sfn2011

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: Custom Conjugates, Blank-SAP (Cat. #IT-21)

ATS Poster of the Year Winner

Krajewski SJ, Smith MA, Williams H, Ciofi P, Lai JY, Mcmullen NT, Rance NE (2011) Ablation of NK3 receptor-expressing KNDy neurons in the rat arcuate nucleus using [MePhe7]Neurokinin B-Saporin. Neuroscience 2011 Abstracts 712.09. Society for Neuroscience, Washington, DC.

Summary: A subpopulation of neurons expressing kisspeptin, neurokinin B and dynorphin (KNDy neurons) has been shown to reside within the arcuate nucleus of many mammalian species. Although these peptides are critical for reproductive function, the precise role of the arcuate KNDy neurons is not fully understood. Here we describe a method to ablate KNDy neurons based on their co-expression of the Neurokinin 3 receptor (NK3R, Burke et al., J. Comp. Neurol, 2006). Saporin, a molecular neurotoxin, was conjugated to [MePhe7]Neurokinin B, a selective NK3R agonist ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). Binding studies revealed that the conjugation of saporin did not alter the affinity of [MePhe7]NKB to NK3R in rat cerebral cortex membranes. To investigate the specificity of this conjugate for ablation of NK3R neurons, stereotaxic surgery was used to bilaterally inject [MePhe7]NKB-SAP into the arcuate nucleus of female rats. Control rats were injected with saporin conjugated to a scrambled peptide (Blank-SAP, Advanced Targeting Systems). Rats were sacrificed 31-34 days later and the brains were processed for immunohistochemical studies. Nissl stained sections from [MePhe7]NKB-SAP-treated rats showed no signs of inflammation at the injection sites and no qualitative changes in cell density compared to Blank-SAP control rats. Immunohistochemistry revealed near-complete loss of NK3R-immunoreactive (ir) neurons throughout the arcuate nucleus of [MePhe7]NKB-SAP rats. When the injection site was dorsal to the arcuate nucleus, there was also variable loss of NK3R-ir cells in the lateral hypothalamus and zona incerta. In the arcuate nucleus, [MePhe7]NKB-SAP injections resulted in a 98% and 94% reduction in the number of kisspeptin and neurokinin B-ir neurons, respectively, compared to Blank-SAP controls. The number of dynorphin-ir neurons in the arcuate nucleus of [MePhe7]NKB-SAP-treated rats was reduced by 67%, a value consistent with the co-expression of NK3R on dynorphin neurons in our previous study (Burke et al., J. Comp. Neurol, 2006). In contrast, arcuate proopiomelanocortin and neuropeptide Y immunoreactivity were preserved in [MePhe7]NKB-SAP rats. Moreover, there was no difference in GnRH-ir fiber density in the median eminence between the two groups. These results document the utility of [MePhe7]NKB-SAP for selective ablation of NK3R-expressing KNDy neurons in rat hypothalamus. These rats were used to examine the role of KNDy neurons in the estrogen regulation of LH secretion and body weight in the female rat (see Smith et al., Soc. Neurosci. Abstr. 2011).

Related Products: Custom Conjugates, Blank-SAP (Cat. #IT-21)

ATS Poster of the Year Winner

Winter SS, Köppen JR, Stout JM, Cameron HA, Wallace DG, Cheatwood JL (2011) Growth factor infusion increases BrdU-positive cells in the denervated medial septum following 192-IgG-saporin lesion. Neuroscience 2011 Abstracts 331.04. Society for Neuroscience, Washington, DC.

Summary: During the progression of Alzheimer’s Disease, degeneration of basal forebrain structures is associated with a decline in mnemonic function and frequently results in episodes of wandering behavior. Previous work has demonstrated that the septohippocampal cholinergic system uniquely contributes to rat spatial orientation. Enhancement of endogenous adult neurogenesis represents one potential method to restore function to the septohippocampal system. Therefore, we tested the hypothesis that co-infusion of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) would increase the number of new cells in the medial septum following a lesion of the cholinergic system produced by focal injection of the ribosome-inactivating selective immunotoxin 192-IgG-saporin in rats. For this, rats received injections of 192-IgG-saporin into the medial septum. At the same time, a cannula was placed in the lateral ventricle and attached to a subcutaneously-placed osmotic minipump containing either 1) EGF, bFGF, and bromodeoxyuridine (BrdU), or 2) BrdU alone. Infusion of growth factors and BrdU continued for a period of two weeks, at which point the pumps were removed. At 21 days following 192-IgG-saporin injury, rats were perfused following standard protocols. Cryostat sections were collected at 40 microns and were processed via double-fluorescent immunochemistry (IHC) using antibodies against BrdU and doublecortin (DCX). Photomicrographs of BrdU and DCX immunofluorescence were captured under epifluorescence and the number of BrdU-positive and DCX-positive cells was quantified. We detected significantly higher numbers of BrdU-positive cells in the medial septum of rats that received growth factors compared to rats that received BrdU-only (p<0.05). These results indicate that infusion of growth factors following 192-IgG-saporin lesion of the medial septum resulted in an increase in the number of new immature neurons in the medial septum. Studies aimed at determining the fate of these young neurons and their influences on spatial orientation are ongoing.

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

Taxini CL, Bícego K, Takakura A, Moreira T, Gargaglioni L (2011) Noradrenergic neurons of the A5 region play a role on hypoxic ventilatory response in unanesthetized rats. Neuroscience 2011 Abstracts 345.09. Society for Neuroscience, Washington, DC.

Summary: The ventrolateral pons contains the A5 group of noradrenergic neurons which is involved in cardiorespiratory control. These cells are strongly activated by carotid body stimulation and display central respiratory modulation. Recently, we showed that A5 neurons contribute to the cardiorespiratory effects elicited by chemoreflex stimulation in anesthetized rats. In the present study, we assessed the role of A5 noradrenergic neurons on cardiorespiratory responses produce by hypoxia in unanesthetized rats. To selectively destroy noradrenergic neurons, we administered the immunotoxin anti-dopamine β-hydroxylase-saporin (anti-DβH-SAP, 200nL) bilaterally in the A5 region of male Wistar rats (n = 8). Hypoxia (7% O2, 30 min) produced an increase in ventilation (Ve) (1470 ± 141 mLkg-1min-1), respiratory frequency (RF) (179 ± 139 brethsmin-1) and heart rate (484 ± 29 bpm), without affect mean arterial pressure (MAP) in conscious rats. Bilateral destruction of the catecholaminergic A5 neurons reduced the hypoxia-induced hyperventilation (942 ± 110 mLkg-1min-1, p<0.05), increase in RF (139 ± 11 breathsmin-1, p<0.05) and tachycardia (399 ± 39 bpm, p<0.05). These results suggest that A5 noradrenergic neurons contribute to the increase in heart rate, ventilation and respiratory frequency during peripheral chemoreflex stimulation.

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

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

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

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