References

Contreras M, Carrasco M, Riveros M, Quispe M, Valdes J, Torrealba F (2007) Early activation of the tuberomammillary nucleus is a common factor in appetitive behaviors in rats. Neuroscience 2007 Abstracts 842.19/WW11. Society for Neuroscience, San Diego, CA.

Summary: Histamine neurons of the tuberomammillary nucleus show an earlier activation during the appetitive phase of feeding, compared to the other arousal system nuclei. To test if in different appetitive behaviors also these histaminergic neurons become active first, we studied changes in Fos-ir in arousal nuclei during sexual, drinking and drug-seeking behavior. Male rats were exposed to sexually receptive or to non-receptive female rats, allowing sensory but not sexual contact. Receptive females elicited increased sniffing time which positively correlated with Fos-ir in the dorsal raphe, laterodorsal tegmental nucleus, orexin hypothalamic neurons and tuberomammillary nucleus. Non receptive females induced less sniffing and no increased Fos-ir. Other male rats were deprived of water for 48 h and presented with an empty water bottle to induce appetitive behavior. The presentation of an empty water bottle to thirsty rats induced increased approaches to the bottle while they tried to drink. While water deprivation per se increased Fos-ir in the dorsal raphe and the locus coeruleus, the presentation of the bottle increased Fos-ir in the tuberomammillary nucleus and induced a further Fos-ir increase in the locus coeruleus. Other male rats were conditioned to amphetamine (1.5 mg/Kg i.p.) using a place preference task. Conditioned rats, but not rats injected with saline instead of amphetamine, showed a significant preference for amphetamine-paired room and increase in the number of Fos-ir in the tuberomammillary nucleus, orexin hypothalamic neurons and locus coeruleus. To evaluate the importance of the histaminergic neurons in the appetitive phase of these motivated behavior, we lesioned tuberomammillary nucleus using saporin conjugated to the hypocretin 2. The histaminergic neurons lesion blunted the appetitive phase in all motivated behaviors studied, without affecting general motor capacities. Taken together our results indicate that the histaminergic neurons become active at the onset of different motivated behaviors and they are key in the arousal that is essential in motivation. Other arousal nuclei may participate depending on the particular behavior.

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

Loyd DR, Murphy AZ (2007) Lesioning mu opioid receptor-containing neurons in the ventrolateral periaqueductal gray attenuates morphine analgesia in male but not female rats. Neuroscience 2007 Abstracts 921.4/NN15. Society for Neuroscience, San Diego, CA.

Summary: Chronic pain will affect four out of five persons at some point across the lifespan. While the opioid-based narcotic morphine is the most prevalent treatment for chronic pain in clinical settings, it is becoming increasingly clear that morphine produces a significantly greater degree of analgesia in males compared to females. In both somatic and visceral pain models, the ED50 for morphine is generally two-fold higher for females than for males. The midbrain periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) is the primary circuit for opioid-based analgesia. We have recently shown that the PAG-RVM pathway is sexually dimorphic both in its anatomical organization and in its activation during persistent pain. Interestingly, while female rats have a greater number of PAG neurons that project to the RVM, inflammatory pain activates these cells to a greater degree in males. Additionally, systemic morphine inhibits the pain-induced activation of PAG neurons in males, but not females. Sex differences in neuronal activity during pain and morphine analgesia are prominent in the ventrolateral PAG, a region containing a large population of mu opioid receptor-containing neurons. We have recently shown that females have significantly lower levels of mu opioid receptors (MOR) in this region, however it is not known whether sex differences in MOR expression contribute to our observed sex differences in morphine analgesia. To test the role of ventrolateral PAG MOR in morphine analgesia, the cytotoxin saporin conjugated to the MOR agonist dermorphin (Der-Sap) was injected into the ventrolateral PAG to site-specifically lesion MOR-containing neurons. Twenty-eight days later, rats received an intraplantar injection of CFA to induce persistent pain and twenty-four hours later morphine was administered systemically using a cumulative dosing paradigm (1.8 -18mg/kg). Lesions of PAG MOR-containing neurons resulted in a two-fold rightward shift in morphine ED50 values in male rats compared to controls. Interestingly, in females no difference was noted in morphine ED50 for Der-Sap treated females versus controls suggesting that the PAG is not a critical site for morphine analgesia in females. Der-Sap treatment had no significant impact on baseline paw withdrawal latencies or CFA-induced hyperalgesia. These results indicate that the PAG is a primary locus for systemic morphine analgesia in males only and suggests the necessity for the development of sex-specific treatments for persistent pain in females.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Pang K, Sinha SP, Jiao X, Servatius RJ (2007) Understanding the role of non-cholinergic medial septal neurons in learning and memory: Implications for disease- and aging-related impairments. Neuroscience 2007 Abstracts 932.22/WW17. Society for Neuroscience, San Diego, CA.

Summary: The medial septum-diagonal band of Broca (MS) has an important function in learning and memory. Furthermore, degeneration of the MS may contribute to cognitive impairments associated with Alzheimer’s disease and normal aging. Because the MS contains several types of neurons, the neuronal population(s) involved in learning and memory has been actively investigated. Animal studies have mainly focused on the cholinergic neurons that project to the hippocampus. Although complete lesions of the MS or fimbria-fornix transaction leads to spatial memory impairments, selective damage of cholinergic MS neurons produces no or a mild impairment in spatial memory, suggesting an important role of non-cholinergic neurons. Most of these non-cholinergic neurons are GABAergic. Previously, we used low concentrations of kainic acid to examine the importance of non-cholinergic MS neurons in spatial memory. However, a more selective toxin for GABAergic neurons would facilitate research, as it has done for the cholinergic system. In the present study, we use a new GABAergic immunotoxin that combines an antibody to the GABA transporter GAT1 with saporin. GAT1-saporin was administered into the medial septum of male Sprague Dawley rats. Our preliminary results show that GABAergic septohippocampal neurons as assessed by parvalbumin-immunoreactivity were virtually eliminated, while cholinergic neurons were spared in the medial septum. Current work is focused on further characterizing the cell populations affected by GAT1-saporin. Preliminary behavioral results demonstrate that GABA MS lesions did not impair spatial reference memory in the initial acquisition of a water maze task. However, a deficit was observed in reversal learning. Further testing in a procedure where the escape platform moves to a new location every day showed that rats treated with GAT1-saporin were mildly impaired in within-session learning of the new platform location. These preliminary results demonstrate that intraseptal GAT1-saporin is effective in eliminating at least some populations of GABAergic neurons in the MS. Furthermore, the preliminary behavioral results are consistent with our previous results demonstrating that damage of non-cholinergic MS neurons produces a very specific impairment on reversal learning. In summary, GAT1-saporin may be a useful tool to examine the function of GABA MS neurons in learning and memory and their contribution to cognitive impairments in disease and aging.

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ATS Poster of the Year Winner

Rennie KE, Frechette M, Pappas BA (2007) Behavioural consequences of combined cholinergic lesion and chronic cerebral hypoperfusion in rats. Neuroscience 2007 Abstracts 698.16/R26. Society for Neuroscience, San Diego, CA.

Summary: Chronic cerebral hypoperfusion compromises the health of hippocampal neurons, leading to a slowly emerging loss of pyramidal cells accompanied by spatial memory impairments in rats. Recent research suggests that vascular abnormalities resulting in insufficient cerebral blood flow or impaired nutrient delivery to the brain represent a significant risk factor for Alzheimer’s disease (AD) and may contribute to its pathogenesis. AD is also characterized by dysfunction of the forebrain cholinergic system. Since there is evidence that this system is involved in the control of local cerebral blood flow, we hypothesized that there would be synergistic effects of chronic cerebral hypoperfusion and cholinergic dysfunction. Hence, the aim of this study was to determine whether cholinergic dysfunction exacerbates the effects of cerebral hypoperfusion. Female rats were subjected to forebrain cholinergic lesion or control surgery by intraventricular infusion of the immunotoxin 192-IgG-saporin (192S) or phosphate buffered saline (PBS) on postnatal day 7. Six months later the rats underwent permanent bilateral occlusion of the carotid arteries (2VO), which causes moderate, chronic cerebral hypoperfusion, or sham surgery. When exposed to an open field 48, 72 and 96 hours after 2VO or sham surgery, the groups did not differ on measures of overall activity. However, the cholinergic lesion increased the latency to enter the centre area, and reduced both the number of centre entries and the percentage of total distance that was traveled in the inner squares. The lesion effects were mainly seen in the combined 192S/2VO group while 192S or 2VO alone produced only minor behavioural changes. Elevated plus testing 2 weeks after surgery revealed a reduction in open but not closed arm entries due to the cholinergic lesion. Interestingly, the effects of 2VO were dependent on the status of the cholinergic system. 2VO increased open arm entries in the PBS group, but decreased this behaviour in the 192S group. Thus on both the open field and elevated plus maze, the cholinergic lesioned rats displayed more anxious behaviour, particularly after 2VO. Finally, cholinergic lesion produced impairments on the working memory version of the Morris water maze. Again, this effect was most pronounced in the combined 192S/2VO group. This effect is unlikely to be due to motivational or sensorimotor deficits as all groups performed similarly on a cued platform version of the maze. Cholinergic lesion and 2VO appear to act synergistically to produce behavioural alterations, even at relatively early time points after 2VO. Their combined effects on CA1 pyramidal cell viability are currently under examination.

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McAuley J, Pang K (2007) Effects of NBM lesions on selective attention in an interval timing task. Neuroscience 2007 Abstracts 742.9. Society for Neuroscience, San Diego, CA.

Summary: Divided and sustained attention are impaired by damage to the nucleus basalis magnocellularis (NBM), which provides cholinergic and GABAergic input to the neocortex. The present study was performed to further investigate the role of the NBM in attention using a selective attention version of the peak-interval timing procedure. Male Fisher 344 rats were initially trained using a peak interval procedure to time a light stimulus, delivering reward for the first lever press after 12 s. Selective attention was then tested in distracter sessions where random tone bursts and house light flashes were presented on some trials, but not others. These distracter sessions were interleaved with non-distracter sessions that were identical to initial peak-interval training. Preliminary results in normal young rats show that peak times on un-reinforced probe trials with distraction were lengthened as compared to probe trials without distraction in the same session. Moreover, peak times on non-distracter probe trials were similar between distracter and non-distracter sessions. In these preliminary studies, the observed overestimation of time during selective attention testing was transient, supporting the view that attention modulates the rate of an internal clock. Current studies aim to determine the influence of selective cholinergic or GABAergic NBM lesions in this selective attention task.

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Kalinchuk AV, Porkka-Heiskanen T, McCarley RW, Basheer R (2007) Progressive decrease in sleep deprivation-induced extracellular adenosine release and recovery NREM sleep following intracerebroventricular injection of 192 IgG-saporin. Neuroscience 2007 Abstracts 735.10/TT29. Society for Neuroscience, San Diego, CA.

Summary: The basal forebrain (BF) is an important site in the homeostatic regulation of sleep mediated by adenosine (AD) release (Porkka-Heiskanen et al., 1997). The BF comprises different neuronal populations, including cholinergic, GABAergic and glutamatergic cells. Immunotoxin 192 IgG-saporin has been used in several studies to investigate the role of the BF cholinergic vs. non-cholinergic cells in the regulation of spontaneous sleep and homeostatic sleep response after sleep deprivation (SD) but results of these studies are controversial. 2 weeks after local saporin injection into the caudal BF (horizontal diagonal band/magnocellular preoptic area/substantia innominata, HDB/MCPO/SI), recovery sleep is reduced; however, 2 weeks after ICV saporin injection, no changes in recovery sleep occur. We hypothesized that this difference in ICV vs. local effects might be explained by a delayed lesion of the cholinergic cells in the HDB/MCPO/SI area after ICV injection. Consequently, in the same rats, we examined the time course of the effects of ICV-injected saporin on SD-induced BF AD levels and the homeostatic sleep response at both 2 and 3 weeks post-injection. Male rats were ICV injected with saporin (6μg, n=9) or saline (n=5) and implanted with EEG/EMG electrodes and guide cannulae for microdialysis probes targeting the HDB/MCPO/SI. Experimental schedule, performed for each rat at 2 and 3 weeks post-injection, included spontaneous sleep-wake recording for 24h beginning at 8am (7am:7pm L:D) and SD for 6h beginning at 10am followed by recovery sleep at 4pm-8am. AD samples were collected at 30min intervals on SD day from 8am to 8pm. Histology evaluated the extent of cholinergic cell loss and probe locations. 2 weeks after ICV saporin injection, SD induced significant increases in BF AD levels (+126%), NREM recovery sleep duration (+41%) and NREM delta power (+91%). All values were similar to saline-treated animals. However, 3 weeks after ICV saporin injection, SD did not increase BF AD nor NREM recovery sleep, while delta power in NREM sleep had a modest increase (+21%). The changes observed 3 weeks after ICV injection were quantitatively similar to those observed 2 weeks after local BF saporin administration (Kalinchuk et al., 2005). We conclude that the effect of ICV saporin-induced cholinergic lesions follows a slower time course (3 weeks or longer) compared to local BF injections in reducing the SD-induced AD increase and the homeostatic sleep response. Taken together, our present and previous observations imply that cholinergic neurons in the BF play an important role in the regulation of SD-induced AD release and NREM recovery sleep.

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

Wood DA, Patterson N, Fuller P, Sherman D, Saper C, Lu J (2007) Genetic dissection of neural circuitry underlying REM sleep behavior disorder (RBD). Neuroscience 2007 Abstracts 736.28/VV11. Society for Neuroscience, San Diego, CA.

Summary: REM sleep behavior disorder (RBD), a parasomnia typically manifested as dream enactment behavior, may represent an early pathophysiologic manifestation of Lewy body diseases (LBD), such as Parkinson disease and dementia with Lewy bodies. Preclinical investigation of possible underlying neural mechanisms of RBD suggests that a set of glutamatergic neurons located in the sublaterodorsal nucleus (SLD), which project to GABA/glycine interneurons in the ventral horn are responsible for atonia during REM sleep (Lu et al. 2006, A putative flip-flop switch for control of REM sleep, Nature 441, 589-94). Based upon these findings, we hypothesize that a loss of glutamate from these neurons in the SLD produces REM sleep without atonia, an animal equivalent of RBD. To assess this question, we selectively eliminated glutamate release from SLD by injecting adeno-associated virus-Cre recombinase (AAV-Cre) into the SLD of mice with lox P sites flanking exon 2 of the vesicular glutamate transporter 2 (VGLUT2) gene. In addition, we examined the role of the ventromedial medulla (VMM) in REM atonia by injecting orexin-saporin in rats and AAV-Cre into flox-VGAT (vesicular GABA/glycine transporter) and flox-VGLUT2 mice. Consistent with our hypothesis, these data show that loss of the VGLUT2 gene in the SLD produces REM sleep without atonia (walking, running and myoclonic jerking) without alteration of total amount of REM sleep. Furthermore, loss of the VGLUT2 but not the VGAT gene in the intermediate VMM results in myoclonic jerking against the background of tonic atonia during REM sleep. Based upon these observations, we propose that suppression of muscle activity during REM sleep is controlled by the activation of excitatory glutamatergic projections from the SLD (with collaterals targeting the intermediate VMM) and from the intermediate VMM, which terminate at inhibitory interneurons in the spinal cord. Collectively, this work provides novel insight into the control of muscle tone during REM sleep, which may have implications for our understanding of neurological conditions that precede the onset of neurodegenerative disease.

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Gompf HS, Fuller PM, Saper CB, Lu J (2007) Locus coeruleus (LC) is involved in sustaining arousal. Neuroscience 2007 Abstracts 736.3/UU16. Society for Neuroscience, San Diego, CA.

Summary: The locus coeruleus (LC) has traditionally been thought to be involved in arousal; however, lesions of the LC have minimal effects on basal sleep-wake behavior. We propose that the LC instead may be required to sustain arousal under conditions of environmental challenge. To test this hypothesis, we intraventricularly injected saline, or 0.25, 0.5, or 1µg anti-DBH-saporin (DBH-SAP selectively lesions the LC), and implanted EEG/EMG electrodes. On recording days, each animal was paired with a normal rat (social interaction) and presented with novel objects every hour for 5 hours from ZT 6 to ZT 11. We then repeated the same experiment for 2.5 hrs and immunostained tissue for Fos and TH or Fos and DBH. We also repeated the same experiment in rats with unilateral LC lesion by 6-OHDA. During 5 hr of stimulation with novel objects and social interaction (distracting stimuli, DS), controls or partial LC lesioned animals (0.25 µg DBH-SAP) spent 83 ± 8% and 92 ± 4% awake respectively (n = 3 and 4, p = 0.4) whereas animals with complete LC lesions (0.5 and 1 µg) spent significantly less time in wakefulness (59 ± 4% and 66 ± 5% respectively, n = 3 and 4, p = 0.0005). The reduction of wakefulness occurred primarily during the second 30 mins of each hour. Following DS exposure, Fos was highly expressed in the cerebral cortex in both LC lesioned groups and controls. Furthermore, we found a correlation (R2 = 0.79) between the remaining LC neurons and wakefulness under DS. Rats with loss of one LC showed no changes in wakefulness, and Fos was highly and symmetrically induced in the cerebral cortex. Our results suggest that the LC is specifically involved in sustaining arousal.

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Martin MM, Carter LA, Jones JL, Winter SS, Wallace DG (2007) Organization of food protection behavior is differentially influenced by hippocampal and cortical cholinergic deafferentation. Neuroscience 2007 Abstracts 742.6/AAA9. Society for Neuroscience, San Diego, CA.

Summary: Previous work has suggested that rats use temporal information to organize their food protection behaviors. Studies have demonstrated different roles for hippocampal and cortical cholinergic function in processing of temporal information in standard interval timing procedures. The present study examined the role of hippocampal and cortical cholinergic function on the organization of food protection behavior. Long Evans female rats received either injections of 192 IgG-Saporin (SAP) or saline (SHAM) into the medial septum (MS) or nucleus basalis (NB). Subsequent to recovery, rats were placed into an enclosure and provided a hazelnut in the presence of an unoperated conspecific. All rats engaged in dodging or bracing behaviors to prevent the theft of the hazelnut. During a dodge, the rat places the food item in its mouth to use both fore- and hind-limbs to escape the approaching conspecific. In contrast, during a brace, the rat’s forelimbs maintain contact with the food item, and only the hind limbs are used to make shorter lateral movements. Only rats receiving sham lesions displayed a consistent transition from primarily engaging in dodging behavior to primarily engaging in bracing behavior during the consumption of the hazelnut. The MS SAP group displayed a disruption in their temporal organization of food protection behaviors. Although the NB SAP animals displayed impaired responding to the approaching conspecific (resulting in frequent thefts), their food protection behaviors tended to exhibit temporal organization. These results provide further evidence as to the role of the basal forebrain cholinergic system in temporal organization of behavior.

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Wallace DG, Knapp SK, Silver JA, Martin MM, Winter SS (2007) Selective hippocampal cholinergic deafferentation disrupts exploratory trip organization. Neuroscience 2007 Abstracts 743.17/BBB11. Society for Neuroscience, San Diego, CA.

Summary: Rats organize their exploration of an environment around a central location or home base. Movements away from the home base are characterized as a series of slow progressions punctuated by stops. Subsequent to the last stop, the homeward segment is a single, rapid progression associated with a consistent temporal pacing of linear speeds. Observing these characteristics of exploratory behavior independent of environmental cue availability or familiarity has supported rats’ use of self-movement cues generated after departing the home base to estimate the distance and direction back to the home base. The current study investigated the effects of selective hippocampal cholinergic deafferentation on home base establishment and exploratory trip organization. Long Evans female rats either received injections of 192 IgG-Saporin (SAP) or saline (SHAM) into the medial septum. Subsequent to recovery, rats were placed on a large circular table with access to a refuge under complete dark conditions (infrared cameras and goggles were used to visualize the rat). Although all rats established a home base in the refuge, impairments in exploratory trip organization specific to the homeward segment were observed in SAP rats. Specifically, SAP rats displayed inconsistent temporal pacing of homeward segment linear speeds. These observations are consistent with a role for hippocampal cholinergic function in processing self-movement cues.

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