Abstracts from Society for Neuroscience (SFN) San Diego, CA • November 3-7, 2007

43 entries found for : sfn2007

Survival, integration and differentiation of human neural stem cells transplanted into an animal model of cholinergic degeneration

Cusulin C, Aztiria E, Cacci E, Battaglini PP, Kokaia Z, Leanza G (2007) Survival, integration and differentiation of human neural stem cells transplanted into an animal model of cholinergic degeneration. Neuroscience 2007 Abstracts 779.5/E7. Society for Neuroscience, San Diego, CA.

Summary: The recently characterized F7B cell line, derived from human fetal cortex (Cacci et al, 2007) has been observed able to differentiate in vitro into different glial and neuronal subtypes. However, no much data is available about its capacity for differentiation in vivo. In the present work, F7B cells were grafted to the medial septum of newborn (P8) intact rats and to littermates that had been subjected to selective cholinergic deafferentation at P4 using the 192 IgG-saporin immunotoxin. The animals were sacrificed 1 or 3 months after grafting and the dissected brains were processed for immunocytochemistry, using cell-specific (HuNu and GFP) and differentiation markers (Dcx, GFAP, NeuN, and HuD). Overall, grafted F7B cells exhibited an excellent ability to survive and differentiate into the host tissue environment. Survival rate varied among the groups, being consistently higher when the cells were grafted into lesioned, as opposed to intact, animal. Moreover, a better survival was seen at 1 month, compared to 3 months post-grafting, regardless the lesion condition. Interestingly, the presence of a cholinergic depletion in the recipient appeared to affect differentiation of grafted F7B cells. In fact, higher numbers of Dcx+ and HuD+ cells were scattered within the grafts placed in lesioned animals, as compared to controls. In lesioned, but not intact animals, at 3 months post-grafting, sparse F7B cells were found to express the mature neuronal marker NeuN. On the other hand, grafted F7B immunoreactive for GFAP were similarly detected in all transplanted animals. F7B cells appear to be feasible for transplantation, being able to survive and differentiate into a developing brain, and to positively respond to the new environment created by a lesion. Further studies are warranted to test their actual capacity for functional integration.

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

Cholinergic saporin lesions in the nucleus accumbens attenuate alcohol drinking

Camp MC, Alcantara AA (2007) Cholinergic saporin lesions in the nucleus accumbens attenuate alcohol drinking. Neuroscience 2007 Abstracts 809.4/X26. Society for Neuroscience, San Diego, CA.

Summary: Identifying specific cell types and the plasticity that occurs within those neuronal networks that lead to alcohol drinking is critical for the development of improved treatment programs for alcoholism. We have previously reported that cholinergic neurons of the nucleus accumbens (NAc) undergo alcohol-induced intracellular and receptor neuroadaptations. These changes may facilitate acetylcholine (ACh) release, which in turn modulate the neuronal circuits that underlie alcohol drinking. We postulate that alcohol-induced neuroplasticity of the cholinergic system facilitates alcohol drinking whereas removal of ACh influences attenuates alcohol drinking. Cholinergic cell lesion studies investigating a direct causal role of these neurons in drug abuse and memory have reported their importance in reinforcement, conditioned place preference, reward-related procedural learning and working memory. The effects of accumbal cholinergic lesions on alcohol drinking, however, have not been examined. To test a direct causal link between cholinergic cells and alcohol drinking, the present study employed bilateral cholinergic cell lesions in the NAc and measured alcohol drinking in C57BL/6J mice. The effects of cholinergic ablation on alcohol drinking were examined following a Drinking in the Dark model of binge drinking and on motor impairment using the fixed speed rotarod test. Cholinergic ablations were produced by microinjections of mu p75-saporin into the NAc. After recovery, animals were given 20% (v/v) ethanol in water for 4 hours a day in the home cage starting 3 hours after lights off for 1 month. Cholinergic cell eliminated mice showed a 26% decrease in alcohol drinking compared to saline microinjected controls (p<0.05) and fell from the rotarod 24% sooner than controls (p<0.05). The present study provides evidence of a direct causal link between cholinergic cells and alcohol drinking. These results suggest that increased ACh release facilitates binge drinking, whereas removal of ACh signaling attenuates drinking. These findings provide the basis for accumbal cholinergic-targeted pharmaceutical and behavioral treatment programs designed to attenuate alcohol binge drinking.

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

Changes in neurokinin-1 receptor expression in populations of spinal lamina I neurons in rats lacking non-peptidergic nociceptive fibers

Saeed AW, Ribeiro-Da-Silva A (2007) Changes in neurokinin-1 receptor expression in populations of spinal lamina I neurons in rats lacking non-peptidergic nociceptive fibers. Neuroscience 2007 Abstracts 821.4/FF19. Society for Neuroscience, San Diego, CA.

Summary: Neurokinin-1 receptor (NK-1r)-containing lamina I projection neurons are deeply involved in the transmission of pain-related information to the brain. Previous studies have shown that lamina I neurons can be classified morphologically into fusiform, pyramidal and multipolar cells, and that these types differ in functional properties, with the pyramidal type being non-nociceptive. Our laboratory has shown not only a considerable increase in immunoreactivity for the NK-1r in animals with CFA-induced arthritis but also a de novo expression of these receptors by pyramidal neurons (Almarestani et al., Soc.Neurosci.Abstr., Program # 249.11, 2006). Based on this, we deemed it interesting to study whether pyramidal neurons would also express NK-1r in an animal model in which we have previously shown a drastic increase in NK-1r expression but no augmented nociceptive responses. To achieve this, we injected, under anesthesia, saporin (SAP) conjugated to the lectin IB4 into the left sciatic nerve of male Sprague Dawley rats to selectively lesion the non-peptidergic nociceptive C-fibers. Animals were sacrificed from 2 weeks to 2 months post-lesion. Horizontal sections of spinal segments L5 and L6 were cut and processed for IB4 binding and NK-1r immunoreactivity using immunofluorescence. Examination of IB4-SAP treated rats at several time points post-lesion revealed increased expression of NK-1r by lamina I cells of the fusiform and multipolar types on the side ipsilateral to the lesion, compared to the contralateral side and to controls. However, pyramidal cells seldom expressed the NK-1r in both control and lesioned animals. Surprisingly, we also observed a direct innervation of lamina I neurons by IB4-positive neurons in control animals, which did not occur ipsilaterally in lesioned animals. These observations support the concept that increased activity by the peptidergic nociceptive afferents may be important in the maintenance of nociceptive responses in the absence of non-peptidergic fibers.

Related Products: IB4-SAP (Cat. #IT-10)

Antinociceptive effects of lumbar intrathecal neuropeptide y-saporin

Kline IV RH, Lemons LL, Wiley RG (2007) Antinociceptive effects of lumbar intrathecal neuropeptide y-saporin. Neuroscience 2007 Abstracts 821.5/FF20. Society for Neuroscience, San Diego, CA.

Summary: Spinal intrathecal (i.t.) neuropeptide Y (NPY) has been shown to be antinociceptive in the rat. Using lumbar i.t. NPY, coupled to the ribosomal inactivating protein, saporin, to selectively destroy spinal dorsal horn cells that express NPY receptors, we sought to determine the effect of this lesion on nocifensive behaviors in the hotplate and formalin tests and on NPY1R staining in the lumbar dorsal horn. Twenty Sprague Dawley male rats were injected i.t. with either saline or 500ng NPY-sap and then were tested on the hotplate for 30 days. Fifteen Long Evans female rats were injected i.t. with either saline or NPY-sap (500ng or 750ng) and then tested on the hotplate for two weeks followed by hindpaw formalin injection. In order to assess responses mediated by C or A-delta thermal nociceptors, hotplate testing used three temperatures: 44C (600 sec trial duration), 47C (200 sec trials), and 52C (first response or 30 sec). In male rats, lumbar i.t. NPY-sap increased hindpaw withdrawal latencies to 44, 47 and 52C, with the greatest effect on 44C. NPY-sap also reduced the total amount of hindpaw lick/guard responding (duration and number of responses) on the 44 and 47C hotplates. Female rats injected with 750ng of NPY-sap showed a decrease in the number of hindpaw lick/guard events on the 44C hotplate. Female rats also showed a decrease in the total number of hindpaw lick/guard events during the interphase (7-21min) and phase II (22-90min) of the formalin test. Additionally operant thermal place preference testing (45C vs 12C) was compared to hotplate reflex testing. Selectivity of NPY-sap was assessed by immunocytochemistry for cells expressing NPY1R and non-selectivity was assessed by staining for NK-1R. Based on the above findings we conclude that selective destruction of dorsal horn NPY1R-expressing neurons produces decreased thermal nociception to a range of noxious heat and also decreases responses to persistent noxious chemical stimulation during the formalin test. In summary, reduced nocifensive behaviors after NPY-sap were more prominent when assessing responses elicited by input from predominately C fiber activation (44C and formalin). This study was supported by the Department of Veterans Affairs.

Related Products: NPY-SAP (Cat. #IT-28)

Immunotoxin lesion of cholinergic nucleus basalis magnocellularis neurons in Lister hooded rats impair performance in a delayed matching-to-place task

Savage S, Ogren S, Olson L, Mattsson A (2007) Immunotoxin lesion of cholinergic nucleus basalis magnocellularis neurons in Lister hooded rats impair performance in a delayed matching-to-place task. Neuroscience 2007 Abstracts 840.1/TT24. Society for Neuroscience, San Diego, CA.

Summary: Central cholinergic systems play an important role in various aspects of cognition, and deficits in cortical cholinergic function have been implicated in the cognitive impairments associated with normal aging and dementia. Cholinergic dysfunctions have also been implicated in several neuropsychiatric disorders, including schizophrenia. Though cognitive dysfunctions, such as impaired working memory, are observed in Alzheimer, as well as schizophrenic patients, the cholinergic mechanisms behind these dysfunctions are not well characterized in animal models. To investigate whether specific cortical cholinergic deficits will affect spatial learning and memory functions, we lesioned the basalo-cortical cholinergic system by stereotaxic infusion of the immunotoxin 192 IgG-saporin in the nucleus basalis magnocellularis (NBM) of adult male Lister hooded rats. Learning and memory was assessed using a delayed matching-to-place (DMP) paradigm in the water maze. We found that animals with cholinergic denervation of neocortex were impaired in the DMP-task. Thus, while the sham-operated animals rapidly learned the task without prior training, saporin-treated rats showed impairment during the initial three days of testing. By the end of the testing period, the lesioned animals had acquired the task. However, the cholinergically denervated animals showed a performance deficit throughout the duration of the experiment with higher trial latencies and longer distance traveled to find the platform as compared to the controls. They also seemed to employ a different strategy to find the hidden platform as compared to control animals. Whether the deficits after cholinergic lesions to the NBM seen in the present experiment are mnemonic and/or attentional in nature remains to be elucidated.

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

Early activation of the tuberomammillary nucleus is a common factor in appetitive behaviors in rats

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)

Lesioning mu opioid receptor-containing neurons in the ventrolateral periaqueductal gray attenuates morphine analgesia in male but not female rats

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)

Understanding the role of non-cholinergic medial septal neurons in learning and memory: Implications for disease- and aging-related impairments

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.

Related Products: GAT1-SAP (Cat. #IT-32)

ATS Poster of the Year Winner

Transplant of hypocretin neurons into the lateral hypothalamus of a narcolepsy rat model

Millan-Aldaco D, Arias-Carrion O, Palomero-Rivero M, Drucker-Colin R, Murillo-Rodriguez E (2007) Transplant of hypocretin neurons into the lateral hypothalamus of a narcolepsy rat model. Neuroscience 2007 Abstracts 779.2/E4. Society for Neuroscience, San Diego, CA.

Summary: Narcolepsy, a disabling neurological disorder, is characterized by excessive daytime sleepiness, sleeps attacks, sleep fragmentation, and cataplexy. This sleep disorder has been linked to a loss of neurons into the lateral hypothalamus (LH) containing the neuropeptide hypocretin (HCRT). Our group has developed an experimental model in rats that mimics several aberrant behaviours observed in human narcolepsy. The bilateral administration of the neurotoxin hypocretin-2-saporin (HCRT2-SAP) into the LH of rats destroys most of the HCRT neurons (~90%) leading to develop narcolepsy as evaluated using EEG/EMG means. In order to replace the HCRT lost neurons by the local injection of the HCRT2-SAP, a suspension of cells from the hypothalamus obtained from rat pups (3-5 days old) were processed for grafting and stained with GFP. This cell suspension was injected into the LH of lesioned rats and they were sacrificed 21 days post-transplant. The brain was cut and sections containing LH were processed for HCRT immunohistochemistry as well as for the presence of HCRT-immunoflorescence neurons. We were able to differentiate the HCRT transplanted neurons into the LH of lesioned rats. Importantly, they were present at the target area 21 days after implant. These somata were similar in size and appearance to adult rat HCRT-immunoreactive neurons. Our results are very promising since the present study indicates that HCRT neurons obtained from rat pups can be grafted into a host brain and graft survived during 21 days. This experimental approach definitely addresses the possibility to replace HCRT neurons in narcolepsy in order to reverse this disease.

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Prefrontal cortical norepinephrine depletion does not impair spatial working memory in rats

King M, Jentsch JD (2007) Prefrontal cortical norepinephrine depletion does not impair spatial working memory in rats. Neuroscience 2007 Abstracts 645.16/CCC18. Society for Neuroscience, San Diego, CA.

Summary: The midbrain dopamine neurons are thought to encode a reward prediction error signal (Schultz et al., 1997; Bayer & Glimcher, 2005). Parkinson's disease (PD) is characterized by a loss of nigral dopamine neurons. Dopaminergic drugs including the dopamine precursor L-Dopa and D2 receptor agonists are taken to relieve disease symptoms. We hypothesized that patients with moderate PD (1) show atypical reinforcement learning off dopaminergic medication due to dopamine neuron loss, and (2) show more normal reinforcement learning on dopaminergic drug therapy. We developed a method to rapidly assess reinforcement learning in human subjects (Rutledge et al., SfN 2005) adapted from matching law tasks used in monkeys (Sugrue et al., 2004; Lau & Glimcher, 2005). On each trial, subjects choose one of two animated crab traps. Rewards (crabs worth $0.10) were scheduled for the two targets with different independent rates. Scheduled rewards remained available until the associated target was chosen, as in the original matching law experiments (Herrnstein, 1961). After a 5-minute training period, subjects completed 800 trials as we varied reward probabilities across blocks. PD patients (n=19) completed one session on and one off dopaminergic medication. Age-matched controls (n=21) and healthy young subjects (n=20) completed one session. We found that young and elderly control subjects had similar reinforcement learning rates, but learning rates were reduced in PD patients (when tested off medication). Learning rates in the same PD patients were restored to control levels when dopaminergic drugs were administered. We also found that the reinforcement-independent strategies of our subjects were influenced by dopamine. Young subjects tended to alternate targets independent of reward history. In contrast, elderly subjects (who suffer some dopamine neuron loss) had a tendency to perseverate in their choices. This tendency was increased in PD patients (off medication), but restored to control levels when dopaminergic drugs were administered. This effect on choice is not explained by existing models of dopamine function. These data support a role for dopamine in human reinforcement learning. Future models of decision making in reinforcement learning tasks must also account for a reward-independent effect of dopamine on choice behavior.

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

On the survival of nestin-expressing neurons in the cholinergic basal forebrain after an immunolesion with 192-IgG-saporin

Nguyen WT, Buhalog A, Hendrickson M, Kalil RE (2007) On the survival of nestin-expressing neurons in the cholinergic basal forebrain after an immunolesion with 192-IgG-saporin. Neuroscience 2007 Abstracts 674.5/D24. Society for Neuroscience, San Diego, CA.

Summary: Nestin is a class VI intermediate filament protein that is widely accepted as a marker for uncommitted neural progenitor cells. However, we have described a class of cells in the cholinergic basal forebrain of the adult rat and human that express markers associated exclusively with neurons, e.g., NeuN, β-III tubulin, and choline acetyl transferase (ChAT) and also express nestin. We have termed these cells nestin-expressing neurons (NENs). To explore the possibility that the expression of nestin by NENs might provide a neuroprotective effect, we administered the immunotoxin 192-IgG-saporin (192-saporin). The toxin consists of a ribosome-inactivating protein coupled to a monoclonal antibody directed against the p75 nerve growth factor receptor (p75 NGFr). As a result, 192-saporin selectively destroys cells expressing this receptor, such as most of the cholinergic neurons in the basal forebrain. Two micrograms of 192-saporin in 6 µL of saline were injected unilaterally into the lateral ventricle of the brain in each of four adult Sprague-Dawley rats. Following a six day survival period, the rats were deeply anesthetized, perfused with 4% paraformaldehyde, and the brains were sectioned and immunostained for nestin and ChAT. After confirming that NENs, which were identified by the co-expression of nestin and ChAT, express the p75 NGFr, we determined the number of NENs in the medial septum and in the nucleus of the diagonal band as a percentage of all ChAT-positive neurons in these nuclei in rats treated with 192-saporin and in controls. We found no statistically significant difference in the proportion of NENs between rats that had received 192-saporin and controls. This result indicates that for the dose of 192-saporin and survival period used in these experiments, the expression of nestin does not confer a neuroprotective effect. Experiments using lower doses of 192-saporin and shorter survival times are underway.

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

Neonatal cholinergic lesion and environmental enrichment:behavior, neurogenesis and CA1 cytoarchitecture

Frachette M, Rennie K, Pappas BA (2007) Neonatal cholinergic lesion and environmental enrichment:behavior, neurogenesis and CA1 cytoarchitecture. Neuroscience 2007 Abstracts 691.9/M9. Society for Neuroscience, San Diego, CA.

Summary: The effects of neonatal cholinergic lesion and environmental enrichment on rat behaviour and hippocampal morphology were determined. Rats were injected with the immunotoxin 192 IgG- saporin (192S) on postnatal day 7, selectively lesioning forebrain cholinergic neurons as shown by their loss of acetylcholinesterase staining and p75NTR immunoreactive (IR) neurons. After weaning, the rats were placed in enriched or standard housing for 42 days. Enriched rats, regardless of whether or not they had received 192S, subsequently showed significantly enhanced performance on the working memory version of the Morris water maze. The lesion had no effect on spatial learning. However, the lesion significantly reduced doublecortin (DCX) IR cells in the dentate gyrus, indicating reduced hippocampal neurogenesis. Enrichment did not affect the number of DCX IR cells in lesioned rats whereas there was an apparent trend towards increased cells in non-lesioned rats. The number of DCX IR neurons in the enriched and impoverished lesion groups were identical and both were significantly less than the average for the enriched non-lesioned mean, suggesting that the lesioned rats were resistant to the effects of enrichment on neurogenesis. As shown by quantitative analysis of Golgi stained CA1 neurons, the cholinergic lesion affected CA1 cell morphology, reducing apical branches and total basal branch length. This was not prevented by enrichment. There were also a number of other effects selective for certain branches but these effects tended to be observed equally often in impoverished and enriched rats. In other words, the consequences of the cholinergic lesion were immune to the housing condition. Enrichment had several effects on hippocampal cytoarchitecture but these were selective for certain branch orders rather than global alterations. The most interesting consequence of enrichment, in terms of its implication for synapse density and information processing capability, was the increased spine density and spine number observed on some branches of the apical tree. This was evident only in the non-lesioned rats. Thus, neonatal cholinergic forebrain lesion reduces dentate gyrus neurogenesis, alters CA1 dendritic morphology but has no effect on spatial learning/memory. It also renders rats unresponsive to the effects of enrichment on dentate gyrus neurogenesis, CA1 dendritic spine morphology but not spatial learning/memory.

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

Behavioural consequences of combined cholinergic lesion and chronic cerebral hypoperfusion in rats

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.

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

Progressive decrease in sleep deprivation-induced extracellular adenosine release and recovery NREM sleep following intracerebroventricular injection of 192 IgG-saporin

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)

Genetic dissection of neural circuitry underlying REM sleep behavior disorder (RBD)

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.

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

Locus coeruleus (LC) is involved in sustaining arousal

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.

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

Neuronal mechanisms underlying the cognitive symptoms in a model of schizophrenia: prefrontal cholinergic inputs are necessary for attentional performance following repeated exposure to amphetamine

Young D, Howe WM, Martinez V, Bruno JP, Sarter M (2007) Neuronal mechanisms underlying the cognitive symptoms in a model of schizophrenia: prefrontal cholinergic inputs are necessary for attentional performance following repeated exposure to amphetamine. Neuroscience 2007 Abstracts 606.9/GG1. Society for Neuroscience, San Diego, CA.

Summary: The neuronal and cognitive effects of repeated exposure to amphetamine (AMPH) model important aspects of schizophrenia. Our prior results indicated that the attentional performance of AMPH-pretreated animals was maintained by abnormally high levels of prefrontal acetylcholine (ACh) release, and that the disruption of attentional performance by AMPH challenges was associated with a failure of the prefrontal cholinergic input system to respond to task onset (Kozak et al. 2007). The present experiment was designed to demonstrate that prefrontal cholinergic inputs are necessary for the (residual) attentional performance following repeated AMPH exposure. As removal of cortical cholinergic inputs per se disrupts attentional performance, we tested the hypothesis that limited prefrontal cholinergic deafferentation, which does not affect baseline attentional performance, prevents the establishment of normal performance following AMPH pretreatment. Rats were trained to perform a sustained attention task requiring the detection of visual signals and the discrimination between signal and non-signal events. Bilateral infusions of small concentrations and volumes of the immunotoxin 192 IgG-saporin into the medial prefrontal cortex did not affect the animals’ baseline performance. After re-establishing stable baseline performance, animals were pretreated with either saline or AMPH in accordance with an established, non-neurotoxic, escalating dosing regimen (1-10 mg/kg, twice daily over 40 days; Robinson et al. 1988). Animals were tested daily throughout the experiment. Following completion of the pretreatment regimen, the attentional performance of sham-lesioned controls recovered slowly over three weeks of continued training/testing. In contrast, performance recovery of deafferented, AMPH-pretreated animals was robustly attenuated and failed to reach pre-treatment levels. Collectively, these results indicate the necessary role of the prefrontal cholinergic input system in mediating the residual attentional performance of repeated AMPH. Therefore, pro-cholinergic treatments are predicted to benefit the attentional performance of schizophrenics. Repeated AMPH serves as a useful model to investigate the neuronal mechanisms underlying the cognitive symptoms of schizophrenia.

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

Organization of food protection behavior is differentially influenced by hippocampal and cortical cholinergic deafferentation

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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Galanin-like peptide stimulates feeding and sexual behavior via dopaminergic fibers within the medial preoptic area of adult male rats

Ganapini V, Powers F, Kuper K, Taylor A, Fraley GS (2007) Galanin-like peptide stimulates feeding and sexual behavior via dopaminergic fibers within the medial preoptic area of adult male rats. Neuroscience 2007 Abstracts 626.14/VV17. Society for Neuroscience, San Diego, CA.

Summary: Galanin-like peptide (GALP) is a hypothalamic neuropeptide known to regulate both food intake and sexual behaviors in adult male rats. We have demonstrated that ICV GALP administration elicits a significant fos response within the mPOA; thus, we feel that GALP stimulates feeding and reproduction by actions within the mPOA. Recent data from our and other labs have led us to suspect that GALP effects on these behaviors are due to activation of tuberoinfundibular dopaminergic neurons that terminate within the mPOA. To test the hypothesis that GALP activates mPOA dopaminergic systems, we utilized an immunolesion technique to eliminate dopaminergic fiber input specific to the mPOA via a dopamine-transporter specific toxin (DATSAP, n = 8) and compared to control injections (SAP, n = 8). All animals were sexually experienced adult male Long Evans rats. We first tested their response to a sexually-primed female rat. DATSAP-treated male rats showed a significant (p <0.001) reduction in male sexual behavior compared to SAP controls. We found that elimination of dopamine fibers within the mPOA significantly (p < 0.001) eliminated all aspects of male sexual behavior under normal mating paradigms. Injections of GALP (5.0 nmol) significantly increased (p < 0.01) male sex behavior in SAP control male rats but GALP was unable to stimulate the expression of these behaviors in DATSAP-treated rats. ICV GALP significantly (p < 0.05) stimulated fos within the mPOA of SAP rats but not in DATSAP-treated male rats. There was no significant difference in 24 hr food intake between SAP and DATSAP rats. However, the orexigenic effect of ICV GALP was significantly (p < 0.001) attenuated in DATSAP-treated male rats compared to SAP controls. These data suggest that GALP activates feeding and sexual behaviors in male rats by stimulating dopaminergic neurons that terminate within the mPOA.

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Selective hippocampal cholinergic deafferentation disrupts exploratory trip organization

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.

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

Effects of NBM lesions on selective attention in an interval timing task

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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Histaminergic regulation of energy homeostasis in the Siberian hamster

I’Anson H, Jethwa PH, Tanna GA, Pattinson LM, Ebling FJP (2007) Histaminergic regulation of energy homeostasis in the Siberian hamster. Neuroscience 2007 Abstracts 629.17/YY20. Society for Neuroscience, San Diego, CA.

Summary: We tested the hypothesis that posterior hypothalamic histaminergic (HA) activity regulates energy homeostasis in the Siberian hamster during long day (breeding season) photoperiods. Adult male Siberian hamsters were given bilateral injections of the retrogradely transported ribosomal toxin, saporin, conjugated to orexin-B receptor antibody (OXSAP, 200 nl, 92 ng/ul) into the posterior hypothalamus (PH) to selectively destroy HA neurons, the majority of which possess orexin-B receptors. Controls were injected with unconjugated saporin (sham). Metabolic rate (VO2 ml/kg0.75/h), ingestive behavior and locomotor activity were monitored using the comprehensive lab animal monitoring system (CLAMS, Columbus instruments). Body weight was significantly decreased by day 12 post-surgery in OXSAP compared with sham hamsters and remained significantly lower throughout the 5 month study, even though food intake was comparable between groups. At 3 months post-surgery, OXSAP food intake was significantly higher in the dark (p< 0.05) and significantly lower in the light phase (p<0.05), but not different overall between groups. In addition, the frequency of feeding bout tended to be lower during dark and light phases compared with sham hamsters (p=0.07). Lower body weight with no overall change in food intake suggests an increase in energy expenditure in the OXSAP hamsters. Consistent with this interpretation, locomotor activity in OXSAP hamsters tended to be higher during the dark phase (p=0.09), but not in the light phase. In addition, metabolic rate was significantly higher during the first two hours of the dark phase compared with sham hamsters (p<0.05), and tended to be higher during the entire dark phase (p=0.08). During a second CLAMS study (4 months post-surgery), metabolic rate was monitored following injection of an H3 receptor antagonist (thioperamide, 30 mg/kg, ip) as a probe to determine if any significant HA cell loss had occurred. Metabolic rate was significantly lower during the first 2 hours after thioperamide in sham hamsters, but not in OXSAP hamsters, suggesting that HA regulation of energy balance had been compromised by the OXSAP lesion. Immunohistochemical results confirmed 63-96% loss of HDC-immunoreactivity in magnocellular neurons of the posterior hypothalamus in the OXSAP group. These data support the hypothesis that posterior hypothalamic HA neuron activity modulates metabolic activity during the breeding season in the Siberian hamster, although it is likely that ablation of additional neuronal phenotypes which express orexin-B (e.g. MCH) may contribute to the observed metabolic effects.

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Walking the Plank: Role of the medial septum in distance estimation

Winter SS, Martin MM, Wallace DG (2007) Walking the Plank: Role of the medial septum in distance estimation. Neuroscience 2007 Abstracts 743.21/BBB15. Society for Neuroscience, San Diego, CA.

Summary: Controversy surrounds the role of the septohippocampal system in spatial orientation. Recent work has demonstrated that selective cholinergic deafferentation of the hippocampus impairs use of self-movement cues while sparing environmental cue use. Self-movement cues are generated from changes in position or direction. The current study examines the role of the septohippocampal cholinergic system in processing of self-movement cues related to changes in position or distance estimation in a food hoarding task. The probability of food hoarding has been shown to be influenced by travel distance and time to consume the food item. Long Evans female rats received either injections of 192 IgG-Saporin (SAP) or saline (SHAM) into the medial septum. Subsequent to recovery, rats were placed in a refuge on a 15 cm wide plank and allowed to traverse the plank to collect food pellets located at the end. Both the distance to the food pellet (2.4 vs. 4.8 m) and size of the food pellet (190, 500, 1000 mg) were varied across days. Differences in food hoarding probability were observed between groups. SAP rats were less likely to modify their food hoarding probability in response to changes in plank length relative to SHAM rats. These results are consistent with selective hippocampal cholinergic deafferentation producing deficits in processing self-movement cues related to distance estimation.

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

Intact delayed nonmatching-to-sample in monkeys with combined lesions of the temporal cortical cholinergic system and the fornix

Gaffan D, Baxter MG, Browning PGF (2007) Intact delayed nonmatching-to-sample in monkeys with combined lesions of the temporal cortical cholinergic system and the fornix. Neuroscience 2007 Abstracts 341.11. Society for Neuroscience, San Diego, CA.

Summary: Rhesus monkeys were pre-operatively trained in truly trial-unique delayed nonmatching-to-sample (DNMS) in an automated apparatus. They were then divided into a control group (n=3) and an experimental group (n=3) and received injections into the inferior temporal cortex of either saline (controls) or the selective cholinergic immunotoxin ME20.4-saporin (experimentals). A postoperative DNMS test showed no significant impairment in the experimental group, both groups performing at their pre-operative level. Both groups then underwent a second surgery to transect the fornix. Again, there was no significant impairment in DNMS, both groups performing at their pre-operative level. If the lesions are confirmed histologically then these results are in marked contrast to our findings with scene learning, in which monkeys with the same combined lesion as those in the present experimental group were severely impaired. However, a number of recent studies have shown that tasks with temporally complex events extended over trials, like DNMS, discrimination learning set, and serial reversal set, depend on a short-term prospective memory strategy that is supported by the interaction of temporal cortex with prefrontal cortex. Thus, the performance of DNMS does not require the laying down of new long-term memories.

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Lesion of intercalated (ITC) amygdala neurons interfere with extinction of classically conditioned fear responses

Likhtik E, Popa D, Apergis-Schoute J, Fidacaro GA, Pare D (2007) Lesion of intercalated (ITC) amygdala neurons interfere with extinction of classically conditioned fear responses. Neuroscience 2007 Abstracts 426.6/HHH29. Society for Neuroscience, San Diego, CA.

Summary: The acquisition of conditioned fear responses (CRs) is thought to involve the potentiation of synapses conveying information about the conditioned stimulus (CS) to the basolateral (BLA) amygdala. Expression of CRs would depend on the transfer of potentiated CS inputs by the BLA to the central amygdala (CE). In contrast, the mechanisms of extinction remain controversial. It has been proposed that ITC neurons, which receive BLA inputs and generate feedforward inhibition in CE, are in a key position to mediate extinction. In this view, NMDA-dependent potentiation of BLA inputs to ITC cells during extinction training, would dampen the impact of CS-related BLA activity on CE neurons, inhibiting CRs. However, this idea is difficult to test because ITC cells occur in small, lateromedially dispersed clusters, making conventional lesioning methods inadequate. Here, we took advantage of the fact that, compared to the rest of the amygdala, ITC cells express a much higher concentration of mu opioid receptors (muORs). As a result, we could lesion them by performing local injections of a peptide-toxin conjugate (demorphin conjugated to saporin, D-Sap) that selectively targets cells expressing muORs. Control rats received injections of saporin conjugated to a blank peptide (B-Sap). On Day 1, intact rats were subjected to a standard cued fear conditioning protocol in context A. On Day 2, they received 20 CS alone presentations in a different context (B). On Day 3, rats then received either D-Sap or B-Sap injections in the ITC cell masses. One week later, extinction recall was tested in context B with 10 CS alone presentations. Compared to control (B-Sap) rats (n=10), ITC-lesioned rats (n=5) had an extinction deficit (ANOVA, F=11.687, p = 0.005). Post-hoc t-tests comparing % time freezing during the first five or last five CSs revealed that rats with ITC lesions had significantly higher freezing levels throughout the extinction recall test (p<0.002 for both tests). These differences were not attributable to a non-specific increase in freezing or anxiety levels as exploratory behaviors in a novel open field in control and ITC-lesioned rats were indistinguishable. Overall, these results indicate that ITC cells are involved in the expression of extinction.

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Severe visual learning impairments in monkeys with combined but not separate lesions of the temporal cortical cholinergic system and the fornix

Browning PG, Gaffan D, Baxter MG (2007) Severe visual learning impairments in monkeys with combined but not separate lesions of the temporal cortical cholinergic system and the fornix. Neuroscience 2007 Abstracts 341.7. Society for Neuroscience, San Diego, CA.

Summary: A dense amnesia can be produced in the monkey by sectioning the anterior temporal stem, amygdala and fornix, a procedure which deafferents temporal cortex from modulatory inputs from the midbrain and basal forebrain. The present experiment investigated the neurochemical specificity of these severe learning impairments by selectively destroying cholinergic projections to the entire inferior temporal cortex by making multiple injections of the immunotoxin ME20.4-saporin into the inferior temporal cortex bilaterally. Six male macaque monkeys were preoperatively trained to learn new object-in-place discrimination problems each day until they could rapidly learn many such problems within a testing session. The monkeys then underwent surgery and received either injections of immunotoxin (n=3) or injections of saline (n=3). Both groups of monkeys were unimpaired when postoperative and preoperative performance were compared. Each monkey then underwent a second surgery to transect the fornix. After this surgery monkeys who had previously received injections of immunotoxin into temporal cortex showed a severe learning impairment, whereas monkeys who had previously received injections of saline showed a mild impairment. Monkeys with the combined immunotoxin plus fornix lesion were also severely impaired at concurrent object discrimination learning. These results suggest that different neuromodulatory inputs to inferior temporal cortex may act in concert to support cortical plasticity in visual learning such that the loss of acetylcholine only is not sufficient to disrupt normal learning behavior. The results also suggest that in monkeys, as in humans with Alzheimer’s disease, severe memory impairments occur only when a loss of acetylcholine projections to cortex is accompanied by organic tissue damage.

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Hypocretin/orexin neuronal loss increases adult neurogenesis

Arias-Carrion O, Hernandez-Martinez H, Drucker-Colin R (2007) Hypocretin/orexin neuronal loss increases adult neurogenesis. Neuroscience 2007 Abstracts 456.14/C7. Society for Neuroscience, San Diego, CA.

Summary: Adult neurogenesis in the subventricular zone (SVZ) is subjected to physiological regulation and can be modified by brain injuries. The sleep disorder narcolepsy may now be considered a neurodegenerative disease, as there is a massive reduction in the number of neurons containing the neuropeptide, hypocretin (HCRT). In the present study, we investigate the relationship between hypocretin neuronal loss and adult neurogenesis. The neurotoxin, hypocretin-2-saporin (HCRT2-SAP), was administered bilaterally to the lateral hypothalamus (LH) to lesion HCRT neurons. Five weeks after HCRT2-SAP administration a loss of HCRT-ir neurons into LH was produced. In normal animals, a high density of HCRT-ir fibers was found in the septum and was poor in the corpus callosum and striatum. These densities decreased in lesioned animals. To label dividing cells, we used 5-bromo-2′-deoxyuridine (BrdU). BrdU was injected twice daily during days 10-14 after lesion, saline or control procedure. Animals were killed at 3 weeks after the last BrdU injection. Experimental depletion of HCRT in rats increases precursor cell proliferation in the SVZ and subependimal layer of 3rd ventricle. However, we don't find BrdU/HCRT double-labeled cells in the subependimal zone or LH. These observations suggest that the HCRT is a negative factor in adult neurogenesis.

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Cholinergic depletion of prefrontal cortex does not impair episodic memory or strategy implementation in rhesus monkeys

Baxter MG, Kyriazis DA, Croxson PL (2007) Cholinergic depletion of prefrontal cortex does not impair episodic memory or strategy implementation in rhesus monkeys. Neuroscience 2007 Abstracts 341.9. Society for Neuroscience, San Diego, CA.

Summary: The prefrontal cortex is involved in regulating multiple aspects of memory, decision-making, and cognitive control. Cholinergic input to prefrontal cortex is thought to be involved in supporting its functions. To examine this hypothesis we tested 4 rhesus monkeys (3 male) with cholinergic depletion of ventrolateral prefrontal cortex (N=2) or the entire prefrontal cortex, excluding its medial aspect (N=2). Selective cholinergic depletion was produced by multiple injections of the immunotoxin ME20.4-saporin (0.02 ug/ul) into the prefrontal cortex. These monkeys were tested on two tasks that each require frontal-inferotemporal interaction, as well as an intact ventrolateral prefrontal cortex. The first, strategy implementation, requires monkeys to apply different choice strategies to different categories of objects in order to maximize the rate of reward delivery, and engages decision-making and cognitive control. The second, scene memory, is a test of episodic memory in which monkeys rapidly learn 20 new object-in-place scene discrimination problems within a single test session. Cholinergic depletions of prefrontal cortex, whether they were limited to ventrolateral prefrontal cortex or included the whole of lateral and orbital prefrontal cortex, were without effect on either strategy implementation or new scene learning relative to each monkey's preoperative performance. Thus, episodic memory and strategy implementation can proceed normally even with severely disrupted cholinergic input, so loss of cholinergic input on its own cannot explain impaired prefrontal function in conditions such as Alzheimer's disease. Acetylcholine may work in tandem with other neuromodulators to affect prefrontal cortex function; alternatively, it may only be involved in very specific aspects of cortical function, for example representational plasticity.

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Ketamine-induced gating deficit of hippocampal auditory evoked potentials in rats is alleviated by medial septum inactivation and antipsychotic drugs

Ma J, Tai S, Leung LWS (2007) Ketamine-induced gating deficit of hippocampal auditory evoked potentials in rats is alleviated by medial septum inactivation and antipsychotic drugs. Neuroscience 2007 Abstracts 498.12/GG19. Society for Neuroscience, San Diego, CA.

Summary: Gating of sensory responses is impaired in schizophrenic patients and animal models of schizophrenia. Ketamine, an N-methyl-D-aspartate receptor antagonist, is known to induce schizophrenic-like symptoms in humans. In this study, we investigated some conditions underlying ketamine’s effect on the gating of auditory responses in the hippocampus of freely moving rats. Gating was measured by the ratio of the second-click response (P2) to the first-click response (P1), or P2/P1, with P1 and P2 measured as peak amplitudes. Ketamine (1, 3 or 6 mg/kg s.c.) dose- dependently increased P2/P1 ratio as compared to saline (s.c.). P2/P1 ratio in saline injected rats was 0.48 + 0.05 and was 0.73 + 0.17 in ketamine (3mg/kg) treated rats. Pre-inactivation of the medial septum with GABAA receptor agonist muscimol (0.25 μg/0.6 μL) or systemic administration of antipsychotic drugs, including chlorpromazine (5 mg/kg i.p.), haloperidol (1 mg/kg i.p.) or clozapine (7.5 mg/kg i.p.), decreased P2/P1 to values comparable to normal rats without drug injection. Infusion of muscimol in the medial septum or injection of antipsychotic drug alone did not affect the P2/P1 ratio. However, rats with selective lesion of the septohippocampal cholinergic neurons (by 192-IgG saporin) showed significant higher P2/P1 (0.86 + 0.10) than that of sham lesioned rats (0.26 + 0.07), but ketamine’s effect in increasing P2/P1 ratio was preserved. It is suggested that the septohippocampal cholinergic inputs participate in normal auditory gating in the hippocampus whereas the entire medial septum mediates ketamine-induced deficit of hippocampal auditory gating. In addition, the effectiveness of various antipsychotic drugs in antagonizing ketamine-induced impairment of auditory gating confirms the validity of this animal model of schizophrenia. (Supported by NSERC grant and CIHR grant 15685).

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Destruction of NPY receptor expressing neurons in the arcuate nucleus causes obesity and hyperphagia without increasing lateral hypothalamic orexigenic peptide gene expression

Li A-J, Dinh TT, Ritter S (2007) Destruction of NPY receptor expressing neurons in the arcuate nucleus causes obesity and hyperphagia without increasing lateral hypothalamic orexigenic peptide gene expression. Neuroscience 2007 Abstracts 524.20/BBB20. Society for Neuroscience, San Diego, CA.

Summary: NPY-SAP, a conjugate of neuropeptide Y (NPY) and the ribosomal inactivating toxin, saporin (SAP), is a compound that selectively lesions NPY receptor-expressing neurons. Previously we showed that injection of NPY-SAP into the hypothalamic arcuate nucleus (ARC) induces hyperphagia and obesity in rats. To further investigate the mechanisms responsible for NPY-SAP-induced obesity, we injected NPY-SAP or blank-saporin (B-SAP) control into the ARC and subsequently examined the expression of two orexigenic neuropeptide genes in the lateral hypothalamic area (LHA), which is densely innervated by ARC neurons. Our hypothesis was that loss of leptin-sensitive neurons in the ARC in the NPY-SAP injected rats would lead to increased expression of orexigenic neurons elsewhere in the hypothalamic feeding circuitry. Body weight gain and food intake were dramatically increased in the NPY-SAP group. In addition, expression of NPY and cocaine- and amphetamine-regulated transcript (CART) mRNA was significantly reduced in the ARC of obese rats, indicating a loss of NPY receptor-expressing NPY and CART neurons in this region. In contrast, NPY and CART gene expression in the dorsomedial hypothalamic nucleus was unchanged in NPY-SAP rats, indicating that the NPY-SAP-induced lesion was limited to the ARC. However, contrary to our hypothesis, expression of the orexigenic neuronpeptides, melanin-concentrating hormone (MCH) or prepro-orexin mRNA in LHA was not enhanced, but was slightly reduced in the NPY-SAP rats. These results indicate that an enhancement of MCH or orexin expression in the LHA is not necessary for the hyperphagia and obesity observed after NPY-SAP lesions in the ARC. Supported by PHS grant #DK 40498.

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Molecular basis of violent behavior: The role of NK1 receptors

Haller J, Toth M, Zelena D, Halasz J (2007) Molecular basis of violent behavior: The role of NK1 receptors. Neuroscience 2007 Abstracts 531.22/GGG24. Society for Neuroscience, San Diego, CA.

Summary: Background. Neurons expressing Neurokinin1 receptor (NK1 or Substance P receptor) are abundant in limbic areas crucial for different emotional behaviors. In recent years, NK1 receptor blockers were proposed for the treatment of anxiety and depression. Moreover, in two different laboratory models, NK1 receptor blockade was successfully used to decrease violent components of aggression related behaviors in Wistar rats (Biol. Psychiatry, 2007, in press). In the above study, the NK1 receptor blockade reduced the number of more violent hard bites, while the number of soft bites was unaltered. Aggressive encounters were accompanied by a marked activation of neurons expressing NK1 receptors in the medial amygdala and in the hypothalamic attack area, where the highest number and proportion of activated NK1 positive neurons were found. Aim / Methods. We evaluated the precise role of neurons expressing NK1 receptors in the hypothalamic attack area during resident/intruder test. These neurons were selectively eliminated by a Substance P conjugated saporin bilateral microinjection into the hypothalamic attack area. After a week recovery, lesioned and vehicle treated control residents were faced with a smaller untreated opponent in their home cages for 20 min. The brains of the residents were later removed to assess the site of injection and the extent of the lesion. Results. In lesioned Wistars, the bilateral microinjection resulted in a complete and selective disruption of NK1 positive neurons in the hypothalamic attack area. Compared to vehicle injected controls, the number of hard bites toward unfamiliar residents showed a marked decrease (almost a complete abolition) in the lesioned group. The latency of hard bites was significantly increased compared to vehicle injected controls. The number of bite attacks was also reduced, but this reduction was mainly secondary to the dramatic reduction in the number of hard bites. Conclusions. Our data show that hypothalamic neurons expressing NK1 receptors are involved in the control of aggressiveness, especially in the expression of violent attacks. These data confirm and support earlier results that NK1 antagonists - beyond anxiety and depression - may also be useful in the treatment of aggressiveness and violence.

Related Products: SP-SAP (Cat. #IT-07)

Vestibular activation stimulates cholinergic system in the hippocampus

Tai S, Ma J, Leung L (2007) Vestibular activation stimulates cholinergic system in the hippocampus. Neuroscience 2007 Abstracts 399.21/OO13. Society for Neuroscience, San Diego, CA.

Summary: The vestibular system has been suggested to participate in spatial navigation, a function ascribed to the hippocampus. We examined the mechanisms that induced hippocampal theta, a 4-10 Hz rhythm in the electroencephalogram (EEG), during vestibular activation in rats. Freely behaving rats were rotated at various speeds, on a vertical axis, in the light or dark. Hippocampal EEGs were recorded by implanted electrodes in hippocampal CA1, and analyzed by spectral analysis. A clear hippocampal theta rhythm was induced during immobility by rotations at different speeds (20-70 rpm). The rotation-induced theta was abolished, in light and dark settings, by muscarinic cholinergic receptor antagonist atropine sulfate (50 mg/kg i.p.) but not by atropine methyl nitrate (50 mg/kg i.p.), which did not pass the blood-brain barrier. Rotation-induced theta was attenuated in rats in which the cholinergic neurons in the medial septum (MS) were lesioned by 192 IgG-saporin (0.14 µg/0.4 µl infused bilaterally into the MS 10-20 days prior to the experiments). Cholinergic lesion in the MS was confirmed by a depletion of MS neurons that stained positively for choline acetyltransferase and an absence of acetylcholinesterase histochemical staining in the hippocampus. Bilateral lesion of the vestibular receptors (by 0.1 ml intratympanic injection of 300 mg/ml sodium arsanilate) also attenuated the rotation-induced theta rhythm. Vestibular lesion was confirmed by the contact righting test where lesioned rats will “walk” upside down on a Plexiglas sheet placed in contact with the soles of the feet while intact rats will right themselves immediately. In summary, an atropine-sensitive hippocampal theta is activated by septohippocampal cholinergic neurons which are in turn activated by vestibular stimulation. Vestibular-activated septohippocampal cholinergic activity is likely an important component of spatial navigation.

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

Effects of spatial cueing on visual discrimination performance of rats following loss of basal forebrain corticopetal cholinergic neurons

Burk JA, Altemose KE, Lowder MW (2007) Effects of spatial cueing on visual discrimination performance of rats following loss of basal forebrain corticopetal cholinergic neurons. Neuroscience 2007 Abstracts 424.4/HHH3. Society for Neuroscience, San Diego, CA.

Summary: Basal forebrain corticopetal cholinergic neurons are necessary for normal attentional performance. However, the exact attentional task parameters that are sufficient for inducing deficits following loss of basal forebrain corticopetal cholinergic neurons remain unclear. In Experiment 1, rats were trained to perform a spatially cued visual discrimination task (press a lever under an illuminated panel light) with explicit attentional demands removed. Animals then received infusions of the cholinergic immunotoxin, 192IgG-saporin, or saline into the basal forebrain. All animals were then tested in the same task trained before surgery and then task parameters, including the duration of visual signals and the inter-trial interval, were manipulated in order to tax attentional processing. Lesioned animals exhibited an initial increase in response latencies immediately following surgery but this deficit was not observed when task demands were increased. When the task was modified to remove spatial cueing, by presenting visual signals or no signal from a centrally-located panel light, lesioned animals exhibited an increase in lever press latencies compared to sham-lesioned animals. In Experiment 2, rats were trained in a visual discrimination task that, within each session, had blocks of trials with or without spatial cueing, using procedures similar to Experiment 1. After receiving intra-basalis infusions of 192IgG-saporin or saline, animals were tested for 12 sessions in the same task trained before surgery followed by one session in which the inter-trial interval was decreased. Lesioned animals did not exhibit deficits immediately following surgery, but did show elevated lever press latencies compared to sham-lesioned animals when the inter-trial interval was decreased. There was a trend for this lesioned-induced deficit to be more pronounced when spatial cues were not present. These experiments indicate that cortical acetylcholine is critical for maintaining normal visual discrimination performance when spatial cueing is not available. We speculate that, under conditions in which spatial cueing is unavailable, the lesion-induced increased lever press latencies reflect a disruption in recalling rules for an appropriate response.

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

192 IgG-saporin lesions of the cholinergic basal forebrain disrupt selective attention and trace conditioning but spare delay and long-delay conditioning

Butt AE, Amodeo D, Flesher M, Marsa K, Holt R, Lladones R, Olney R, Haynes J, Kinney-Hurd BL, Dach N (2007) 192 IgG-saporin lesions of the cholinergic basal forebrain disrupt selective attention and trace conditioning but spare delay and long-delay conditioning. Neuroscience 2007 Abstracts 424.9/HHH8. Society for Neuroscience, San Diego, CA.

Summary: Recent research suggests that Pavlovian trace conditioning, but not delay conditioning, is a form of declarative memory that requires attention, where both of these processes depend on specific brain systems. For example, past research has shown that although amnesiac humans with damage to the hippocampus (HPC) acquire a normal conditioned response (CR) in delay conditioning paradigms, where the conditioned stimulus (CS) and unconditioned stimulus (US) partly overlap, they fail to acquire the CR in trace conditioning paradigms, where the CS and US are separated in time. Others have shown that the anterior cingulate cortex (ACC) in rats and the medial prefrontal cortex (mPFC) in rabbits are similarly necessary for trace but not delay conditioning. The basal forebrain cholinergic system (BFCS) has projections to the mPFC, ACC, and HPC. Given that each of these regions is critical for trace but not delay conditioning, we hypothesized that lesions of the BFCS using 192 IgG-saporin (SAP) would selectively impair trace but not delay or long-delay appetitive conditioning in rats. Additionally, given evidence suggesting BFCS involvement in attention, it was hypothesized that the addition of varying levels of distraction during the trace conditioning task would cause progressively greater degrees of impairment in the BFCS lesion groups compared to controls. In contrast, neither the control groups nor the BFCS lesion groups were expected be negatively affected by the addition of a distracting stimulus in the delay and long-delay conditioning tasks. Rats received bilateral SAP lesions or sham lesions of the BFCS prior to conditioning with a white noise CS and sucrose pellet US in either a delay, long-delay, or 10 s trace conditioning paradigm. Separate groups of BFCS lesion and control rats were subjected to varying levels of visual distraction (flashing house light) in each paradigm; no distraction, low distraction (continuously blinking light), and high distraction (intermittent, unpredictable flashing light). Results supported our hypotheses, with the BFCS lesion groups showing normal delay and long-delay conditioning regardless of level of distraction. In contrast, trace conditioning was impaired in the BFCS lesion groups, with progressively greater degrees of impairment occurring with greater levels of distraction, and complete disruption of learning in the high distraction condition. Together, these experiments suggest that the BFCS is necessary for normal trace conditioning, and that the BFCS is critically involved in selective attention tasks where animals must discriminate relevant stimuli from distracting background stimuli.

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

Effects of systemic bicuculline on the formalin-induced nociceptive response in the lip and c-Fos expression in the SP-Saporin-treated rats

Masawaki A, Sugiyo S, Shimoda T, Sakai Y, Ohyamaguchi A, Uehashi D, Moritani M, Yoshida A, Niwa H, Takemura M (2007) Effects of systemic bicuculline on the formalin-induced nociceptive response in the lip and c-Fos expression in the SP-Saporin-treated rats. Neuroscience 2007 Abstracts 186.16/RR16. Society for Neuroscience, San Diego, CA.

Summary: This study examines the effect of systemic bicuculline (2 mg/kg, ip) on formalin-induced pain-related behavior in the lip (PRB; face scrubbing behavior) and c-Fos expression in the trigeminal nucleus caudalis (SpVc) 2hrs after formalin injection and 2-4 weeks after intra cisterna magna (i.c.m.) injection of substance P (SP) conjugated to neurotoxin, saporin (SP-Sap; 3 µM, 5 µl), blank-Sap- or saline-treatment. In SP-Sap-treated rats, the number of NK-1- immunoreactive (NK-1-IR) neurons in lamina I of the SpVc decreased compared with that of saline- or blank-Sap-treated rats. In SP-Sap-treated rats, PRB at phase 2 decreased compared with that of saline- or blank-Sap-treated rats. In SP-Sap-treated rats, the number of c-Fos-IR cells in the VcI/II decreased compared with that in the saline- or blank-Sap-treated rats. In saline- and blank-Sap- treated rats but not SP-Sap-treated rats, systemic bicuculline decreased the number of PRB at phase 2. These results indicate that i.c.m. injection of SP-Sap eliminates NK-1-bearing neurons in L1 of SpVc, and that NK-1-bearing neurons in the SpVc have pivotal role in formalin-induced PRB at phase 2 and c-Fos expression in the SpVc. The decremental effects of systemic bicuculline on the formalin-induced nociceptive responses at phase 2 and c-Fos expression in the VcI/II are secure in the presence of NK-1 receptor bearing neurons in the Vc.

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Developmental origins of ventral medullary NK1r neurons

Gray PA, Vandunk C (2007) Developmental origins of ventral medullary NK1r neurons. Neuroscience 2007 Abstracts 187.15/SS7. Society for Neuroscience, San Diego, CA.

Summary: Breathing is a fundamental neural behavior generated by neurons within the brainstem. In the adult rat, bilateral injection of substance P conjugated to saporin (SSP) in the preBötzinger Complex (preBötC) eliminates normal breathing. Unilateral injection produces sleep disordered breathing and eliminates the effects of excitatory amino acid injection on breathing and blood pressure. SSP injection in the RTN region, in contrast, blunts chemosensitivity. The extent these different effects are due to ablation of anatomically, functionally, or genetically distinct populations are unknown. Further, the extent NK1R expression identifies unique populations in neonatal mice is unknown. Transcription factors are fundamental to determining the properties of neurons and it has been proposed a “combinatorial code” of transcription factor expression defines each distinct functional population. From a genome-scale analysis of over 1000 transcription factors and transcriptional co-factors we identified several genes expressed in respiratory regions of the brainstem. Using immunohistochemistry, we analyzed gene expression patterns of NK1R expressing neurons of the ventral medulla in embryonic and neonatal mice. We find there are at least four genetically distinct, partially overlapping populations of NK1R expressing neurons in the ventral medulla. These differ in the transcription factors they express, their onset of NK1R expression, and their co-expression of peptides and g-protein coupled receptors. At the level of the preBötC, there are two populations of NK1R expressing neurons derived from distinct developmental progenitor populations. One population corresponds to the preBötC as defined in rats and co-expresses the peptide somatostatin. A second population is continuous with, and genetically identical to the subCVLM population important for respiratory-cardiovascular coordination. Both populations co-express the somatostatin 2a receptor (SST2aR). At the level of the RTN/pFRG, there are also two populations of NK1R expressing neurons that are derived from similar developmental precursors as the preBötC. Neither of these populations expresses SST2aR. Whether either of these populations corresponds to the RTN or pFRG is currently unknown. These data suggest both the preBötC and RTN/pFRG regions contain developmentally related NK1R expressing populations. Further, they identify the developmental origin of preBötC neurons known to be essential for normal breathing and provide a foundation for understanding the genetic origin of an important neural circuit.

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Noradrenergic denervation of dorsal medial prefrontal cortex (mPFC) modulates paraventricular hypothalamic responses to acute restraint stress

Radley JJ, Sawchenko PE (2007) Noradrenergic denervation of dorsal medial prefrontal cortex (mPFC) modulates paraventricular hypothalamic responses to acute restraint stress. Neuroscience 2007 Abstracts 198.4/ZZ13. Society for Neuroscience, San Diego, CA.

Summary: The dorsal mPFC is implicated in restricting the hypothalamo-pituitary-adrenal (HPA) axis response to acute emotional stress via its influence on neuroendocrine effector mechanisms represented in the paraventricular hypothalamic nucleus (PVH). The afferents that provide for mPFC engagement during stress may include ascending noradrenergic projections from the locus coeruleus (LC), which are known to be stress-responsive and capable of modulating attentional mechanisms and other aspects of mPFC function. Arguing against such a role for LC-mPFC projections is evidence that LC lesions attenuate PVH/HPA responses to acute emotional stress. Here we sought to clarify the role of the noradrenergic innervation of mPFC in acute stress-induced PVH activation. Rats received injections of the axonally transported catecholamine immunotoxin, anti-dopamine-β-hydroxylase (DBH)-saporin, centered in the prelimbic area, or sham injections of IgG-saporin or saline. 14 days later, rats were subjected to 30 min of restraint and perfused 2 h later. Immunohistochemical localization of the immediate-early gene product, Fos, was employed as an index of cellular activation in PVH. Cell counts revealed that acute stress reliably provoked marked increases in the number of Fos-labeled neurons in the PVH of all restrained groups relative to unstressed controls. Among stressed groups, anti-DBH-saporin lesions in the dorsal mPFC decreased activational responses in the PVH by 30%, relative to sham-lesioned animals, that were focused in the hypophysiotropic (dorsal medial parvocellular) subdivision. DBH immunostaining revealed a virtually complete noradrenergic denervation of the prelimbic area in immunotoxin-treated animals, while the ventral mPFC (infralimbic area) remained intact. Cortical fields dorsally and laterally adjoining the target area displayed a partial denervation, consistent with local collateralization of prelimbic-projecting LC neurons. While these data are consistent with a role for LC-mPFC projections in facilitating restraint-induced PVH engagement, it remains to be determined how this perspective may be reconciled with prior evidence indicating that mPFC serves normally to inhibit PVH/HPA responses to acute emotional stress.

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

Amygdalar neuropeptide Y (NPY) signaling modulates stress-induced reductions of food intake in Balb/cJ mice

Sparrow AW, Lowery EG, Thiele TE (2007) Amygdalar neuropeptide Y (NPY) signaling modulates stress-induced reductions of food intake in Balb/cJ mice. Neuroscience 2007 Abstracts 270.10/X24. Society for Neuroscience, San Diego, CA.

Summary: The existing literature suggests that NPY signaling in the amygdala modulates anxiety-like behaviors and ethanol consumption in rodents, but does not modulate food intake. On the other hand, NPY signaling within the hypothalamus controls food intake but does not influence anxiety-like behavior. Based on these observations, the current study tested the hypothesis that attenuation of NPY signaling within the amygdala would increase anxiety-like behavior and augment stress-induced increases of ethanol consumption while at the same time have no effect of feeding behavior. To address this hypothesis, male Balb/cJ were given bilateral injection (48 ng/5-min/side) into the central nucleus of the amygdala (CeA) of NPY conjugated to the neurotoxin saporin (NPY-SAP) or saporin alone (Blank-SAP). NPY-SAP is a ribosome inactivating neurotoxin that targets and kills cells expressing NPY receptors. After recovery, mice were first tested for anxiety-like behavior using the zero maze test. They were then given access to 8% (v/v) ethanol versus water in a two-bottle test. After ethanol intake stabilized, half the NPY-SAP and Blank-SAP mice were subjected to a 5-min forced swim stress sessions, once a day over 5-days. Ethanol, water and food consumption were measured for 4-weeks following the forced swim procedures. At the end of the experiment, ethanol was removed for two-weeks and all mice were given a 24-hour open-field locomotor activity test. The results showed that mice treated with NPY-SAP in the CeA spent significantly less time in the open portion of the zero maze reflecting elevated anxiety-like behavior. Contrary to predictions, neither neurotoxin treatment nor stress condition altered ethanol intake. Interestingly, NPY-SAP treated mice that experienced forced swim stress consumed significantly less food than non-stressed NPY-SAP treated mice and stress and non-stressed mice treated with the Blank-SAP. Reduced feeding by NPY-SAP stressed mice was not associated with reduced body weight, suggesting possible alterations of energy metabolism. Further, reduced feeding was not attributable to reductions of activity. This study provides novel evidence that amygdalar NPY signaling modulates feeding/energy balance in mice with a history of stress exposure.

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ERK1/2 regulates microglia-neuron signaling and pain by PGE2 following SCI

Hains BC, Zhao P, Waxman S (2007) ERK1/2 regulates microglia-neuron signaling and pain by PGE2 following SCI. Neuroscience 2007 Abstracts 287.16/LL14. Society for Neuroscience, San Diego, CA.

Summary: We recently showed that microglia become activated after experimental SCI and dynamically maintain hyperresponsiveness of spinal cord nociceptive neurons and pain-related behaviors. Mechanisms of signaling between microglia and neurons that help to maintain abnormal pain processing are unknown. In this study, adult male Sprague Dawley rats underwent T9 spinal cord contusion injury. Four weeks after injury when lumbar dorsal horn multireceptive neurons became hyperresponsive and when behavioral nociceptive thresholds to mechanical and thermal stimuli were decreased, we tested the hypothesis that prostaglandin E2 (PGE2) contributes to signaling between microglia and neurons. Immunohistochemical data showed specific localization of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), an upstream regulator of PGE2 release, to microglial cells and a neuronal localization of the PGE2 receptor E-prostanoid 2 (EP2). Enzyme immunoassay analysis showed that PGE2 release was dependent on microglial activation and ERK1/2 phosphorylation. Pharmacological antagonism of PGE2 release was achieved with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor PD98059 and the microglial inhibitor minocycline. Cyclooxygenase-2 expression in microglia was similarly reduced by MEK1/2 inhibition. PD98059 and EP2 receptor blockade with AH6809 resulted in a decrease in hyperresponsiveness of dorsal horn neurons and partial restoration of behavioral nociceptive thresholds. Selective targeting of dorsal horn microglia with the Mac-1-SAP immunotoxin resulted in reduced microglia staining, reduction in PGE2 levels, and reversed pain-related behaviors. On the basis of these observations, we propose a PGE2-dependent, ERK1/2-regulated microglia-neuron signaling pathway that mediates the microglial component of pain maintenance after injury to the spinal cord.

Related Products: Mac-1-SAP rat (Cat. #IT-33)

Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN) and central respiratory chemoreception in rats

Stornetta RL, Takakura AC, Moreira TS, Mulkey DP, Bayliss DA, Guyenet PG (2007) Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN) and central respiratory chemoreception in rats. Neuroscience 2007 Abstracts 230.9. Society for Neuroscience, San Diego, CA.

Summary: The RTN contains glutamatergic interneurons that are strongly activated by CO2 via acidification. These chemosensitive neurons are non-catecholaminergic and they express the transcription factor Phox2b. Although RTN chemoreceptors innervate selectively the brainstem regions that contain the respiratory rhythm and pattern generator (CPG), it is not yet clear whether these neurons drive inspiration or expiration, pump or airway muscles, autonomic circuits or all of the above. To determine whether RTN neurons drive inspiration, we examined whether their selective destruction modifies the CO2 sensitivity of the phrenic nerve discharge (PND) in anesthetized vagotomized rats. Using electrophysiological recordings in vivo and in slices, we found that the chemosensitive neurons of RTN express substance P receptors. We also found that these cells can be destroyed by local injection of a substance P agonist conjugated with saporin (SSP-SAP). The kill rate of RTN chemoreceptors was determined by counting the number of residual Phox2b-expressing non-catecholaminergic neurons present in this structure 15 days after toxin injection. Unilateral injection of 0.6 ng SSP-SAP destroyed 75% of the presumptive chemoreceptors on the injected side only. The lesion was selective because nearby neurons such as facial motoneurons, catecholaminergic and serotonergic cells were spared. SSP-SAP also spared the ventral respiratory column caudal to RTN except for a small amount of damage in the Bötzinger region closest to RTN. Unilateral lesion of the Phox2b-expressing neurons of RTN had no effect on PND and on respiratory chemoreception. However, in such rats, a single injection of the GABA-mimetic muscimol into the contralateral intact RTN instantly eliminated PND. After muscimol, PND did not usually reappear in the presence of hypercapnia up to 10% end-expiratory CO2. However, PND could typically be reactivated by strong stimulation of peripheral chemoreceptors which suggests that the respiratory oscillator had remained functional after muscimol. Unilateral injection of a lower dose of SSP-SAP (0.15 ng) had no effect on the Phox2b-expressing neurons of RTN. In such rats, unilateral injection of muscimol into the contralateral RTN had no detectable effect on PND and central chemoreception.

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Protection from dendritic atrophy with testosterone following partial motoneuron depletion: dose-dependence in males and efficacy in females

Coons KD, Wilson RE, Sengelaub DR (2007) Protection from dendritic atrophy with testosterone following partial motoneuron depletion: dose-dependence in males and efficacy in females. Neuroscience 2007 Abstracts 56.11/S5. Society for Neuroscience, San Diego, CA.

Summary: Partial depletion of motoneurons from the highly androgen-sensitive spinal nucleus of the bulbocavernosus (SNB), or the more typical somatic motoneuron population innervating the quadriceps muscles, induces dendritic atrophy in remaining motoneurons. Treatment with testosterone (T) is neuroprotective, and dendritic atrophy following partial motoneuron depletion is attenuated in both populations. In the present study, we examined the dose-dependency of T effects in male rats, as well as its potential efficacy in females. Motoneurons innervating the bulbocavernosus/levator ani (BC/LA) or vastus medialis muscles were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, saporin-injected rats were given T implants designed to produce plasma titers ranging from 0.75 to 5.0 ng/ml or left untreated. Four weeks later, motoneurons innervating the contralateral BC or the ipsilateral vastus lateralis muscles were labeled with cholera toxin-conjugated HRP, and dendritic arbors were reconstructed in 3 dimensions. Partial motoneuron depletion resulted in dendritic atrophy in remaining SNB and quadriceps motoneurons (40% and 36% of normal length, respectively). T treatment attenuated this atrophy in a dose-dependent manner, with maximum effectiveness at 2.0-2.5 ng/ml (the normal adult physiological level). This dosage of T resulted in SNB dendritic lengths that did not differ from those of intact control males. In contrast, although dendritic atrophy in quadriceps motoneurons was attenuated by the same dosage of T, resultant dendritic lengths were 60% of normal length, and did not improve further with higher levels of T. Neuroprotective effects of T treatment were also assessed in quadriceps motoneurons in female rats (adult female rats lack the SNB neuromuscular system). As described above, motoneurons innervating the vastus medialis muscles were selectively killed by saporin injection, and females were given T implants (resulting in plasma levels of 2.0-2.5 ng/ml) or left untreated. Four weeks later, motoneurons innervating the ipsilateral vastus lateralis muscles were labeled with cholera toxin-conjugated HRP, and dendritic arbors were reconstructed. As in males, partial motoneuron depletion in females resulted in dendritic atrophy (52% of normal length) in remaining quadriceps motoneurons, and this atrophy was attenuated (70% of normal length) with T treatment. Together, these findings suggest that the neuroprotective effects of T on dendrites are achieved with dosages within the normal physiological range, and furthermore can be observed in motoneurons of both male and female rats.

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Poster: Recognition of novel objects and their location in rats with selective cholinergic lesion of the medial septum

Cai L, Johnson DA (2007) Poster: Recognition of novel objects and their location in rats with selective cholinergic lesion of the medial septum. Neuroscience 2007 Abstracts 92.21/TT2. Society for Neuroscience, San Diego, CA.

Summary: The goal of this project was to determine whether cholinergic neurons projecting from the medial septum (MS) to the hippocampus play a role in novel object recognition or location. The specific aim was to determine whether lesion of cholinergic neurons in MS by the selective cholinergic neurotoxin 192IgG-saporin (SAP) would induce retrograde amnesia and/or anterograde amnesia for a novel object and/or its location. Male SD rats were tested in an object recognition paradigm. The time the rats spent examining old and novel objects was measured. Infusion of SAP into medial septum was performed 2 days after a one week pre-surgery training. Fourteen days after surgery post-surgery retention testing for retrograde object memory was carried out. Then 3 days later, a new acquisition training and retention testing for anterograde memory was started. One-way ANOVA and Fisher’s exact test were used for statistical analysis. There were no significant differences in the exploration ratios between the control group without surgery and the CSF surgical group. The mean exploration ratios for both groups demonstrated retention of memory for the novel object and its placement. SAP infusion into the MS failed to induce a deficit in retrograde amnesia 2 days after training, but did show a strong trend for anterograde amnesia for novel object recognition and a significant association with anterograde amnesia for object location. Conclusions were that cholinergic neurons of MS were not involved in retrograde object memory 2 days before the infusion of SAP and may or may not be necessary for anterograde object memory formation, but cholinergic neurons of the MS were involved in anterograde spatial memory formation for novel objects.

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

Dendritic atrophy following partial motoneuron depletion: time course of recovery and protection with testosterone

Ferguson AS, Sengelaub DR (2007) Dendritic atrophy following partial motoneuron depletion: time course of recovery and protection with testosterone. Neuroscience 2007 Abstracts 56.24/S18. Society for Neuroscience, San Diego, CA.

Summary: In male rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) project to the bulbocavernosus and levator ani (BC/LA) muscles, and both the motoneurons and their target muscles are highly androgen-sensitive. We have previously demonstrated that partial depletion of motoneurons from the SNB induces dendritic atrophy in remaining motoneurons, and that treatment with testosterone (T) is neuroprotective against this atrophy. In the present study, we assessed dendritic atrophy after partial motoneuron depletion in SNB motoneurons at a variety of time points, to determine its time course and pattern with and without T treatment. Motoneurons innervating the BC/LA muscles in gonadally intact males were selectively killed by intramuscular injection of cholera toxin-conjugated saporin. Simultaneously, saporin-injected males were given T implants (45mm) or left untreated. At 2, 4, 6, or 10 weeks after motoneuron depletion, motoneurons innervating the contralateral BC were labeled with cholera toxin-conjugated HRP, and dendritic arbors were reconstructed in 3 dimensions. As previously reported, partial motoneuron depletion resulted in dendritic atrophy in remaining SNB motoneurons. While motoneuron depletion occurs within 7 days after saporin injection, dendritic atrophy in remaining SNB motoneurons progresses linearly over several weeks, with a decrease of 32% present at 2 weeks after motoneuron depletion, and a decrease to 66% at 4 weeks. Evidence of recovery in dendritic lengths was observed at 6 weeks post depletion (only 43% decreased), and by 10 weeks SNB dendritic lengths had returned to those of normal, intact males. Treatment with T altered the pattern of dendritic atrophy. While initial dendritic atrophy was similar to that of untreated saporin-injected males (29% decreased at 2 weeks post motoneuron depletion), T treatment attenuated dendritic atrophy. Four weeks after motoneuron depletion, SNB dendritic lengths had not declined further in T-treated males (32% decreased), and were now 102% longer than those of untreated, saporin-injected males. These findings suggest that SNB dendrites undergo a protracted atrophy and subsequent recovery following partial motoneuron depletion, and that the neuroprotective effects of T attenuate the magnitude of the induced atrophy.

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