targeted-toxins

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.

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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)

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.

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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.

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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.

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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.

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