sfn2007

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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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)

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.

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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.

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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.

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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)

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.

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

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

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

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

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

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ATS Poster of the Year Winner

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

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

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