sfn2010

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)

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)

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)

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)

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)

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)

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)

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)

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)

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)