sfn2009

Carvalho AF, Reyes AS, Van Bockstaele EJ (2009) The role of limbic norepinephrine in cannabinoid-induced aversion. Neuroscience 2009 Abstracts 449.3/V29. Society for Neuroscience, Chicago, IL.

Summary: The endocannabinoid system has been implicated in diverse physiological mechanisms including modulation of pain and analgesia, learning and memory and feeding, among others. Thus, targeting the cannabinoid system has risen to the forefront in the development of novel treatments for a number of pathophysiological processes. Consistent with this, agonists of the cannabinoid receptor type 1 (CB1R) have been successfully used in the treatment of severe anorexia in patients with AIDS and in alleviating nausea and vomiting in patients undergoing chemotherapy. However, significant side effects have been observed in clinical trials raising concerns regarding the potential clinical utility of cannabinoid-based agents. Disturbances in mood and affect, including paranoia, anxiety and nervousness, have been reported in patients. 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 contribute to the expression of unwanted side effects. We have previously shown an increase in anxiety-like behaviors in rats receiving repeated administration of cannabinoid agonists. This increase in anxiety was accompanied by increases in indices of noradrenergic activity. In the present study, we investigated whether norepinephrine in the limbic forebrain of rats is required for cannabinoid-induced aversion using an immunotoxin lesion approach combined with behavioral analysis using a place conditioning paradigm. Male Sprague Dawley rats received bilateral injections of a ribosomal toxin, saporin (SAP) conjugated to an antibody that specifically recognizes the enzyme dopamine-beta-hydroxylase (DSAP), into the limbic forebrain. Control rats received saporin alone. As previously reported, administration of the synthetic cannabinoid receptor agonist, WIN 55,212-2 (3.0mg/kg), induced aversion in a place conditioning paradigm in SAP-only treated rats. The rats’ spatial memory was also evaluated using the Morris Water Maze. Depletion of norepinephrine using DSAP in specific limbic regions impaired cannabinoid-induced aversion to WIN 55,212-2 without affecting learning and memory processes. Taken together, noradrenergic projections to the limbic forebrain may be critical in the manifestation of aversive behaviors associated with cannabinoid agonist exposure.

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

Freiria-Oliveira AH, Blanch GT, De Paula PM, Colombari E, Menani JV, Colombari DS (2009) Role of A2 noradrenergic neurons and angiotensinergic mechanisms on hypotension induced by hemorrhage. Neuroscience 2009 Abstracts 467.18/DD70. Society for Neuroscience, Chicago, IL.

Summary: The A2 catecholaminergic neurons in the commissural subdivision of the nucleus tractus solitarii (cNTS) are activated by hemorrhage. However, the role of these neurons on the cardiovascular adjustments to hemorrhage is not fully understood. In the present study we investigated the effects of A2 noradrenergic neuron lesion alone or combined with the blockade of angiotensinergic mechanisms on the recovery of blood pressure after hemorrhage. Male Holtzman rats (280-320 g) anesthetized with ketamine combined with xylazine were submitted to lesions of dopamine-beta-hydroxilase (DβH)-containing neurons in the cNTS achieved with injections of anti-DβH-saporin (12.6 ng/60 nl, n=6-8) or sham lesions (injection of immunoglobulin-G-saporin, 12.6 ng/60 nl, n=6). Changes in blood pressure to hemorrhage were tested 30 days after lesions. Immunohistochemistry for tyrosine-hydroxilase was performed to confirm the efficacy of DβH neuron lesion in the cNTS. Two days before tests, femoral artery and vein were cannulated under ketamine and xylazine anesthesia. Hemorrhage consisted in four blood withdrawals (2 ml/300 g body weight, every 10 min) in conscious rats. Immediately after the 4th blood withdrawal, the hypotension was similar in A2-lesioned and sham-lesioned rats (-62 ± 7 mmHg and -73±7 mmHg, respectively). However, A2-lesioned rats rapidly (20 min) recovered from hypotension (-7±2 mmHg), while sham rats did not completely recover from hypotension until the end of experiment (60 min after the 4th blood withdrawn, -20±3 mmHg). The pre-treatment with losartan (angiotensin type 1 receptor antagonist, 10 mg/kg of body weight, iv) impaired the recovery of blood pressure by A2-lesioned rats (-29 ± 4 mmHg and -28 ± 3 mmHg, 20 and 60 min after the 4th blood withdrawal). In sham rats, the treatment with losartan also reduced the partial recovery of blood pressure at the end of the test (-39±6 mmHg, vs. sham control: -20±3mmHg), however, losartan did not affect the hypotension 20 min after the 4th blood withdrawal (-30± 6 mmHg vs. sham control: -35 ± 9 mmHg). The results suggest that A2 noradrenergic neuron lesion in the cNTS facilitates the recovery of hypotension after hemorrhage, probably increasing the action of angiotensinergic mechanisms.

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

Macleod JE, Ackerman CM, Bucci DJ (2009) Contributions of the medial prefrontal cortex to negative occasion setting. Neuroscience 2009 Abstracts 477.2/FF61. Society for Neuroscience, Chicago, IL.

Summary: The medial prefrontal cortex of rats has a role in many aspects of cognitive function, and especially forms of inhibitory learning. Recent research has revealed heterogeneous functions of the prelimbic (PL) and infralimbic (IL) regions of the medial prefrontal cortex in modulating response inhibition. In a recent study, we tested the effects of separate neurotoxic lesions of the PL or IL in a serial feature negative discrimination paradigm (negative occasion setting). Rats received daily training sessions consisting of 16 trials: on 4 trials in each session, a tone was presented and followed by food reward; on the remaining trials, the tone was preceded by a visual stimulus and not reinforced. Our results indicate that PL but not IL is necessary for learning the discrimination. A second study was conducted to investigate the effects of these lesions on rats that were first extensively trained in this task. We found that rats that had been trained for 30 days prior to receiving PL or IL lesions were still able to perform the task as well as controls. Therefore, PL lesions disrupt acquisition but not performance of a serial feature negative discrimination. This same task has been used in our laboratory to investigate the effects of nicotine on learning. We have shown that nicotine-treated rats exhibit greater discrimination between the two trial types as evidenced by less frequent responding during non-reinforced trials, and learn the discrimination in fewer sessions than control rats. In addition, rats receiving nicotine showed an increase in rearing behavior during the presentation of the light, suggesting nicotine enhanced attention to the visual stimulus. One possible critical site of action for nicotine’s effects is the medial prefrontal cortex. Research in other laboratories utilizing other training procedures suggest that cholinergic activity in the medial prefrontal cortex is critical for attending to behaviorally relevant stimuli, and have implicated the rat PL in visual attention as well as inhibiting prepotent, goal oriented responses. We investigated the contribution of the cholinergic PL to learning the serial feature negative discrimination task by training rats that had received infusions of 192-IgG-saporin into PL to remove cholinergic input from the basal forebrain. No differences between control and lesion rats were observed. Taken together, the results suggest that PL is necessary for acquisition of a serial feature negative discrimination, although the basal forebrain cholinergic input into this region is not required to sufficiently learn the task.

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

Datta S, Chatterjee K, Kline IV RH, Wiley RG (2009) CCK receptor- expressing dorsal horn neurons: Role in pain and morphine analgesia. Neuroscience 2009 Abstracts 265.13/Z37. Society for Neuroscience, Chicago, IL.

Summary: Spinal intrathecal cholecystokinin (CCK) has anti-opiate activity, and the CCK antagonist, proglumide potentiates opiate analgesia. In the present study, we sought to determine the effects of selectively destroying CCK receptor-expressing lumbar dorsal horn neurons using the targeted cytotoxin, CCK-saporin on reflex and operant nocifensive responses to heat, and on the actions of systemic morphine and naloxone. Exp. 1: Adult, female rats were injected into the lumbar CSF with either 1500 ng of CCK-sap (n=7) or blank (control nonsense peptide)-saporin (n=6). Exp. 2: rats were pre-injected intrathecally with 1 ug of proglumide (CCK antagonist) followed by 1500 ng CCK-sap (n=4) or only CCK-sap (1500 ng; n=4). Rats were then tested on the hotplate at 44°C and 47°C and on an operant thermal preference task (TPT) using a shuttle box where the floor on one side was 15°C and the other 45°C. Morphine was tested in the TPT using 0, 0.5, 1.5 and 2.5 mg/kg s.c. 4-8 weeks post-toxin. Naloxone (0 vs 0.8 mg/kg s.c) was also tested in the TPT. In Exp. 1, the CCK- sap group showed decreased hotplate reflex responses, but decreased time on the 45°C side in the TPT. In Exp. 2, CCK-sap only rats also showed greater heat aversion in the TPT. In both Exps, CCK-sap groups demonstrated greater heat aversion (less analgesia) than either control group after morphine in the TPT. After naloxone, both control groups, but not the CCK-sap rats, showed increased heat aversion (hyperalgesia). We interpret these results as showing that selective destruction of CCK receptor- expressing superficial dorsal horn neurons increases nocifensive reflex responses to aversive heat and produces thermal hyperalgesia while decreasing the effects of both morphine and naloxone suggesting a complex role for CCK receptor-expressing dorsal horn neurons in modulation of nociception and opiate drug action.

Related Products: CCK-SAP (Cat. #IT-31)

Vetrivelan R, Anaclet C, Fuller PM, Yoshida K, Lu J, Saper CB (2009) Comparison of sleep-wake changes after lesions of two sleep-promoting cell groups in the preoptic region in rats. Neuroscience 2009 Abstracts 277.2/EE14. Society for Neuroscience, Chicago, IL.

Summary: Previous studies have shown that two cell groups within the preoptic region viz., median preoptic nucleus (MnPO) and ventrolateral preoptic nucleus (VLPO) show c-Fos expression during spontaneous sleep. However, while lesions of the VLPO have been shown to cause sleep loss, the effects of MnPO lesions on sleep have not been available to date. We therefore performed cell-body specific lesions of these two nuclei using the toxin orexin-saporin and studied the spontaneous sleep-wake behavior in rats. We found that the animals with more than 70% cell loss in the VLPO (n=15) showed a 31% increase in wakefulness (61.03±1.15% in VLPO-lesioned animals vs 46.53±0.55% in controls, P <0.001) and a concomitant reduction in non rapid eye movement (NREM) sleep and REM sleep. There was also a significant reduction in the average NREM sleep episode duration (120±6.57 Sec Vs 143.04 ± 4.53 sec in control animals, P < 0.01) in rats with VLPO lesions. On the other hand, lesions involving 80-90% cell loss in the MnPO (n=6) produced a moderate 15% increase in wakefulness (53.8±1.09% vs 46.53±0.55% in controls. P<0.001). Although the NREM sleep episode duration was reduced in these animals (126 ± 6.61 Sec vs 143.04 ± 4.53 sec in control animals, P = 0.06), it did not reach statistical significance. The extent of the lesions in the present study was estimated by an individual blind to the experimental conditions and the sleep results. Although specific cell groups (MnPO or VLPO) were carefully targeted, partial damage (10-20%) to the other cell group was often encountered. Nevertheless, our results clearly demonstrate that while the MnPO plays an important role in the regulation of sleep, the VLPO plays a substantially greater role.

Related Products: Orexin-B-SAP (Cat. #IT-20)

Morin LP, Studholme KM (2009) Saporin lesions that target suprachiasmatic cells bearing NPY receptors eliminate or greatly impair circadian rhythm generation and entrainment. Neuroscience 2009 Abstracts 278.7/EE49. Society for Neuroscience, Chicago, IL.

Summary: General destruction of the SCN caused by electrical lesions produce loss of circadian rhythmicity and entrainment. More specific, cell-directed lesion methods, such as the use of NMDA as a neurotoxin, have not been successful. Here, we describe the use of the ribotoxin, Saporin (SAP), to kill specific types of SCN neurons and show the effects of such selective lesions on the hamster circadian locomotor rhythm. Adult male golden hamsters were injected bilaterally with 200 nL of a SAP/neuropeptide conjugate into the SCN bilaterally. The neuropeptides were neuropeptide Y (NPY), cholecystokinin (CCK) or substance P (SP). NPY terminals are distributed throughout the SCN; CCK and SP cells are present in the SCN and there have been NPY and SP receptors described in the hamster SCN. SAP/NPY (N=10) treatment caused arrhythmicity in 4 animals under LD conditions and 4 others became arrhythmic when transferred to DD. Arrhythmicity occurred in 1/9, 0/8 and 0/10 animals treated with SAP/CCK, SAP/SP or vehicle. There was also a significant effect of treatment on the level of variability of the activity records as indicated by precision of activity onset (p<.008) and approximate entropy analysis of disorder within the running record (p<.004). The SAP/NPY group accounted for nearly all the between-group variability. The histology showed a large decrease in the number of SCN cells, but there were many cells remaining after SAP/NPY treatment. Care was taken to determine that the remaining cells were, in fact, neurons. Also, the brains of lesioned animals retained reasonably intact RHT, GHT and 5HT input pathways. Normal histology evaluated for NeuN, a neuronal antigen, showed that an unexpected pattern of NeuN-IR cells in the SCN of normal animals, with the majority of such neurons found in an area that includes the SCNce and the region dorsolateral. NeuN was heavily co-localized with calbindin-IR in cells of the SCNce, but not with VP- or VIP-IR. This distribution of SCN cells containing NeuN-IR was approximately the same in both mice and hamsters. Conclusions: (1) SAP/NPY lesions many, but not all SCN neurons; (2) Such lesions result in massive degradation of circadian rhythmicity; (3) The three main SCN input pathways remain essentially intact after SAP treatment; (4) NeuN-IR neurons are distributed in a novel pattern in the SCN of both mouse and hamster; (5) Presently unidentified SCN cells bearing NPY receptors are likely to be critical to the generation of cohesive circadian rhythms, whereas those bearing SP or CCK receptors are minimally, if at all involved.

Related Products: CCK-SAP (Cat. #IT-31), SSP-SAP (Cat. #IT-11), NPY-SAP (Cat. #IT-28)

Janke KL, Fazelinik S, Roland JJ, Servatius RJ, Servatius RJ, Servatius RJ, Pang K (2009) Role of the medial septum on navigational strategy and shifting between strategies: Effects of selective cholinergic and GABAergic lesions. Neuroscience 2009 Abstracts 283.5/EE134. Society for Neuroscience, Chicago, IL.

Summary: Cholinergic and GABAergic neurons are major components of the septohippocampal pathway, and comparisons between the two neuronal populations are important for understanding the function of medial septum-vertical limb of the diagonal band (MSDB). Recently, we have been investigating the importance of MSDB neurons in cognitive flexibility. Cognitive flexibility is commonly examined in reversal of stimulus-reward associations and attention set shifting. The present studies examine whether selective lesions of cholinergic or GABAergic MSDB neurons impair shifting between egocentric and allocentric navigation strategies. Sprague Dawley rats were administered saline, GAT1-saporin or 192-IgG saporin into the MSDB to produce no damage, selective GABAergic damage or selective cholinergic damage, respectively. Lesion verification will be performed using immunocytochemistry at the end of the studies. In a plus maze, rats started in one of two arms opposite each other (i.e., north and south arms) randomized across trials. On any single trial, the arm opposite the starting arm was blocked forming a T-maze. Rats have a choice of entering one of the remaining 2 arms (east or west arms) for food reinforcement. During the acquisition phase of the first study, rats were reinforced to enter a particular arm (east or west: allocentric response) regardless of their starting location. After they reached criteria (10 consecutive correct choices), the goal location was either reversed (east to west) or shifted to an egocentric response strategy (left or right turn). Animals that received either GAT-1-saporin (.26 ug/ul) or 192-IgG saporin (.217ug/ul) lesion reached criteria faster than saline treated rats. No significant effects of either lesion were observed on spatial reversal or strategy shifts. However, qualitative assessment of the damage suggests that GAT1-saporin may have produced an incomplete lesion. Therefore, a second study using GAT1-saporin at .325 ug/ul was conducted. For this study, half of the rats were trained on an egocentric strategy and the other rats are reinforced for an allocentric response. When rats reached criteria, half of each group was trained in a reversal learning or strategy shift. Preliminary data show that rats treated with GAT1-saporin or saline learned the initial egocentric or allocentric strategy at a similar rate. However, animals were faster to reach criteria in the allocentric condition than the egocentric condition. Reversal learning and strategy shifting in the second study is currently being assessed. The results of this study will provide important insight into the role of the MSDB in learning and cognitive flexibility.

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

Roland JJ, Janke KL, Gluck MA, Beck KD, Pang KCH, Servatius RJ (2009) Selective cholinergic and GABAergic lesions of the medial septum slows acquisition of the classically conditioned eyeblink response in rats. Neuroscience 2009 Abstracts 283.6/EE135. Society for Neuroscience, Chicago, IL.

Summary: Both human and animal studies have demonstrated that the hippocampus is not essential for the acquisition of delay eyeblink conditioning. However, nonselective medial septal damage, in both rabbits and humans, impaired acquisition of delayed eyeblink conditioning, as well as latent inhibition of eyeblink conditioning. The medial septum provides a major cholinergic and GABAergic afferent projection to the hippocampus, and the effects of medial septal damage is widely believed to occur through its connections to the hippocampus. Cholinergic muscarinic antagonists impaired delay eyeblink conditioning when administered systemically or directly into the hippocampus. Computational models also predicted the lack of effects on delay conditioning or latent inhibition of eyeblink conditioning caused by interference of the cholinergic septohippocampal system Recent studies have suggested that the GABAergic septohippocampal system may be a major site of action for scopolamine. Therefore, the current study examined the effect of selective cholinergic or GABAergic medial septal lesions on the classically conditioned eyeblink response. Adult male Sprague-Dawley rats received either a sham, cholinergic (192-IgG saporin) or GABAergic (GAT1-saporin) lesion in the MS/DB. Two weeks later, all animals were implanted with stimulating and recording electrodes in the periorbital muscle. Following recovery, all animals received three consecutive days of delay eyeblink conditioning. Each daily session consisted of 100 paired CS-US (conditional stimulus – unconditioned stimulus) trials with an average intertrial interval (ITI) of 30 seconds. The CS was a 500ms tone which co-terminated with the US, a 10ms, 10V periorbital stimulation. Our preliminary results shows that both cholinergic and GABAergic lesions impaired acquisition of delayed eyeblink conditioning, as compared to the sham-lesioned group. However, after three days of training all three treatment groups reached the same asymptotic performance. Future studies will assess the effects of combined cholinergic and GABAergic lesions and the effects of these septal lesions on latent inhibition of the conditioned eyeblink response.

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

Lima T-Z, Blanco MM, Bueno MA, Dos Santos Junior JG, Bargieri DY, Mello LE (2009) The influence of cholinergic degeneration on the progression of Alzheimer’s disease and its action in determining the outcome of lithium treatment. Neuroscience 2009 Abstracts 139.26/D36. Society for Neuroscience, Chicago, IL.

Summary: A substantial loss of cholinergic innervation in the hippocampus and cerebral cortex is universally accepted as a typical feature of Alzheimer’s disease (AD). Cholinergic deafferentation is an often, but not a constant phenomenon in AD and its contribution to the progression of disease is not completely understood. The present work was aimed to evaluate the effect of cholinergic deafferentation on cognitive decline and on Amyloid-b (A_) metabolism and how this outcome is modulated by lithium. To this end rats were subjected to neonatal intracerebroventricular injection of 192 IgG-saporin (an immunotoxin selective to cholinergic neurons). Three months after surgery animals were evaluated in Morris Water Maze (MWM) and then entered a three months long lithium (LiCl) or control treatment. At the end of treatment, animals were once again tested in MWM and their cerebral cortical A_ levels were measured. We found that working memory impairment arises earlier and it is also more severe than reference memory related deficits. The cognitive performance was only slightly influenced by LiCl treatment. Furthermore we found that the cholinergic denervation produced by neonatal IgG-sap infusion did not modify A_ levels or its aggregation state. Moreover lithium increased the levels of A_1-42 despite decreasing the amount of A_1-40, an effect that is dependent of cholinergic integrity. These data suggest that the contribution of cholinergic deafferentation, which occurs over the progression of disease, to the amyloigenesis is likely to be modest in AD brain. In addition the effects of lithium treatment presented here imply in avoiding its use as prophylactic propose for AD and in AD cases without marked cholinergic degeneration.

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

Wiley RG, Kline IV RH, Lemons LL (2009) Role of galanin receptor-expressing dorsal horn neurons in nocifensive reflex responses to heat. Neuroscience 2009 Abstracts 170.17/X19. Society for Neuroscience, Chicago, IL.

Summary: Spinal intrathecal (i.t.) galanin has been reported to be antinociceptive in some situations. Using lumbar i.t injections of galanin, coupled to the ribosomal inactivating peptide, saporin, to selectively destroy spinal dorsal horn cells that express galanin receptors, we sought to determine the role of galanin receptor-expressing dorsal horn neurons in reflex nocifensive hotplate behavior. Rats were injected into lumbar CSF with either 500 ng or 750 ng Gal-sap or saline, then tested over several weeks on the hotplate at 44o, 47o and 52oC. Gal-sap increased hindpaw withdrawal latencies only to 44oC and decreased the amount of responding on both 44o and 47oC hotplates. Morphine (5 mg/kg, s.c.) twenty minutes before 44°C hotplate testing slightly increased initial response latency and significantly decreased responding of the control rats. The antinociceptive effect of morphine in the Gal-sap rats was approximately additive with the antinociceptive effect of Gal-sap. Mustard oil applied to the dorsal hindpaws significantly increased responding on the 44°C hotplate in control rats, but produced less of an increase in Gal-sap rats. Topical capsaicin to hindpaw plantar skin reduced control, but not Gal-sap, responses on the 44°C hotplate. These results suggest a role for galanin receptor-expressing dorsal horn neurons in modulation of nociception that is unique, different from several other types of dorsal horn neurons and suggests a strategy for augmenting opiate drug effect.

Related Products: Galanin-SAP (Cat. #IT-34)