targeted-toxins

Blanco-Centurion CA, Shiromani PJ (2008) Effects of lesions of three downstream targets of orexin/hypocretin neurons does not support the “flip-flop switch” model. Neuroscience 2008 Abstracts 586.2/SS35. Society for Neuroscience, Washington, DC.

Summary: Hypocretin (HCRT) neurons heavily innervate the cholinergic neurons in the basal forebrain (BF), histamine neurons in the tuberomammillary nucleus (TMN) and the noradrenergic locus coeruleus (LC) neurons, three populations that have traditionally been implicated in arousal. Based on the innervation HCRT neurons may regulate arousal by driving these downstream arousal neurons. Here we directly test this hypothesis by simultaneously lesioning these neurons using three saporin-conjugated neurotoxins. Forty four adult male Sprague-Dawley rats received stereotaxically (under anesthesia) delivered microinjections of three different saporin-conjugated neurotoxins as follows: HCRT2-saporin (250 ng/L in 0.25 µL) to lesion TMN neurons; anti-DBH-saporin (1 μg/μL in 0.25 μL) to destroy noradrenergic LC neurons; and 192-IgG-saporin (2 μg/μL ICV; 3μL) to kill the BF cholinergic neurons. Control rats were injected with pyrogen-free saline solution. Rats that had triple lesion the neuronal loss was as follows: -89.2% of ChAT-BF, -75.4% of ADA-TMN and -93.3% of DBH-LC). Surprisingly, in these rats three weeks after lesion the daily levels of wake were not changed. However, rats with lesions of two (ChAT+LC) or three (Chat+TMN+LC) neuronal populations had fewer arousals (<40sec) and a more stable sleep architecture (fewer transitions between states) compared to non-lesioned saline rats. These results are contrary to predictions of the “flip-flop” model. From these data and evidence from knockout mice, we hypothesize that the LC, histamine TMN, and BF cholinergic neurons serve to rapidly awaken a sleeping brain, and with it turn on cognitive function, attention, vigilance, and if necessary the “flight-or-fight” response. Hyperactivity of these neurons may underlie the hyperarousal in PTSD.

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

Martin MM, Weathered SL, Wagner SJ, Wallace DG (2008) Galanthamine does not attenuate attentional or temporal impairments subsequent to cholinergic deafferentation of the cortex or hippocampus. Neuroscience 2008 Abstracts 441.12/T4. Society for Neuroscience, Washington, DC.

Summary: The role of the basal forebrain cholinergic system in early symptomology of dementia of the Alzheimer’s type (DAT) remains an area of intense debate. Although involvement of the basalocortical cholinergic system in attentional processing has been established, function of the septohippocampal cholinergic system remains to be determined. A recent study demonstrated a double dissociation between these systems in the organization of rat food protection behavior that may parallel the attentional impairments and temporal disorientation observed during the early stages of DAT. The current study sought to examine whether an acetylcholinesterase inhibitor currently used for the treatment of DAT (i.e., galanthamine) could attenuate these deficits. Consistent with previous research, intraparenchymal injections of 192 IgG-Saporin into the nucleus basalis or medial septum area in female Long Evans rats produced dissociable effects on the organization of food protection behavior. Specifically, nucleus basalis lesions selectively reduced the number of successful food protection behaviors; whereas, medial septum lesions selectively disrupted the temporal organization of food protection behavior. These impairments were not attenuated by the administration of 3 mg/kg s.c. galanthamine twice daily. Results of this study suggest that the modest benefits afforded by galanthamine administration in DAT patients may not reflect improved attention or temporal orientation. Continued studies aimed at understanding the neural dysfunction underlying these deficits may lead to the development of novel therapeutic agents for DAT.

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

Botly LC, De Rosa E (2008) Cholinergic deafferentation of the neocortex with 192 IgG-Saporin impairs feature binding in rats. Neuroscience 2008 Abstracts 418.2. Society for Neuroscience, Washington, DC.

Summary: The binding problem refers to the fundamental challenge of the central nervous system to integrate sensory information registered by distinct brain regions to form a unified neural representation of a stimulus. While the cognitive mechanisms and functional neuroanatomy of feature binding have been well examined by the human cognitive literature, the neurochemistry of feature binding remains unknown. We contend that acetylcholine (ACh) is critical for feature binding given this neuromodulator’s presumed role in modulating attention, and the well-established importance of attention to feature binding. Using systemic pharmacology in rats, we have previously established a critical role for ACh in feature binding at encoding, but have yet to identify the target brain regions cholinergic input must reach for successful feature binding to occur. Given the recognized importance of the frontal and parietal cortices to attentional processing, we hypothesized that cholinergic deafferentation of the neocortex would impair feature binding in a similar manner to that of systemic cholinergic blockade. To test this hypothesis, rats received bilateral 192 IgG-Saporin lesions of the nucleus basalis magnocellularis (NBM) of the basal forebrain. Relative to sham-lesioned rats, NBM-lesioned rats were significantly impaired at acquiring a crossmodal Feature-Conjunction (FC) task, while their ability to retrieve the FC task and to acquire a crossmodal Feature-Singleton (FS) task remained intact. These findings provide further support for our cholinergic attentional hypothesis of feature binding and reveal the importance of neocortical cholinergic input from the basal forebrain to the feature binding encoding process.

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

Sugiyo S, Uehashi D, Masawaki A, Ohyamaguchi A, Abe T, Yonehara N, Takemura M (2008) Intra cisterna magna and Rostral ventromedial medulla injection of anti-Serotonin transporter-Saporinpertussis enhanced somatotopically different c-Fos expression and pain related behaviour in the medullary dorsal horn in rats. Neuroscience 2008 Abstracts 369.11/KK23. Society for Neuroscience, Washington, DC.

Summary: The rostral ventromedial medulla (RVM) is a key center in descending pain modulator, which contain serotonergic neurons having descending projectional terminals in the trigeminal caudal nucleus (Vc; medullary dorsal horn). The functional significance of serotonergic neurons in the RVM is largely unknown. Pretreatment with anti IgG serotonin transporter conjugated with neurotoxin, saporin (anti-SERT-SAP; Advanced Targeting Systems) selectively eliminates cells bearing serotonin transporter, namely serotonergic neurons. 2-4 weeks after injection of anti-SERT-SAP (0.5 µM, 10 nl) into the RVM, the number of serotonin-immunoreactive (IR) cells in the RVM significantly decreased. Formalin injection (1,25% in saline) into the upper lip induced biphasic nociceptive pain-related behavior (PRB). In the rats anti-SERT-SAP-pretreated into the RVM, showed decreased the number of formalin-induced PRB at 1st and 2nd phase compared with the Blank-SAP-pretreated control. 2-4 weeks after intra cisterna magna (CM) pretreatment of anti-SERT-SAP(5 µM, 5 µl), the number of serotonin-IR cells in the RVM also reduced. In stark contrast to the results of pretreatment into the RVM, anti-SERT-SAP-pretreated rats into the CM increased the number of formalin-induced PRB at 1st and 2nd phase. These results indicate that serotonergic neurons in the RVM are constituted by two groups, 1) having pronociceptive function and 2) antinociceptive function projecting to the superficial layers of the Vc.

Related Products: Anti-SERT-SAP (Cat. #IT-23)

Moreira TS, Takakura AC, Stornetta RL, Guyenet PG (2008) Selective lesion of retrotrapezoid Phox2b-expressing neurons attenuates the central chemoreflex in rats. Neuroscience 2008 Abstracts 383.3/RR70. Society for Neuroscience, Washington, DC.

Summary: Injection of the neurotoxin saporin-substance P (SSP-SAP) into the retrotrapezoid nucleus (RTN) attenuates the central chemoreflex in rats. Here we ask whether these deficits are caused by the destruction of a type of pH-sensitive interneuron that expresses the transcription factor Phox2b and is non-catecholaminergic (Phox2b+TH-). We show that RTN contains around 2100 Phox2b+TH- cells. Injections of SSP-SAP into RTN destroyed Phox2b+TH- neurons but spared facial motoneurons, catecholaminergic and serotonergic neurons and the ventral respiratory column caudal to the facial motor nucleus. Two weeks after SSP-SAP, the apneic threshold measured under anesthesia was unchanged when fewer than 57% of the Phox2b+TH- neurons were destroyed. However, destruction of 70 ± 3.5 % of these cells was associated with a large rise of the apneic threshold (from 5.6 to 7.9% end-expiratory pCO2). In anesthetized rats with unilateral lesions of around 70% of the Phox2b+TH- neurons, acute inhibition of the contralateral intact RTN with muscimol instantly eliminated phrenic nerve discharge (PND) but normal PND could usually be elicited by strong peripheral chemoreceptor stimulation (8/12 rats). Muscimol had no effect in rats with an intact contralateral RTN. In conclusion, the destruction of the Phox2b+TH- neurons is a plausible cause of the respiratory deficits caused by injection of SSP-SAP into RTN. At least 70% of these cells must be killed to cause a severe attenuation of the central chemoreflex under anesthesia. The loss of an even greater percentage of these cells would presumably be required to produce significant breathing deficits in the awake state.

Related Products: SSP-SAP (Cat. #IT-11)

Mack SO, Wu M, Xu G (2008) Chemoresponsiveness of the hypothalamic paraventricular nucleus (PVN) is influenced by neuropeptide Y. Neuroscience 2008 Abstracts 383.7/RR74. Society for Neuroscience, Washington, DC.

Summary: The hypothalamic paraventricular nucleus (PVN) initiates autonomic responses to stress and behavioral changes. Neuropeptide Y-containing neurons primarily from the arcuate nucleus and, to a lesser extent, from the brainstem innervate preautonomic oxytocin-containing neurons in the PVN. The role of the PVN in mediating the effects of NPY on energy balance has been studied extensively; however, whether NPY influences respiratory drive via the PVN is not known. Previously, we demonstrated that stimulation of the PVN modulates cardiorespiratory responses via oxytocinergic innervation of neurons in the rostral ventrolateral region of the medulla oblongata where rhythm generating neurons are located. In this study, we selectively lesioned neurons in the PVN bilaterally with neurotoxin neuropeptide Y-saporin (NPY-SAP; 50 ng/100 μl per side) or blank-saporin (control vehicle). Both groups of rats showed similar (P>0.05) increases in body weight gain and intake of food and water over an 8 week period after lesioning. Core body temperature, measured at the same time every day, was also similar for both groups (P>0.05). At 4 weeks post lesioning, NPY-SAP treatment had no effect (P>0.05) on respiratory frequency (fR), tidal volume (VT) and minute ventilation (VE ) in awake, unrestrained animals breathing room air. During exposure to a hypercapnic challenge (5% CO2) for 10 minutes, fR (135 ± 7 vs 114 ±5 breaths min-1) and VE (154 ±13 vs 114± 5 ml min-1 100g-1 ) for the treated animals were significantly elevated (P<0.05) above responses for the control rats. Tidal volume for the treated (1.1 ±0.06) and the control (1.0 ± 0.03) groups was not different (P>0.05). Sensitivity to CO2 with respect to fR in the treated animals reached a peak at 4 weeks and declined thereafter over the next 4 weeks. While there were no apparent changes in morphology or number of parvocellular oxytocin-containing neurons 4 weeks after lesioning, abnormal morphology and a significant (P<0.05) reduction in oxytocin immunoreactive cells were prominent by 8 weeks post treatment. These findings indicate that NPY plays a role in modulating the respiratory response to hypercapnic stress through oxytocin neurons in the PVN. Further studies are needed to determine whether alterations in this pathway may be involved in the onset of hypoventilation associated with obesity.

Related Products: NPY-SAP (Cat. #IT-28)

Holland PC, Angeli N, Lasseter H, Wheeler DS (2008) Unilateral lesions of lateral hypothalamic orexin neurons impair surprise-induced enhancements of learning. Neuroscience 2008 Abstracts 387.16/SS63. Society for Neuroscience, Washington, DC.

Summary: Recent evidence indicates that hypothalamic orexin (hypocretin) neurons are importantly involved in arousal, aspects of learned motivational function, and the cholinergic mediation of sustained attention and the enhanced detection of weak but significant cues. Here we examined the role of these neurons in the modulation of attention in the associative learning of rats. The surprising omission of an expected event can enhance attention to cues present at the time of surprise, and hence facilitate subsequent learning about those cues. In previous research, we showed that circuitry including the amygdala central nucleus (CeA), the substantia nigra pars compacta, cholinergic neurons in the substantia innominata/nucleus basalis, and portions of the medial prefrontal and posterior parietal cortex, form a network essential for this surprise-enhanced learning. In the present study, rats received orexin-saporin lesions of the lateral hypothalamus (LH) in one hemisphere and ibotenic acid lesions of CeA in the other. Because most projections between LH and CeA are ipsilateral, this combination of lesions functionally disconnects CeA from LH orexin neurons. Rats in three control groups received unilateral lesions of LH or CeA (with sham lesions of the other region) or sham lesions of both regions. The rats were then trained in a task in which attention was manipulated by shifting a predictive relation between two cues. First, all rats received serial light-tone pairings, half of which were followed by food. Next, for half of the rats in each lesion condition the tone was omitted on nonreinforced trials, whereas the remaining rats continued to receive the same light-tone trials as before. Finally, attention to the light was assessed by measuring the rate of learning a new light-food relation. If the rats were surprised by the omission of tone during the previous phase, then attention to the light would be enhanced, resulting in faster acquisition of light-food conditioning. Consistent with previous findings, sham-lesioned rats and rats with unilateral CeA lesions showed this surprise-induced enhancement. By contrast, rats with unilateral LH lesions showed no such enhancement of learning, but otherwise performed comparably to controls. Notably, damage to CeA contralateral to the LH damage produced no additional impairment. Thus, LH orexin neurons play an important role in the surprise-induced enhancement of attention and learning, but not solely by their interactions with CeA.

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

Lecourtier L, Leroux E, Cosquer B, Cassel JC (2008) A synergistic role for GABAergic and cholinergic neurons of the medial septum in spatial reference memory processing: assessment with the Morris water-maze and a novel double-H maze in rats. Neuroscience 2008 Abstracts 389.10/TT38. Society for Neuroscience, Washington, DC.

Summary: The medial septum – from which GABAergic and cholinergic neurons project to the hippocampus – might be one of the key structures involved in hippocampal-dependent spatial memory processing. Indeed, lidocaine-induced septal inactivation disrupts encoding and retrieval, but not consolidation of a spatial memory in the water maze. Furthermore, the activation of septal 5-HT1A receptors prevents encoding but neither immediate consolidation nor retrieval of such a memory. As i) 5-HT1A receptors are located on most GABAergic and a significant part of cholinergic neurons of the medial septum, and ii) highly selective cholinergic lesions in the medial septum weakly affect spatial learning, it is possible that normal spatial memory processing depends on a cooperation between cholinergic and GABAergic neurons of this region. To address this possibility using selective lesions, 192 IgG-Saporine (IgG group) or Orexine-Saporine (OREX group) was infused into the septum to damage cholinergic or GABAergic neurons, respectively. In a third group (IgG/OREX group), both lesions were combined. Sham-operated rats were used as controls. The lesion effects were assessed on locomotor activity and on acquisition/retrieval of two water-maze tasks, the Morris maze and the double-H maze. In separate groups, retrieval was tested at three post-acquisition intervals in the Morris maze, namely 1, 5 and 25 days, and two post-acquisition periods in the double H maze, namely 5 and 25 days. Only the combined lesion resulted in nocturnal hyperactivity. In the Morris water-maze, at 25 days, there was no retrieval, whatever the group; whereas at the 1-day delay all groups retrieved the platform, at the 5-day delay, IgG and OREX rats showed normal performance while IgG/OREX rats were impaired. In the double-H maze, at the 5-day delay all groups remembered the platform location. These effects will be compared to those found at the 25-day delay (experiment currently running). Our data show that the GABAergic and cholinergic neurons of the septum synergistically contribute to the regulation of hippocampal-dependent (declarative-like) spatial memory processing.

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

Jeong D, Hwang Y, Chang J (2008) The effect of electrical stimulation in the rats for dementia animal model. Neuroscience 2008 Abstracts 340.11/M2. Society for Neuroscience, Washington, DC.

Summary: Forebrain Cholinergic neurons in the nucleus basalis meynert (NBM) project primarily to the neocortex, and those in the medial septum project to the hippocampus and make a role in memory function. A case study was reported that electrical stimulation of the hypothalamus improves hippocampus dependent memory function. The hypothesis of this study is that electrical stimulation of NBM would induce memory enhancement by effect on neocortex and/or hippocampus. Animal models were induced by selective immunolesion of cholinergic neurons. The cholinergic immunotoxin 192 IgG-saporin was injected in lateral ventricle. 192 IgG-saporin injected rats were compared with Dulbecco’s phosphate bufferd saline (DPBS) injected rats. Neurological deficit and functional outcome were determined by immuohistochemistry using anti-choline acetyl transferase antibody and Morris water maze behavioral test. DBS electrode was implanted in NBM and Stimulation parameters are selected from animal stimulation test. Extent of the cholinergic lesion was showed in the basal forebrain complex region at 192 IgG-saporin injected rats. 192 IgG-saporin injected rats were severely impaired in the probe test of the water maze test. We observe that NBM stimulation induced memory enhancement in dementia models through the behavioral test. Therefore, our animal DBS system could be a useful instrument for investigation of dementia.

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

Burjanadze M, Beselia G, Chkhikvishvili N, Kotaria N (2008) Learning strategy selection in the water maze in medial septal electrolytic and selective cholinergic neurons lesioned rats. Neuroscience 2008 Abstracts 389.5/TT33. Society for Neuroscience, Washington, DC.

Summary: In this experiment the ability of medial septal electrolytic, selective ACh lesioned (by immunotoxin 192 IgG-saporin) and sham-operated rats, to learn the location of a visible, as well as submerged platform in a water maze was investigated. A total of 36 male outbred albino rats were used in the study. All experiments were approved by the Animal Care and Use Committee of the Institute and were in accordance with the principles of laboratory animal care. The rats’ responses in the competition test were classified as either cue or place directed, based on the swim path for those trials. Sham-operated rats acquired both the visible and hidden platform versions of the task, but when required to choose between the spatial location they had learned and the visible platform in a new location, majority of them swam first to the old spatial location. The medial septal electrolytic lesioned rats acquired the visible platform version of the water maze task but failed to learn the platform location in space. When the visible platform was moved to a new location they often swam directly to it. The medial septal selective ACh lesioned rats, as well as sham-operated, acquired the platform location in space. Sham-operated and selective ACh lesioned rats identified as place responders, had significantly more accurate searches during hidden platform training, providing additional evidence of their effective use of a place learning strategy rather than medial septal electrolytic lesioned rats. These findings suggest that the septo-hippocampal system is essential for accurate spatial learning and suggest its role in processing information about the spatial environment, but deficits observed after septal electrolytic lesions cannot be accounted solely to the loss of hippocampal ACh and raised the unexpected possibility that hippocampal ACh is not essential for all types of hippocampal-dependent memory.

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