported catecholamine immunotoxin that selectively lesions norepinephrine and epinephrine neurons that innervate the injection site. Furthermore, 4V injection of glucosamine increased Fos expression in catecholamine populations responsible for key glucoregulatory responses. These results demonstrate that glucokinase in hindbrain catecholamine neurons is a mediator of food intake and possibly a transduction mechanism for stimulation of glucoregulatory feeding by these neurons.

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

Roach EB, Hussain Shuler MG (2011) Cholinergic reinforcement and temporal learning in rodent visual cortex. Neuroscience 2011 Abstracts 608.16. Society for Neuroscience, Washington, DC.

Summary: The idea that neuromodulators act as reinforcement signals has an intricate scientific history, including the well-characterized analogue of prediction error relayed by midbrain dopamine neurons. Neuromodulators released from discrete nuclei are poised to broadcast to many brain regions at once, and so it is an appealing concept to investigate other neuromodulatory systems within a reinforcement learning framework. Reward timing activity, a neural reflection of operantly learned stimulus-reward intervals in the primary visual cortex (V1), offers a tractable in vivo model to examine the role of candidate neuromodulators in temporal reward learning. Reward timing was first characterized in rats trained to lick a delivery tube to receive water rewards, where stimulation to one eye indicated reward availability after x licks, while stimulation to the other eye required y licks. Simultaneously recorded activity in V1 indicated that single unit responses evolve from reporting only visual characteristics to showing persistent increased/decreased firing or peak activity corresponding to the time of anticipated reward. Individual neurons report one interval or the other, even those with binocular peri-stimulus responses, arguing that reward timing is learned locally within V1. Theoretical work suggests that the local expression of reward associated intervals requires an interaction between the visually-evoked network response and a reinforcement signal conveying the time of reward. Based on anatomical and neurophysiological evidence, we hypothesized that cholinergic input from the basal forebrain (BF) could provide such a reward signal to V1. To test its necessity, BF cholinergic innervation in V1 was lesioned — using the selective neurotoxin 192 IgG-saporin — prior to changing the experimental policy between cues and associated reward delays. This allowed an examination of two potential roles for BF cholinergic input: in expressing previously learned intervals and in acquiring information about new intervals. We found that neurons from saline-infused controls, but not lesioned animals, shifted as a population to report the new, behaviorally relevant intervals (Kolmogorov-Smirnov, p < 0.05). Importantly, neurons from lesioned animals continued to report the previously learned intervals, suggesting that BF cholinergic input is required to learn, but not express, reward timing. These results support the notion that acetylcholine released from BF afferents acts as a reinforcement signal that guides cortical network plasticity.

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

Paolone G, Lamy D, Sarter M, Lee T (2011) Cognitive performance-associated increases in cholinergic neurotransmission also serve as a circadian signal to sustain performance-induced diurnal activity patterns. Neuroscience 2011 Abstracts 610.12. Society for Neuroscience, Washington, DC.

Summary: Daily practice of a sustained attention task (SAT) during the light phase of the light/dark cycle causes a stable, entrained, diurnal behavioral activity pattern (Gritton et al. 2009). As SAT performance is mediated by increases in cortical cholinergic neurotransmission, this experiment assessed levels of acetylcholine (ACh) release across the light and dark cycle of animals that previously performed the SAT at a fixed time. Circadian behavioral activity was recorded, and prefrontal ACh release was measured, using microdialysis, beginning on the third day following the last SAT session. SAT practice took place in either the light phase [ZT4], the dark phase [ZT16], or in a constant light condition [LL]. A control group practiced a daily fixed interval [FI-9] schedule of reinforcement at ZT4. A second control group was handled at randomly selected times but was neither water-deprived nor performed a task [NP]. Dialysates were collected, in a new environment, for 180 min total, beginning 90 min before the onset of prior task practice and again during the equivalent time period twelve hours later. For all animals, ACh release levels were higher during the dark phase. In SAT-performing animals, ACh levels increased for 45 min at ZT4 and ZT16. In addition, the ZT4 animals’ behavioral activity was robustly increased during this interval. Animals trained at ZT 4 reversed back to a nocturnal activity pattern 8-10 days after cessation of SAT practice, coinciding with the loss of the task time-synchronized cholinergic activity. In order to determine the necessity of these prior task period-synchronized release events for maintaining diurnal activity patterns, basal forerbain cholinergic neruons were lesioned by intra-basalis infusion of 192 IgG-saporin. As was expected, this lesion impaired SAT performance. Furthermore, following cessation of daily SAT practice, prior performance-period synchronized cholinergic release events were abolished in lesioned animals. Moreover, the lesion triggered a rapid post-performance return to a nocturnal acitvity pattern. Collectively, these results indicate that SAT performance-associated increases in prefrontal cholinergic activity not only support SAT performance but also contribute to cognition-induced diurnality. Furthermore, circadian control of cholinergic activation optimizes task performance as well as the generation of a cholinergic zeitgeber signal. In conclusions, the brain’s clocks and increases in cortical cholinergic neurotransmission interact bidirectionally to sustain cognitive performance and performance-evoked diurnal activity patterns.

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

Talman WT, Nitschke Dragon D, Jones S, Moore SA, Lin L-H (2011) Cardiovascular dysfunction and cardiac injury result from selective glial damage in the nucleus tractus solitarii. Neuroscience 2011 Abstracts 664.14. Society for Neuroscience, Washington, DC.

Summary: In man, extensive CNS dysfunction as may occur after subarachnoid hemorrhage may lead to cardiac damage and cardiac arrhythmias. We have shown that highly selective and restricted lesions of the nucleus tractus solitarii (NTS) may lead to similar cardiac and cardiovascular compromise. For example, using conjugates including the cytotoxin saporin (SAP) to selectively damage NTS neurons that express NK1 receptors or those that express tyrosine hydroxylase (TH) leads to cardiac dysfunction and associated lability of arterial pressure. In continuing efforts to better characterize cellular changes produced by introducing into the NTS conjugates containing SAP, we have studied the effect of anti-dopamine-beta-hydroxylase (anti-DBH)-SAP, stabilized substance P (SSP)-SAP, SAP (unconjugated), blank-SAP (non-targeted peptide conjugate), IgG-SAP (non-targeted immunoglobulin conjugate), and 6-hydoxydopamine (6-OHDA) as a control without SAP injected into NTS. We assessed effects of the injected agents both on cellular markers [NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), gamma-aminobutyric acid (GABA) receptor type a and b, neuronal nitirc oxide synthase (nNOS), TH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), glial fibrillary acidic protein (GFAP), connexin 43 (Cx43), DBH and protein gene product 9.5 (PGP 9.5)] and on cardiovascular and cardiac function. We have found that each compound containing SAP (including blank-SAP, IgG-SAP, unconjugated SAP) led to loss of GFAP and Cx43 immunofluorescent labeling in the NTS as well as lability of arterial pressure, cardiac arrhythmias, and cardiac myocytolysis. Those outcomes occurred despite neuronal specificity for each of the SAP conjugates. For example, anti-DBH-SAP led to a decrease in TH and DBH staining as well as a profound loss in GFAP and Cx43. In contrast, SSP-SAP led to loss of NK1 as well as GFAP, Cx43, and glutamate receptor markers but did not lead to loss of DBH or GABA. SSP-SAP also caused a loss in PGP9.5 which was not observed in all other agents. SAP and blank-SAP, on the other hand, led to loss of GFAP and Cx43 while 6-OHDA led to loss of TH and DBH, increased GFAP and decreased Cx-43. We are still investigating the effects of 6-OHDA on lability of arterial pressure and cardiac events but preliminary data suggest that, in doses used, it led to loss of TH and DBH but did not lead to either lability or cardiac events that were seen with each of the conjugates containing an SAP moiety. This study suggests that glial dysfunction may alone interefere with cardiovascular control through the NTS and may lead to cardiac damage and cardiovascular dysfunction.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11), Mouse IgG-SAP (Cat. #IT-18), Blank-SAP (Cat. #IT-21), Saporin (Cat. #PR-01)

Carr F, Géranton SM, Hunt SP (2011) The role of descending facilitation in the initiation and maintenance of mechanical hypersensitivity following inflammation. Neuroscience 2011 Abstracts 702.10. Society for Neuroscience, Washington, DC.

Summary: Central sensitisation is the key mechanism involved in the generation of mechanical hypersensitivity associated with tissue injury. Dorsal horn excitability is subject to regulation by descending modulation via the rostral ventromedial medulla (RVM) and enhanced descending facilitation under conditions of persistent nociceptive input contributes to the maintenance of mechanical hypersensitivity in chronic pain states. Depletion of mu-opioid receptor expressing (MOR+) cells of the RVM and depletion of spinal serotonin have been used previously to demonstrate the contribution of descending facilitation to the maintenance of neuropathic pain. Here we have used the same ablation techniques to investigate the contribution of descending pathways to the initiation and maintenance of mechanical hypersensitivity associated with ankle joint inflammation. Male Sprague-Dawley rats (215-220g at the time of injection) received bilateral microinjections of the selective cytotoxin dermorphin-saporin (1.5pM each side). 28 days later the animals received either an injection of 10μl Complete Freund’s Adjuvant (CFA) to the left ankle joint or underwent a sham procedure. Mechanical hypersensitivity of the hindpaw plantar surface was assessed using von Frey hairs from 2 hours up to 8 days post CFA injection. In a separate group of rats (160-180g at the time of injection) depletion of spinal serotonin was out carried out by intrathecal administration of 5,7-dihydroxytrptamine (5,7-DHT). Animals received either 10 μl of 5,7-DHT in saline (6μg/μl) or vehicle control. 6 days later animals received either CFA injection or underwent a sham procedure and mechanical hypersensitivity was assessed as in the dermorphin-saporin experiment. Depletion of the MOR+ cells of the RVM and of spinal serotonin was confirmed using immunohistochemistry. Dermoprhin-saporin pre-treatment resulted in significantly increased paw withdrawal thresholds from 6 hours up to 8 days following CFA injection (p < 0.01, ANOVA with repeated measures). In contrast depletion of spinal serotonin by 5,7-DHT led to a smaller attenuation of mechanical hypersensitivity at 24 hours and 48 hours following inflammation (LSD post hoc test, p < 0.01) but did not result in significantly increased paw withdrawal thresholds at the earlier time points.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Smith MA, Williams H, Krajewski SJ, Mcmullen NT, Rance NE (2011) Arcuate NK3 receptor-expressing KNDy neurons are essential for estrogen modulation of LH secretion and body weight in the female rat. Neuroscience 2011 Abstracts 712.07. Society for Neuroscience, Washington, DC.

Summary: Arcuate kisspeptin, neurokinin B, and dynorphin (KNDy) neurons have been proposed to mediate estrogen negative feedback in multiple species. To determine if these neurons are essential for this feedback, we ablated KNDy neurons in the arcuate nucleus of female rats using [MePhe7]Neurokinin B, a selective NK3 receptor (NK3R) agonist, conjugated to Saporin ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). The specificity of this conjugate for NK3R-expressing KNDy neurons is described in a separate abstract (see Krajewski et al., Soc. Neurosci. Abstr. 2011). Twenty-four female rats were ovariectomized (OVX) and received bilateral arcuate microinjections of either [MePhe7]NKB-SAP or a scrambled peptide conjugated to Saporin (Blank-SAP controls). 20-23 days later, animals were implanted with s.c. silastic capsules containing 17β-estradiol (E2), and animals were sacrificed 11 days later. Blood samples for RIA of serum LH were taken at time of OVX and injections (baseline), 20-23 days post-OVX, and 11 days after E2-treatment. Because OVX and E2-treatment have well-described effects on body weight, animals were weighed at the same three time points. In control animals, OVX induced a 13-fold rise in serum LH, which returned to baseline 11 days after E2 replacement. In contrast, OVX had no effect on serum LH in [MePhe7]NKB-SAP animals. There was a small decrease in serum LH 11 days after E2 replacement in [MePhe7]NKB-SAP animals, but the magnitude of this change was much less than seen in control animals. Control animals also exhibited a 20% increase in body weight 20-23 days after OVX, followed by a significant reduction after E2 replacement. Surprisingly, neither OVX nor E2 replacement affected body weight in [MePhe7]NKB-SAP-treated animals. Rather, these animals showed a steady increase in body weight throughout the experiment, at rates comparable to intact female rats or OVX rats treated with E2 (Williams et al., Endocrinology, 2010). Immunohistochemical studies showed near-complete destruction of KNDy neurons in the arcuate nucleus of [MePhe7]NKB-SAP animals. There was preservation of proopiomelanocortin and neuropeptide Y immunoreactivity in the arcuate nucleus and GnRH-immunoreactive fibers in the median eminence. These data provide compelling evidence that arcuate KNDy neurons play an essential role in estrogen negative feedback on LH secretion as well as the estrogen modulation of body weight.

Related Products: Blank-SAP (Cat. #IT-21), NKB-SAP (Cat. #IT-63)

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