targeted-toxins

Saenz CM, Borowski T, De Lacalle S (2008) Contrasting effects of estrogen on memory tasks in young female rats. Neuroscience 2008 Abstracts 794.17/UU7. Society for Neuroscience, Washington, DC.

Summary: Sleep deprivation may lead to behavioral alterations and it has been associated with a hyperalgesic state in human beings and animal models. The tricyclic antidepressant amitriptyline can be used as an analgesic drug in patients and in chronic pain animal models that are not improved with classical analgesics, such as spinal nerve injury induced model of peripheral neuropathy. The pain hypersensitivity following both paradoxical sleep deprivation (PSD) and peripheral nerve injury shares common spinal mechanisms, which involve at least the glutamate receptors and nitric oxide. In this way, we evaluated the effects of amitriptyline pretreatment in the thermal hyperalgesia observed in paradoxical sleep deprived rats. Amitriptyline (10 and 30 mg/Kg) or saline were administered i.p. during 11 days to male Wistar rats (n = 7/group, 250 – 350 g). In the last 3 or 4 days of treatment the animals were submitted to 72 or 96 hours of PSD, respectively, or remained in home cages, being subsequently evaluated for their thermal sensitivity on a hot plate test (52oC or 46oC), 1 or 24 hours after the last drug administration. In order to verify if the results observed in the highest withdrawal latencies were due to a reduction on the locomotor activity rather than an analgesic effect, the number of squares crossed in an open field arena during 5 minutes, subsequently to the hot plate test was counted. The results demonstrated that paw withdrawal latency response to 52oC was significantly lower in paradoxical sleep deprived rats than controls (-37%, p<0.05). This hyperalgesic effect was also detected in animals pre-treated with 10 mg/kg (-41%, p<0.05) or 30 mg/Kg (-53%, p<0.05) of amitriptyline. At the highest dose, both groups presented a higher withdrawal threshold when compared to their respective saline groups (+185%, p<0.05 and +112%, p<0.05; control and sleep deprived rats, respectively). However, in the open field test a decrease in the number of squares crossed in control animals was observed (-52%, p<0.05), but not in sleep deprived rats (-3%, p>0.05). When the animals were allowed to recover for 24h from sleep deprivation, the pre-treatment with amitriptyline (10 mg/Kg) was not able to prevent the hyperalgesic state (-60%, p<0.05). Even with lower thermal stimulus (46oC) and sleep deprivation period (72h), the difference between control and sleep deprived animals could still be detected (-40%, p<0.05), with no changes after an amitriptyline 10 mg/Kg pre-treatment (-43%, p<0.05). Overall, these findings highlight that thermal pain hypersensitivity induced by PSD was not prevented by amitriptyline pre-treatment, as observed in other models of inductive pain.

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

Savage ST, Oberg J, Pernold K, Mattsson A (2008) Enhanced sensitivity to phencyclidine following cortical cholinergic denervation. Neuroscience 2008 Abstracts 842.7/X2. Society for Neuroscience, Washington, DC.

Summary: Alterations in cholinergic signaling in the brain have been implicated as a contributing factor in the pathogenesis of schizophrenia. We have recently shown that cholinergic denervation of cortex cerebri by stereotaxic infusion of the immunotoxin 192 IgG-saporin in the nucleus basalis magnocellularis in adult rats, leads to an enhanced sensitivity to amphetamine. Thus, saporin lesioned rats show a marked increase in locomotor activity, as well as a potentiated dopamine release in nucleus accumbens when challenged with amphetamine. We hypothesize that the loss of cortical cholinergic input alters the activity of cortical glutamatergic neurons and in turn, their regulation of subcortical dopamine neurons. We have previously shown that this cortical cholinergic denervation leads to an increased locomotor response to the NMDA receptor antagonist phencyclidine (PCP), suggesting that disruption of cortical cholinergic activity can lead to disturbances of glutamatergic transmission. In current studies we are investigating attention and memory functions of rats with cholinergic denervation of neocortex using the novel object recognition task. Preliminary data from these investigations shows impairment in performance under PCP-challenge in saporin lesioned rats as compared to sham lesioned controls. These results indicate that cortical cholinergic deficits, in addition to leading to a dramatic potentiation of the locomotor response to PCP, can also lead to an enhanced sensitivity to PCP-induced cognitive impairments. Using pharmacological magnetic resonance imaging (MRI) we are investigating possible spatiotemporal differences in brain activation in rats with cortical cholinergic deficits following administration of PCP. Preliminary data have provided indications of a greater activation in cortical areas in saporin lesioned rats as compared to sham lesioned controls following PCP-challenge. Evaluations of possible alterations in social behavior following cortical cholinergic denervation are ongoing. Social interaction will be investigated under normal conditions, as well as after PCP-challenge. Preliminary results from these studies together with our previous results indicate that loss of cortical acetylcholine can lead to alterations in glutamatergic signaling. These observations are compatible with a possible role of cholinergic deficits in schizophrenia, and provide a possible link between different hypotheses of the disorder.

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

Coons KD, Sengelaub DR (2008) Protection from dendritic atrophy with testosterone following partial motoneuron depletion: Timing and duration of treatment, functional correlates in motor activation. Neuroscience 2008 Abstracts 556.23/CC10. Society for Neuroscience, Washington, DC.

Summary: We have previously demonstrated that partial depletion of motoneurons innervating the quadriceps muscles induces dendritic atrophy in remaining motoneurons; this atrophy can be attenuated in a dose-dependent fashion, and in both male and female rats, with testosterone (T) treatment. In the present study, we examined (1) how the timing and duration of T treatment affect its ability to attenuate induced atrophy in remaining quadriceps motoneurons, and (2) the effects of induced atrophy and T treatment on subsequent motor function in male rats. Motoneurons innervating the vastus medialis muscles were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Rats were then treated with supplemental T at different times post-saporin injection (immediately, or at 2 or 3 weeks), or for different durations (1, 2, 3, or 4 weeks) or left untreated. All T treatments consisted of subcutaneous implants designed to produce plasma titers in the normal physiological range. Following treatment, the morphology of motoneurons innervating the ipsilateral vastus lateralis muscles was examined using retrograde labeling with cholera toxin-conjugated HRP. In a separate set of rats, quadriceps motoneuron activation was assessed using peripheral nerve recording. Motoneuron morphology and motor activation were also assessed in a group of untreated normal males. Partial motoneuron depletion resulted in dendritic atrophy in remaining quadriceps motoneurons. Treatment with T attenuated this atrophy, but in a time-sensitive manner. Four weeks of T treatment (delivered immediately post-saporin), or two weeks of T treatment (after a delay of two weeks post-saporin) were both effective in attenuating induced dendritic atrophy. However, dendritic atrophy in animals with immediate T treatment of shorter durations or longer delays in the start of treatment was comparable to that of animals who received no supplemental T. Consistent with the morphological changes, partial motoneuron depletion in otherwise untreated males resulted in deficits in motor activation: activation of quadriceps motoneurons required greater stimulus intensities and resulted in decreased amplitudes of motor nerve activity. Importantly, just as observed for dendritic morphology, these changes were attenuated by treatment with supplemental T. These results demonstrate that the neuroprotective effect of T on motoneuron morphology is more dependent on the timing of treatment than on its duration, and also provide a functional correlate of the morphological effects of that treatment, further supporting a role for T as a neurotherapeutic agent in the injured nervous system.

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

Ramalingam V, Fuller PM, Lu J, Saper CB (2008) Changes in energy metabolism after ventrolateral preoptic lesions in rats. Neuroscience 2008 Abstracts 586.14/SS47. Society for Neuroscience, Washington, DC.

Summary: The ventrolateral preoptic area (VLPO) is critically involved in the regulation of sleep. For example, lesions of the VLPO have been reported to cause profound insomnia and sleep fragmentation in rats. We evaluated possible changes in energy metabolism and motor behaviors secondary to chronic sleep restriction in VLPO lesioned rats. Under anesthesia (chloralhydrate, 350 mg/kg, i.p.), adult male Sprague Dawley rats (n = 17) received stereotaxic injections of orexin-saporin into the VLPO and were also implanted with EEG/EMG electrodes to assess sleep-wakefulness. Food, water, and body mass measurements were collected for 60 post-lesion days. Sleep-wakefulness was recorded on post-lesion Days 20 and 50. On post-lesion Day 60, the animals were deeply anesthetized and transcardially perfused with 10% formaldehyde. The brains were removed and processed for histological verification of the lesion site. VLPO lesions produced a decrease (34%) in non rapid eye movement sleep (NREM) and a decrease in NREM sleep bout duration (115 ± 5 sec in the VLPO lesioned rats Vs 133 ± 2 in control rats, P < 0.01). The VLPO lesioned animals also exhibited increased food intake when compared to the age-matched controls (0.45 ± 0.004 grams per gram of lean body mass Vs 0.39 ± 0.01 grams per gram of lean body mass, P = 0.05). Food intake (r = 0.90, P<0.001), but not water intake was positively correlated with the amount of sleep loss. Although the weight gain in the VLPO lesioned rats was not statistically different from the controls, it was negatively correlated with the amount of sleep loss in those animals (r = 0.51, P = 0.05). Although the VLPO lesioned animals balanced on the rotatrod for 25% less time than the controls, this did not reach statistical significance, perhaps because the variance was so high in both groups (87 ± 23 seconds Vs 116 ± 25 sec in control rats, P>0.05). The close correlation of sleep loss with changes in food intake and body weight after the VLPO lesions suggests that the increased feeding but lower body weight may be due to the sleep loss, rather than a consequence of damaging neurons adjacent to the VLPO, which would not correlate with sleep loss.

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Thankachan S, Kaur S, Blanco-Centurion C, Sakurai T, Yanagisawa M, Shiromani PJ (2008) Ventrolateral periaquaductal gray (vlPAG): Key area for REM sleep propensity. Neuroscience 2008 Abstracts 586.3/SS36. Society for Neuroscience, Washington, DC.

Summary: In an effort to determine how loss of hypocretin/orexin (HOX) increases REM sleep we have used the neurotoxin, hypocretin-2-saporin (HCRT2-SAP), to lesion HOX receptor bearing neurons. Our efforts have focused on the pons (Blanco-Centurion et al., EJN 19:2741, 2004) since REM sleep is generated from there. Here, we investigate the vlPAG, a region where muscimol robustly increases REM sleep in cats (Sastre et al., Neuroscience, 74:415, 1996), and where HOX might activate GABA neurons that inhibit REM sleep. Lesion of vlPAG neurons with HCRT2-SAP should increase REM sleep. HCRT2-SAP (16.5ng/23nl) or saline (23nl; 0.9%) was injected (glass pipette; isofluorane anesthesia) to the vlPAG area in hypocretin/orexin null mice (HOX null) and in GAD-GFP mice [TgN(GadGFP)45704Swn; to visualize the GABA neurons]. Sleep was recorded 15th and 16th days after the lesion (12:12LD cycle). vlPAG lesion (n=5) significantly (+48.19%) increased REM sleep at night in HOX null mice compared to saline treated HOX null mice (n=7); REM sleep during the day was not changed. Over the 24h period REM sleep was significantly increased (+18.78%). However, cataplexy did not increase. In the GAD-GFP mice vlPAG lesions (n=8) also significantly increased REM sleep at night (+79.4%) compared to saline controls (n=8). The vlPAG lesions caused a significant increase in the number of short bouts (<40sec) of wake, NREM and REM sleep during both day and night. HOX null mice already have highly fragmented sleep, and increased REM sleep at night. Since vlPAG lesions produced a greater sleep fragmentation and increased REM sleep even further suggests that the vlPAG represents a key area, downstream of HOX neurons, in gating REM sleep propensity.

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

Lai YY, Hsieh KC, Nguyen D, Siegel JM (2008) The substantia nigra and the control of sleep. Neuroscience 2008 Abstracts 586.9/SS42. Society for Neuroscience, Washington, DC.

Summary: It has been established that the substantia nigra (SN) is involved in the control of motor activity. However, its role in the regulation of sleep remains unclear. We have previously found that NMDA lesions in the SN suppress sleep in the cat. A recent study demonstrated that lesions of the SN by hypocretin2-saporin result in severe insomnia in the rat. Thus, we hypothesized that activation of the SN by application of either excitatory transmitter analogs/agonists or GABA receptor blockers would induce sleep. Hypocretin had been reported to exert an excitatory effect on SN neuronal activity. The SN receives dense projections from hypocretin neurons. In the current study, we investigated whether microinfusion of hypocretin into the SN would modulate sleep and wakefulness in freely moving rats. Adult male Sprague-Dawley rats were implanted with EEG and EMG electrodes, and a guide cannula targeting the SN. Experiments were conducted one week after the rat had been implanted. The rats were housed individually in sound-attenuated chambers in LD 12:12. Hypocretin-1 was delivered via microdialysis probes (CMA/11) at a rate of 2 μL/min. Each one-hour of hypocretin infusion (ZT4 to ZT5 in the light period) was preceded by a 2-hour baseline period of artificial cerebrospinal fluid (aCSF) infusion and was followed by a 2-hour aCSF infusion. The lower concentration of hypocretin-1 (36 μM, n=2) reduced wakefulness by 19% ± 9.5% and increased slow wave sleep (SWS) by 12.8% ± 2.3% of the baseline level. The higher concentration of hypocretin-1 (72 μM, n=3) reduced wakefulness by 30.5% ± 16.4% of the baseline level and produced an increase in both SWS and REM sleep, by 10.2% ± 2.2% and 63.7% ± 26.6% respectively. The increased sleep induced by both concentrations of hypocretin were also observed in the first post-infusion hour. In conclusion, we found that hypocretin-1 has a sleep-promoting effect in the SN. Our previous study showed that hypocretin (orexin) neurodegeneration occurred in Parkinson’s disease patients. This finding suggests that sleep difficulties in Parkinson’s disease patients may result from a combination of lesions in the SN and the secondary effects of the loss of hypocretin neurons.

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

Carter ES, Newman LA, Galler JR, Mcgaughy J (2008) Effects of cholinergic deafferentation of medial, prefrontal cortex on a cross-modal divided attention task. Neuroscience 2008 Abstracts 590.6/TT58. Society for Neuroscience, Washington, DC.

Summary: Previous research has shown that cholinergic lesions of the nucleus basalis magnocellularis impair cross-modal divided attention (Turchi and Sarter 1997). Cholinergically lesioned rats showed increased response latencies relative to sham-lesioned rats if required to divide attention but did not differ from sham-lesioned rats when tested in a focused attention condition. In the present study, the effects of selective cholinergic depletion of the medial, prefrontal cortex were assessed in the same cross-modal divided attention task (CMDAT). Male, Long-Evans rats were trained on one set of conditional response rules for visual stimuli and another for auditory stimuli. In the CMDAT, rats received 20 trials of either auditory or visual stimuli followed by twenty trials of the alternate modality (focused attention). Within the same session rats received 60 trials of a randomized sequence of all possible stimuli (divided attention). These trials were followed by two additional blocks of focused attention (20 trials/block) so the trial block sequence was Focused Attention 1 (FA1): Divided Attention (DA) : Focused attention 2 (FA2). Subjects received infusions of 192 IgG saporin (pACh-LX) or its vehicle (SHAM-LX) to the prelimbic cortex. Response latencies for all subjects were longer under conditions of divided attention when performance was compared to the first block of focused attention trials. However in pACh-LX rats response latencies in the second block of focused attention trials were longer than in the divided attention trials. The slowed performance in the FA2 block may suggest cognitive fatigue after performing the divided attention trials or may be due to the effects of prolonged time on task. To address this question, a novel sequence of trials DA : FA1 : FA2 was tested. This session showed no difference in response latencies across blocks in either group. The lack of increased response latencies in FA2 suggests the effect in the standard task is related to cognitive fatigue after completion of the DA block and prolonged time on task. The accuracy of pACh-LX rats was decreased in the divided attention block relative to SHAM-LX rats in this block and relative to pACh-LX rats’ own performance in the DA block of the standard task. This loss of accuracy with decreased latency suggests that lesioned rats show a cost of divided attention if no focused attention blocks precede divided attention testing. These data support the hypothesis that cholinergic afferents to the prefrontal cortex mediate divided attention and a loss of these afferents exacerbates cognitive fatigue.

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

Butt AE, Kinney-Hurd BL, Flesher MM, Amodeo DA, Horn LR, Greenfield V, Lladones R, Hernandez G, Loson L (2008) Selective cholinergic lesions of the nucleus basalis magnocellularis disrupt attention in appetitive trace conditioning. Neuroscience 2008 Abstracts 686.14/SS65. Society for Neuroscience, Washington, DC.

Summary: Pavlovian trace conditioning, but not delay conditioning, is a form of declarative memory that requires attention and depends on the medial prefrontal cortex and hippocampus. We have previously shown that selective lesions of the cholinergic basal forebrain projections to the neocortex and to hippocampus disrupt trace conditioning but not delay conditioning. The current experiment examines the contribution of the cortical cholinergic projections of the nucleus basalis magnocellularis (NBM) to the behavioral impairments previously observed following complete basal forebrain lesions involving both the NBM and the hippocampally-projecting medial septum (MS). We hypothesized that selective lesions of the cholinergic NBM neurons would disrupt trace conditioning in a manner similar to that observed following basal forebrain lesions. Additionally, because cholinergic modulation of prefrontal cortex mediates attention in other tasks, we hypothesized that increasing demands on attention in trace conditioning would exacerbate NBM lesion-induced impairments. Rats with bilateral 192 IgG-saporin lesions of the NBM and sham lesion control animals were tested in the trace conditioning paradigm either in the presence or absence of an attention-demanding visual distractor (intermittent, unpredictable flashing light). Rats received 60 trials per day for 10 days, where each trial consisted of a 10 s white noise CS, followed 10 s later by the delivery of a sucrose pellet unconditioned stimulus (US). Conditioned responding was assessed by measuring approach to the food cup. Approach during the CS itself was considered to be non-adaptive, while approach during the trace interval was classified as adaptive responding. Contrary to our hypothesis, results showed that NBM lesions failed to impair acquisition of trace conditioning in the absence of additional attentional demands. These findings suggest that the trace conditioning impairment previously observed following complete basal forebrain lesions were due either to damage to the hippocampally-projecting MS or to a cumulative effect of combined NBM and MS damage. The presence of the visual distractor, however, disrupted acquisition performance in the current experiment as hypothesized. The NBM lesion group in the distracted condition showed excessive non-adaptive responding during CS presentation as compared to controls. The increased attentional load caused by the visual distractor appears to have caused a disinhibition of non-adaptive responding in the NBM lesion group. These results suggest that cholinergic modulation of neocortex is involved in mediating attention during trace conditioning.

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

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.

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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)