targeted-toxins

Schwartz MD, Dittrich L, Fisher SP, Lincoln W, Liu H, Miller MA, Warrier DR, Wilk AJ, Morairty SR, Kilduff TS (2012) Effects of a dual hypocretin receptor antagonist on sleep and wakefulness in rats. Neuroscience 2012 Abstracts 799.23. Society for Neuroscience, New Orleans, LA.

Summary: Benzodiazepine receptor agonists promote sleep by activating GABAA receptors, leading to generalized reduction in cortical activity. They are widely used as hypnotic medications, but have side effects including risk for tolerance and/or dependence, as well as cognitive impairment while under their influence. The excitatory hypocretin (HCRT) neuropeptides promote wakefulness by activating multiple subcortical wake-promoting neurotransmitter systems which, in turn, project to and regulate cortical activity. Blocking HCRT signaling should therefore promote sleep by acting specifically on subcortical brain areas regulating sleep and wake without adversely impacting cortical function. Here, we assessed the ability of the dual HCRT receptor antagonist almorexant (ALM) to promote sleep in rats following ablation of a major sleep-wake regulatory region, the cholinergic basal forebrain (BF). We predicted that ALM would be less effective at inducing sleep in BF-lesioned rats compared to neurologically-intact rats, whereas benzodiazepine-based compounds should be equally as effective in lesioned and intact rats. Male rats received bilateral stereotaxic injections of saline or the selective cholinergic neurotoxin192-IgG-saporin (SAP) directed at the BF and were implanted with telemetry for recording sleep EEG. Following recovery, animals were given increasing doses of ALM, the GABA-A receptor agonist zolpidem (ZOL), or vehicle. Spontaneous sleep/wake regulation and homeostatic recovery from sleep deprivation was also assessed. At baseline, NREM sleep in the dark (active) phase was reduced in SAP rats compared to intact rats; SAP rats also exhibited decreased NREM recovery sleep following 6 h sleep deprivation in the dark phase. Sleep in the light (rest) phase was unaffected by SAP. Analysis of ALM and ZOL administration in these animals is currently in progress.

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

Gulisano M, Parenti R, Gulino R (2012) Neural plasticity in injured spinal cord. Neuroscience 2012 Abstracts 846.09. Society for Neuroscience, New Orleans, LA.

Summary: Sonic hedgehog and Noggin are morphogenetic factors involved in neural induction and ventralization of the neural tube, but recent findings suggest that they could participate in regeneration and functional recovery after injury. Here, in order to verify if these mechanisms could occur in the spinal cord and involve synaptic plasticity, we measured the expression levels of Sonic hedgehog, Noggin, Choline acetyltransferase, Synapsin-I, and Glutamate receptor subunits (GluR1, GluR2, GluR4), in a motoneuron-depleted mouse spinal lesion model obtained by intramuscular injection of Cholera toxin-B saporin. The lesion caused differential expression changes of the analyzed proteins. Moreover, motor performance was found correlated with Sonic hedgehog and Noggin expression in lesioned animals. The results also suggest that Sonic hedgehog could collaborate in modulating synaptic plasticity. Together, these findings confirm that the injured mammalian spinal cord has intrinsic potential for repair and that some proteins classically involved in development, such as Sonic hedgehog and Noggin could have important roles in regeneration and functional restoration, by mechanisms including synaptic plasticity.

Related Products: CTB-SAP (Cat. #IT-14)

Jeong D, Lee J, Lee S, Kim S, Chang J (2012) Spatial memory facilitation by electrical stimulation of the medial septum in rats. Neuroscience 2012 Abstracts 851.01. Society for Neuroscience, New Orleans, LA.

Summary: Recently, deep brain stimulation has been used to treat various neurological disorders. Some studies support that DBS can be a strategy to treat Alzheimer’s disease. The aim of this study was to evaluate the effect of electrical stimulation in the medial septum using rat model mimicking basal forebrain cholinergic deficits of Alzheimer’s disease. Four experimental groups were composed of normal, lesion, lesion + implantation and lesion + stimulation. 192 IgG-saporin (Selective cholinergic toxin, 8ul of 0.63ug/ul) were bilaterally injected into the lateral ventricle. Electrode was stereotactically implanted into the left medial septum (AP +0.6, ML 0.16, DV -6). Stimulation parameters are 50Hz, 120us pulse width and 1 volt. One week after implantation, Stimulation started for 2 weeks. Two weeks after surgery, water maze was performed for 1 week and rats were sacrificed immediately after behavioral test. Features were verified by immunochemistry and AChE assay. During the training trials, latencies of lesion and implantation significantly increased in day3 and day4. In contrast, latency of stimulation group had no differences as compared to normal group but it decreased significantly when compared to lesion group in day4. In the probe test, lesion group had decreases in time in target quadrant, time in platform zone and the number of platform crossing. Although they did not perform as normal group, stimulation group showed tendency of recovery. IHC and AChE assay are ongoing. Spatial memory is associated with hippocampus. We had expected activation of hippocampus by stimulation of the medial septum. We confirmed that stimulation of the medial septum facilitates acquisition and recall of spatial memory. Currently we are studying the effects of medial septal stimulation on the hippocampus.

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

Rossi CA, Lehmkuhle MJ, Dudek FE (2012) Evidence that focal hippocampal interneuron loss disrupts theta- and gamma- band activity. Neuroscience 2012 Abstracts 918.06. Society for Neuroscience, New Orleans, LA.

Summary: Hippocampal theta (6-12 Hz) and gamma (40-100 Hz) activity are oscillatory local field potentials (LFPs) that are thought to play a critical role in the encoding and storage of new information. GABA-ergic interneurons are hypothetically involved in the generation and pacing of these oscillatory patterns of activity. The current study aimed to directly test the hypothesis that interneurons are responsible for local gamma and theta generation in the dorsal CA1 region of the hippocampus in mice. Selective focal interneuron lesions were made by intrahippocampal injection of the targeted neurotoxin SSP-Saporin into dorsal CA1 in the hippocampus of GAD67-GFP transgenic mice. Chronic recording electrodes were also implanted in the lesion area. LFPs were monitored continuously, along with video recordings of the subjects, for a period of several weeks. LFP recordings were analyzed over 24-hour periods for the occurrence of theta- and gamma-band activity. Analysis of LFP data revealed attenuation of both local theta and gamma activity in SSP-saporin-injected animals compared to controls. These results suggest a direct role of GABA-ergic interneurons in the generation of local rhythmic activity in these two frequency bands, and, by extension, an important role in learning and memory processes.

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

Ljubojevic V, Botly L, De Rosa E (2012) Cholinergic contributions to learned attentional suppression in the rat with touchscreens. Neuroscience 2012 Abstracts 729.13. Society for Neuroscience, New Orleans, LA.

Summary: One of the tasks of the attentional system is to filter environmental input according to its behavioral relevance. The neuromodulator acetylcholine (ACh) is thought to play a role in this process because of its ability to boost the signal-to-noise ratio of incoming sensory information. Cholinergic innervation of the attentional system has been shown to be necessary for successful selection of behaviorally-relevant stimuli (signal). However, it is not yet clear if ACh also plays a part in the attentional suppression of behaviorally-irrelevant information (noise). Thus, we examined the effect of cortical cholinergic deafferentation on attentional suppression in rats. To measure attentional suppression, we used a rat analog of the learning-to-ignore (LI) task originally designed for human participants (Dixon et al., 2009). The paradigm consisted of three stages of training (Prime1, Prime2, Probe; 10 sessions per stage), each of which involved stages of visual simultaneous discriminations between two stimuli. In both Prime conditions, individuals learned to respond to target stimuli (A+ and then C+ respectively), while ignoring the same distractor stimulus (B-). During Probe, the previously ignored stimulus became the target (B+) and a novel stimulus (D-) was introduced as a distractor. Eighteen male Long-Evans rats were trained to perform the touchscreen-based LI task. Like the human data, a behavioral decrement (lower accuracy) was observed during the Probe phase of the LI task when compared to Prime 1 and 2, which suggests that the ignored distractor stimulus was suppressed during Prime. We hypothesized that administration of the ACh-specific immunotoxin, 192 IgG-saporin, into the nucleus basalis magnocellularis (NBM) would lead to better performance during Probe condition relative to controls. Accordingly, the rats were subjected to either cholinergic immunotoxic (SAP, N=10) or sham lesion surgery (SHAM, N=8). After 2 weeks of post-surgical recovery, the rats were tested on the LI task with a new stimulus set. The two groups performed comparably during Prime1 and 2, with both SAP and SHAM rats successfully learning the discriminations. As predicted, during Probe SAP rats exhibited significantly less behavioral decrement than controls. Histological analysis revealed that the lesion was chemically and anatomically specific to cholinergic cells in the NBM. This counterintuitive finding suggests that the improved performance during Probe, due to reduced ACh input to the neocortex, was due to inefficient attentional suppression of the behaviorally-irrelevant stimulus.

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

Ortiz-Barajas O, Ramos-Rodriguez J, Berrocoso E, Garcia Alloza M (2012) Limited effect of serotonergic denervation on beta-amyloid and cognitive impairment in APPswe/PS1dE9 mice. Neuroscience 2012 Abstracts 751.12. Society for Neuroscience, New Orleans, LA.

Summary: Alzheimer’s Disease (AD) is a neurodegenerative disease characterized by progressive cognitive and memory impairment. Amyloid-beta (Aβ) deposition, as senile plaques (SP), seems to play a key role in the development and progression of the illness. Moreover SP tend to accumulate in cortex and hippocampus, relevant areas in learning and memory. On the other hand neuronal loss is the pathological feature that best correlates with duration and severity of the illness and at present animal available animal models hardly reproduce the complexity of the disease. We have previously seen that selective cortical and hipocampal cholinergic denervation, using murine p-75 saporin, may worsen cognitive abilities in APPswe/PS1dE9 mice as well as increase SP deposition in denervated areas. In the present work we lesioned 7 months old APPswe/PS1dE9 mice with 1 µl of 5,7-dyhidroxytiptamine (0.16 µg/µl) injected in the raphe nucleus (RN). In order to guarantee selective removal of cortical and hipocampal serotonergic inervation, and protect noardernergic and dopaminergic neurons, animals were i.p. injected with desipramine and nomifensine before surgery. We observed a clear reduction of tryptophan hydroxilase staining in the RN. In the Morris water maze test we observed learning and memory impairment in APPswe/Ps1dE9 mice, without a synergistic effect of the serotonergic lesion. When we assessed SP deposition we did not observe a significant increase of SP in cortex or hipocampus 14 days after the lesion, as we observed after selective cholinergic denervation. Altogether our data suggest that cognitive impairment and induced SP depositioin observed after cholinergic denervation is not achieved when serotonergic system is affected, supporting a selective effect mediated by different neurotransmitter systems. Acknowledgements: MG-A: RYC-2008-02333, ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (PS09/00969), Fundación Dr. Eugenio Rodriguez Pascual, Junta Andalucia Excelencia (CTS-7847).

Related Products: mu p75-SAP (Cat. #IT-16)

Talman WT, Jones S, Nitschke Dragon D, Lin L-H (2012) Selective damage to glia in the nucleus tractus solitarii attenuates cardiovascular reflexes. Neuroscience 2012 Abstracts 524.05. Society for Neuroscience, New Orleans, LA.

Summary: Lesions of the nucleus tractus solitarii (NTS) are known to attenuate or abolish cardiovascular reflex responses. We have previously reported that lesions produced by saporin (SAP) conjugates and focused on neurons that express the neurokinin-1 (NK1) receptor or on other neurons that express both tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH), also attenuate baroreflex function in rats. We found that lesions of both types of neurons also led to loss of glia that stained with glial fibrillary acidic protein (GFAP). Further, we found that injection of SAP alone into the NTS led to loss of GFAP staining while leaving neurons in the region unaffected. Because both of the lesions directed at neurons were made by a toxic conjugate containing SAP, we sought to determine if SAP alone produced changes in cardiovascular reflex function. We found that injection of SAP (3 ng in 100 nl) into the NTS led to loss of the glial marker GFAP as well as connexin 43 (Cx43) immunofluorescent labeling in the NTS but did not affect the neuronal markers NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), neuronal nitric oxide synthase (nNOS), TH, DBH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), NK1, and protein gene product 9.5 (PGP 9.5). In animals treated with bilateral injections of SAP into the NTS, reflex responses were decreased during testing of the baroreflex, the chemoreflex, or the von Bezold Jarisch reflex. Comparable decreases in baroreflex responses were seen in animals treated with SAP alone when compared with other animals treated with SAP conjugates that targeted and concentrated damage to TH/DBH neurons or NK1 neurons in NTS. In contrast, when TH/DBH neurons were targeted by the toxin 6-hydroxydopamine (6-OHDA) lability of arterial pressure did not occur as it did in the other SAP and SAP conjugate studies and reflex responses to the activation of the baroreflex, the chemoreflex, and the von Bezold Jarisch reflex did not differ from control. Furthermore, injections containing SAP or a SAP conjugate, but not those containing 6-OHDA, led to lability of arterial pressure as well as cardiac arrhythmias and cardiac myocytolysis. Our studies cannot exclude a physiological effect of SAP on neurons nor can it exclude an indirect effect of glial damage on NTS neurons. However, the similarity of responses when glia seem to have been targeted alone in contrast to those responses when select neuronal types seem to have been targeted suggests that each of the cardiovascular reflexes relies on intact glia in the NTS for full reflex expression.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11)

Mallory CS, Paolone G, Koshy Cherian A, Blakely RD, Sarter M (2012) Paying attention with a compromised cholinergic system: Attenuated activation of cholinergic neurotransmission in attentional task-performing CHT+/- mice. Neuroscience 2012 Abstracts 536.08. Society for Neuroscience, New Orleans, LA.

Summary: Prefrontal cholinergic neurotransmission is necessary for sustained attentional performance. In rats, prefrontal acetylcholine (ACh) release reaches 140% over baseline during the performance of a sustained attention task (SAT; St. Peters et al., 2011a). SAT performance also increases the density of choline transporters (CHT) in synaptic plasma membranes (Apparsundaram et al., 2005), which we hypothesize is needed to sustain elevations of cholinergic activity and behavioral responses. Here we employed the SAT recently adapted for use in mice (St. Peters et al., 2011b) and developed new techniques that permit monitoring of ACh release via microdialysis of mice performing the SAT in order to determine the impact of genetically manipulated levels of choline transporter capacity. First, reverse dialysis of atropine (50 µM) increased ACh release levels in naive WT mice. In contrast, CHT+/- mice could not sustain these increases, consistent with changes observed in levels of muscarinic receptors in the CHT +/- mice (Bazalakova et al., 2007). However, SAT performance did not differ significantly between WT controls and CHT+/- mice. Furthermore, basal (absolute) levels of ACh release were comparable between strains. However, performance-associated increases in ACh release were strikingly attenuated in CHT+/- mice, reaching 40% over basal levels versus 130% in WT. Performance-associated increases in ACh release in CHT+/- mice were TTX-sensitive, similar to release monitored in WT mice (1 µM via reversed dialysis). To determine whether cholinergic activity was necessary for SAT performance in CHT+/- mice we then removed basal forebrain cholinergic neurons by infusing murine-p75NTR-saporin obtaining similar impairment on SAT performance in both strains. Finally, and because cholinergic activity modulates cortical circuitry primarily via nAChR, mecamylamine (MEC; 50 µM) was reverse dialyzed during SAT performance. WT mice were only moderately impaired in the SAT task, whereas the performance of CHT+/- mice rapidly declined and the performance-associated ACh levels rapidly returned to the pre-task levels. In summary CHT+/- mice are able to perform the basic SAT, despite attenuated levels of cholinergic neurotransmission, likely as a result of compensatory postsynaptic mechanisms. However, their attentional performance and underlying cholinergic signaling exhibit heightened sensitivity to behavioral and pharmacological challenges. Together, these findings suggest that CHT+/- mice are an important model for the impaired cognitive control of attentional performance that is a common symptom of ADHD, schizophrenia and other cognitive disorders.

Related Products: mu p75-SAP (Cat. #IT-16)

Krajewski-Hall SJ, Mittelman-Smith MA, Williams H, Lafrance KJ, Mcmullen NT, Rance NE (2012) A role for kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the regulation of estrous cycles and the estrogen modulation of body temperature. Neuroscience 2012 Abstracts 585.02. Society for Neuroscience, New Orleans, LA.

Summary: We have recently described a method to selectively ablate kisspeptin/neurokinin B/dynorphin (KNDy) neurons using stereotaxic injections of NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (Mittelman-Smith, Endocrinology, 2012). These studies revealed a critical role for arcuate KNDy neurons in tonic gonadotropin secretion, the rise in serum LH after ovariectomy and estrogen modulation of body weight. Here we determine the effects of KNDy neuron ablation on estrous cycles and the estradiol modulation of body temperature. In the first study, stereotaxic injections of NK3-SAP or Blank-SAP were made in the arcuate nucleus of ovary-intact, adult female rats. Rats with nearly complete KNDy-neuron ablation (verified by NKB immunohistochemistry) exhibited constant diestrus and ovarian atrophy, confirming the importance of these neurons in reproductive regulation. In a second experiment, we evaluated the effects of KNDy neuron ablation on the thermoregulatory axis in rats that were ovariectomized (OVX) and then treated with 17β-estradiol (E2). Tail skin temperatures (TSKIN) and core temperatures (TCORE) were recorded in rats throughout the light/dark cycle and during exposure to different ambient temperatures (TAMBIENT) in an environmental chamber. Notably, the average TSKIN of KNDy-ablated rats was consistently lower than control rats, indicative of lower levels of cutaneous vasodilatation. Moreover, KNDy neuron ablation blocked the reduction of TSKIN by E2 that occurred during the light phase in the environmental chamber, but did not affect the E2 suppression of TSKIN during the dark phase. At a high TAMBIENT of 33 C, the mean TCORE of OVX control rats increased to 39.0 C, and was reduced by E2 replacement. In contrast, at this high TAMBIENT, the average TCORE of OVX, KNDy-ablated rats was lower than OVX control rats, and TCORE was not altered by E2 replacement. Because KNDy neurons exhibit dramatic changes in morphology and gene expression in postmenopausal women, we have hypothesized these neurons contribute to the generation of hot flushes. These studies support this hypothesis by providing the first evidence that KNDy neurons participate in the E2 modulation of body temperature and promote cutaneous vasodilatation, one of the cardinal signs of a hot flush.

Related Products: NKB-SAP (Cat. #IT-63), Blank-SAP (Cat. #IT-21)

Anderson ML, Govindaraju KP, Shors TJ (2012) Acetylcholine and Learning: Are they related and does it matter for associating events across time?. Neuroscience 2012 Abstracts 600.12. Society for Neuroscience, New Orleans, LA.

Summary: Decades ago, acetylcholine was considered intrinsic to processes related to attention and/or learning and memory. Much of this was based on its presumed role in dementia associated with Alzheimer’s disease. However, in the last decade or so, this relationship has been questioned and with good reason (Parent & Baxter, 2004). That said, only a few studies have addressed the involvement of acetylcholine in tasks that require an animal to associate stimuli separated in time, such as trace eyeblink conditioning. This type of task is dependent on the hippocampus and is severely disrupted in both patients with Alzheimer’s disease and animal models of the disorder (Kishimoto, 2012; Waddell et al., 2008; Woodruff-Pak & Papka, 1996). In the present study, we hypothesized that animals with minimal Ach input to both hippocampi would not learn whereas those with input into one hippocampus could. The immunotoxin 192 IgG-Saporin was infused into the MSDB to selectively kill cholinergic neurons in Sprague-Dawley rats and then trained with either delay or trace eyeblink conditioning. Delay conditioning requires that the stimuli during training are contiguous in time and is not dependent on the hippocampus. Animals were given 200 trials for four days for 800 trials in total. A complete bilateral MSDB-cholinergic lesion was considered complete if the number of neurons that express choline acetyltransferase was reduced by 75 %. A bilateral lesion of this magnitude prevented early acquisition of the trace response (p<.05). Indeed, none of the animals so far trained reached a learning criterion of 60 % CRs during any session of training. In contrast, animals with a loss of ACh in just one hemisphere were able to learn the CR. Furthermore, preliminary data suggest delay conditioning was unaffected by the loss of ACh from the septum. Finally, animals with half the number of cholinergic neurons were still able to learn trace eyeblink conditioning regardless of whether the damage was bilateral or unilateral. Thus, it would appear that the progressive loss of ACh coincides with the loss of learning potential, especially when that learning requires associations across time. This approach and the experimental results may model the progressive nature of Alzheimer’s disease, in which the loss of neuronal function is slow but cumulative.

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