targeted-toxins

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)

Lemons LL, Wiley RG (2009) Role of galanin receptor-expressing dorsal horn neurons in operant nocifensive responses. Neuroscience 2009 Abstracts 170.18/X20. Society for Neuroscience, Chicago, IL.

Summary: Selective destruction of galanin receptor-expressing dorsal horn neurons using the targeted cytotoxin, galanin-saporin (gal-sap), reduced reflex nocifensive hotplate responses, particularly at 44° C (see adjacent poster). The antinociceptive effect of gal-sap was additive with morphine (5 mg/kg, s.c.) in reducing hotplate responses. While these findings are provocative, inferences about analgesia also require information on cerebral processing of nociceptive information, such as obtained from operant nocifensive responses. We therefore sought to determine the effects of lumbar intrathecal gal-sap on nocifensive operant responses. Thirteen Long Evans female rats were injected with either 500 ng gal-sap or 500 ng blank-sap and tested on the escape test at several temperatures. The escape task consists of a two-chambered box; one side is dark with a thermal floor while the other side is brightly lit with a room temperature shelf. Gal-sap treated rats escaped from the thermal plate to the escape shelf less than controls. The difference was particularly striking at 25°, 38°, 44°, 45°, and 47°C. Morphine effects on escape responses was tested at 44°C. Thirty minutes before testing, rats were injected subcutaneously with either 0, 0.5, 1.5 or 4.5 mg/kg morphine. The testing was done over four days such that every rat was tested at each dosage in a counterbalanced fashion. 1.5mg/kg of morphine significantly reduced the escape duration of the blank-sap control rats, but not the gal-sap rats. The 4.5mg/kg dosage completely eliminated escape responding in both control and gal-sap rats. In summary, Gal-sap rats showed reduced nocifensive reflex responding on the hotplate tests, which was further reduced in essentially additive fashion by 5mg/kg of morphine. The Gal-sap treated rats also showed reduced escape behaviors at 44°C in the operant escape test, but were less sensitive to 1.5 mg/kg of morphine than the control rats. These results differ from the effects of NPY-saporin and dermorphin-saporin, cytotoxins also targeted at dorsal horn interneurons, suggesting that selective destruction of galanin receptor-expressing superficial dorsal horn neurons is analgesic and that galanin-receptor-expressing dorsal horn interneurons play a unique role in nociceptive processing.

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

Jaime S, Perez Cordova MG, Hernandez S, Colom L (2009) Immunolesions of medial septal GABAergic neurons. Neuroscience 2009 Abstracts 241.8/I15. Society for Neuroscience, Chicago, IL.

Summary: Epilepsy is a neurodegenerative condition characterized by spontaneous recurrent seizures that are triggered by excessive electrical activity due to changes in neurological functions. One of the most common forms of epilepsy is Temporal Lobe Epilepsy (TLE) in which seizures originate in limbic structures as hippocampal and/or para-hippocampal areas. Principal cell (i.e. pyramidal cells) activity is indirectly regulated by rhythmic inputs from GABAergic neurons in the septal region of the basal forebrain which selectively innervate inhibitory hippocampal interneurons. In previous studies, using the pilocarpine model of TLE, we have demonstrated that the septum plays an antiepileptic role and that medial septum GABAergic neurons degenerate in the epilepsy process. Thus, damage of medial septum GABAergic neurons may contribute to epileptogenesis. The purpose of this study is to investigate the role of medial septum GABAergic neurons in excitability control and epileptic activity generation. For this purpose, anti-GAT1-SAP (3µL at 325ng/µL) was stereotaxically injected in the medial septum of Sprague Dawley male rats to selectively destroy this neuronal population and investigate the subsequent functional changes. Analysis was performed using stereological approaches which revealed a significant reduction in cell count between treated (anti-GAT1-SAP) and saline-injected control rats (8591.38±941.65 and 25609.87±407.73 respectively; (Student’s t-test; p<0.05). In conclusion, our preliminary results show that the single injections of anti-GAT1-SAP selectively lesions most of the medial septum GABAergic neurons, providing a powerful tool to study the role of these neurons in the control of hyperexcitability states. Studies underway involve the investigation of the functional alterations produced by the selective destruction of MS GABAergic neurons.

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

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)

Mcauley J, Stewart AL, Pang KCH (2009) Role of cholinergic NBM neurons in timing and divided attention. Neuroscience 2009 Abstracts 95.12/EE81. Society for Neuroscience, Chicago, IL.

Summary: The nucleus basalis magnocellularis (NBM) provides cholinergic and GABAergic innervation to the neocortex. In previous studies, non-selective lesions of the NBM using ibotenic acid impaired interval timing and divided attention. Rats with NBM damage produced rightward shifts in peak times, demonstrating overproduction (underestimation) of time. Additionally, NBM damage impaired the ability to divide attention when timing two intervals simultaneously. Damage of the frontal cortex produced similar impairments in timing and divided attention as NBM damage, suggesting the NBM projections to frontal cortex were critical. Currently, the NBM neurons responsible for modulating timing and attention are unknown. The present study will determine the importance of cholinergic NBM neurons in timing and attention using the selective immunotoxin 192-IgG saporin (192-SAP). Sixteen Sprague Dawley rats were first trained on a peak-interval (PI) procedure using fixed-intervals of 12 s and 24 s paired with light and tone stimuli, respectively. During this phase, only one stimulus was presented during a trial (focused attention). Following the initial phase of training, rats were trained on a divided attention version of the peak-interval procedure, in which 2 stimuli were presented simultaneously in a trial and rats timed both intervals in parallel. Rats were administered 192-SAP into the NBM (n = 10) or given SHAM surgeries (n = 6). Following surgery, 192-SAP rats produced a leftward shift in timing with increased variability compared to SHAM rats. These changes in timing were observed in both focused and divided attention conditions, but the effects were larger in divided attention conditions than in focused attention conditions. Results implicate the cholinergic NBM neurons in the modulation of interval timing and divided attention. Current work is verifying the selectivity and efficacy of the 192-SAP administration. Additional studies will examine the role of GABAergic NBM neurons in interval timing and divided attention.

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

Croxson PL, Baxter MG (2009) Multiple neuromodulator depletion interacts with fornix transection to impair episodic memory in monkeys. Neuroscience 2009 Abstracts 98.4/EE128. Society for Neuroscience, Chicago, IL.

Summary: Acetylcholine may play an important role in some aspects of cognitive function, and in particular in episodic memory. However, the role of other neuromodulatory (NM) substances, such as noradrenaline, dopamine, and serotonin, in episodic memory is less well-defined. We tested monkeys on a model of episodic memory in monkeys and carried out specific depletions of different neuromodulators within inferotemporal cortex (IT). Six rhesus macaque monkeys (five male) were trained on an object-in-place scene learning task that models key features of human episodic memory, because learning occurs rapidly (often in a single trial) in the contaxt of unique background scenes. After preoperative testing three monkeys were given injections into IT of the immunotoxin ME20.4-saporin interleaved with injections of 6-hydroxydopamine and 5,7-dihydroxytryptamine. This resulted in depletion of acetylcholine, dopamine, noradrenaline and serotonin throughout IT (group NM+ACh). Three monkeys received the same treatment but omitting the ME20.4-saporin, thus depleting dopamine, noradrenaline and serotonin, but sparing acetylcholine (group NM). Neither group of monkeys (NM+ACh or NM) were impaired in postoperative scene learning. We found previously that addition of fornix transection to depletion of ACh from IT severely impaired scene learning relative to fornix transection alone (Browning et al. 2009, Cerebral Cortex). Therefore we gave each monkey in groups NM and NM+ACh a bilateral fornix transection and performed a further postoperative performance test. As expected, monkeys in group NM+ACh were severely impaired in scene learning following fornix transection. However, monkeys in group NM were also severely impaired in scene learning following fornix transection, despite having no visible damage to cholinergic innervation. Depletion of cholinergic, dopaminergic, adrenergic and serotoninergic innervation of inferotemporal cortex, therefore, is not sufficient to impair monkeys’ performance on an episodic memory task. Furthermore, there is a synergistic interaction between the NM depletion and fornix transection in this task, like that between ACh depletion and fornix transection. This may be due to a general reduction in cortical function after NM depletion, albeit not sufficient to cause episodic memory impairment on its own, which exacerbates the effect of fornix transection. It may point to one or more of these neuromodulators having a role in post-lesion plasticity, a role that is also played by ACh. Importantly, these data suggest that intact cholinergic innervation is not sufficient for post-lesion plasticity.

Related Products: ME20.4-SAP (Cat. #IT-15)