sfn2008

Laplante FP, Dufresne M, Lappi DA, Sullivan RM (2008) Depletion of cholinergic neurons in the nucleus accumbens and its possible involvement in schizophrenic symptomatology. Neuroscience 2008 Abstracts 761.18/FF34. Society for Neuroscience, Washington, DC.

Summary: Schizophrenia is a mental disorder characterized by dysfunctions in several neurotransmitter systems including the central cholinergic system. While alterations in cholinergic neurotransmission have been demonstrated in schizophrenic brains, their biological significance remains to be established. Post-mortem studies of schizophrenic patients have shown a reduction in the density of cholinergic interneurons in the striatum, most prominently in the ventral striatum or nucleus accumbens (N. Acc). Intra-accumbens acetylcholine interacts functionally with the mesolimbic dopaminergic system and is believed to dampen the effects of excessive dopamine (DA) release. Therefore, we hypothesize that a reduction in the density of cholinergic neurons in the N. Acc will be behaviorally relevant, if not causal, to the enhanced (ventral) striatal dopaminergic neurotransmission described in schizophrenia and may contribute substantially to the emergence of schizophrenic symptomatology. In this study we aimed to reproduce in rats a selective reduction in N.Acc. cholinergic cell density, and study the neurophysiological and behavioural consequences of these lesions, relevant to the neuropsychopathology of schizophrenia. A novel saporin immunotoxin coupled with an antibody targeting choline acetyltransferase (ChAT) has been developed. We microinjected this immunotoxin bilaterally (0.5 μg/μl; 0.5 μl) into the N. Acc (core and shell) of adult male Srpague-Dawley rats. Using immunohistochemistry to quantify ChAT staining, we have confirmed that this toxin caused a 40-50 % loss in the number of cholinergic neurons in this region within two weeks post-injection. Lesioned rats exhibited significantly higher spontaneous locomotor activity than control rats and were shown to be hypersensitive to the locomotor activating effects of amphetamine and quinpirole. Furthermore, in separate groups of animals, we have observed in lesioned rats, a reduction in the prepulse inhibition of the acoustic startle response. Taken together, it is proposed that reduction of cholinergic neurons in the N. Acc triggers an enhanced DA responsivity in the N.Acc which may prove highly effective in reproducing behavioral abnormalities analogous to those found in schizophrenia. The neurophysiological consequences of these lesions on DA neurotransmission will be further addressed by measuring both pre- and postsynaptic indices of DA function in this region.

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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.

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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.

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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.

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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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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.

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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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