References

Datta S, Chatterjee K, Kline IV RH, Wiley RG (2008) Lumbar intrathecal CCK-saporin: anatomic and nociceptive effects. Neuroscience 2008 Abstracts 773.4/MM32. Society for Neuroscience, Washington, DC.

Summary: Lumbar intrathecal CCK (cholecystokinin) appears anti-opiate in nocifensive reflex testing and may be important in opiate-resistant neuropathic pain states suggesting a role for CCK receptor-expressing dorsal horn neurons in nociception. In the present study, we sought to determine if selective destruction of CCK receptor-expressing superficial dorsal horn neurons alters pain sensitivity or the analgesic potency of morphine using the targeted cytotoxic conjugate (CCK-sap) of CCK to saporin, a ribosome inactivating protein. 28 adult Sprague Dawley rats were injected via lumbar intrathecal catheter with CCK-sap in doses of 500 ng (n=2), 350 ng (n=3), 700 ng (n=3), 1000 ng (n=4), 1500 ng (n=4), or 3000 ng (n=4). Controls included PBS (n=4) or 1500 ng of plain, unconjugated saporin (n=4). 2 weeks later rats were sacrificed. Lumbar spinal cords were frozen sectioned at 40 µm. One-in-six series of transverse sections at L4-6 were immunostained for CCK. Two rats were injected with 1500 ng of CCK-sap followed by transcardiac aldehyde perfusion in 72 hours. L5 Dorsal root ganglia (DRG) sections were stained with cresyl violet and examined for signs of acute cytotoxicity (chromatolysis and karyohexis). 350 to 1500 ng of intrathecal CCK-sap were well tolerated with no obvious signs of any toxicity. 3000 ng of intrathecal saporin led to motor signs within 72 hours including increased muscle tone, leading to tonic hind limbs extension. Subsequently, twelve Long Evans female rats were tested before and after intrathecal injection of either PBS (n=8) or CCK-sap, 1500 ng (n=4) on: 1 – cold plate (15 °C); 2 – thermal preference shuttle box testing (15/45°C); 3 – hotplate at 44°C, 47°C and 52°C and 4 – thermal preference after morphine (0.5, 1 and 2.5 mg/kg s.c). Anatomical analysis revealed that 1500 ng of CCK-sap decreased CCK immunostaining in the L4-6 Dorsal horn. No acute cytotoxicity was seen in the DRG with1500 ng CCK-sap. Intrathecal CCK-sap was well tolerated at doses ≤1500 ng. CCK-sap produced increased hot side time and decreased crossovers in the thermal preference test. In contrast, CCK-sap decreased latency to first hindpaw lift and increased total responding on the 44 °C hotplate. CCK-sap rats also showed increased hot side time at 45° C at all morphine doses (0, 1 and 2.5 mg/kg s.c.) also with decreased crossovers. We interpret these observations to indicate that CCK-sap produced increased nocifensive reflex responding on the 44° C hotplate consistent with positive modulation of motor responsiveness, and CCK-sap reduced aversion to 45° C heat consistent with an analgesic effect that was additive with morphine.

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Nayate A, Moore SA, Weiss R, Taktakishvili OM, Lin LH, Talman WT (2009) Cardiac damage after lesions of the nucleus tractus solitarii. Am J Physiol Regul Integr Comp Physiol 296:R272-R279. doi: 10.1152/ajpregu.00080.2008

Summary: This work tested the hypothesis that nucleus tractus solitarii (NTS) lesions can lead to fatal cardiac arrhythmias and myocardial lesions. Rats received bilateral injections of 9.4 ng of SSP-SAP (Cat. #IT-11) into the dorsolateral and medial portions of the NTS. Lesioned animals displayed increased arterial blood pressure.

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Rapp KL, Watts AG (2008) Interactions between corticosterone and catecholaminergic afferents in the regulation of neuropeptide gene expression in neuroendocrine CRH neurons in the paraventricular nucleus of the hypothalamus. Neuroscience 2008 Abstracts 782.2/RR7. Society for Neuroscience, Washington, DC.

Summary: Neurons in the medial parvicellular part of the paraventricular nucleus of the hypothalamus (PVH) are responsible for neuroendocrine activation of corticotropes in the anterior hypophysis. While corticotropin-releasing hormone (CRH) is the primary peptide responsible for synthesis and release of adrenocorticotropin hormone (ACTH), vasopressin (AVP) is also effective in stimulating ACTH, which stimulates synthesis & secretion of corticosterone (CORT) from the adrenal cortex. This descending pathway, the HPA axis, is part of the stress axis, as its output of CORT facilitates adaptation to changes in energy. While AVP is synthesized in both parvicellular and magnocellular populations of the PVH, it is the AVP in the parvicellular PVH that colocalizes with CRH and increases after adrenalectomy (ADX). The underlying mechanisms contributing to the CORT regulation of Crh and Avp expression still remain elusive, particularly with regard to the role of neural afferents. A major afferent projection system to the PVH originates from hindbrain catecholaminergic (CA) neuron subpopulations. Using saporin-anti-dopamine beta hydroxylase (DSAP) immunotoxin conjugate, to specifically eliminate CA afferents has revealed the importance of CA projections to PVH for both increased Crh expression, and elevated levels of circulating ACTH & CORT following glycemic challenges. We utilized DSAP-mediated deafferentation, followed by ADX and CORT replacement, to determine the role of CA afferents in mediating effects of circulating CORT on Crh and Avp regulation. Male Sprague Dawley rats (~315g) received acute bilateral microinjections of DSAP stereotaxically delivered into the PVH. A control group received bilateral microinjections of saporin conjugated to a non-targeting mouse IgG (SAP). One week later, rats received ADX and timed-release CORT pellet implants (25, 50 or 100 mg). Seven days post-ADX, rats were killed, and radioimmunoassay of plasma from terminal blood samples revealed significantly higher CORT levels in DSAP- vs. SAP-treated rats in CORT replaced groups: 25 mg (p < 0.001), 50 mg (p < 0.01). In contrast, in situ hybridization revealed significantly increased CRH mRNA levels (p < 0.001) and AVP hnRNA levels (p < 0.02) in DSAP- vs. SAP-treated rats with comparable plasma CORT levels. These results suggest that loss of hindbrain CA afferents contributes to the ability of circulating CORT to regulate Crh and Avp expression. The data implicate synergistic interactions between CORT & PVH neural afferents that provide critical metabolic information from the periphery in the regulation of CRH neuroendocrine neurons. Supported by NINDS. (NS029728)

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Pedersen NP, Anaclet C, Vetrivelan R, Saper CB, Lu J (2008) The retroabducens region is necessary for rapid eye movement (REM) during REM sleep in the rat. Neuroscience 2008 Abstracts 784.15/RR66. Society for Neuroscience, Washington, DC.

Summary: REM sleep is characterized by REMs, atonia of the non-respiratory musculature, and active dreaming during which the electroencephalogram (EEG) is desynchronized in humans and shows increased theta activity in rodents. Surprisingly, the source of the actual REMs during REM sleep is not known, although Pompeiano and Morrison (1965) described the reduction or absence of phasic REM phenomena after electrolytic lesion of the medial and spinal vestibular nuclei in the cat. Using the neurotoxins ibotenic acid and saporin-conjugated anti-orexin B IgG, we systematically placed cell-specific lesions in brainstem candidate structures for the generation of REMs in rats equipped for chronic recording of EEG, electrooculogram, and electromyogram. Lesion of a ‘retroabducens’ area, located immediately caudal and extending ventrally from the abducens nucleus, although leaving the abducens nucleus intact, abolished REMs (as well as waking saccades), without affecting other aspects of REM sleep. Animals with retroabducens lesions showed maintenance of slow oscillations in eye position, characteristic of non-REM or slow wave sleep, throughout REM sleep. Lesions of the medial vestibular nucleus, nucleus prepositus hypoglossi and immediately rostral to abducens did not affect REMs. We hypothesize that the retroabducens area may be required for the generation of saccadic eye movements, similar to the paramedian pontine reticular formation as described in cats and monkeys. The retroabducens region appears to be critical for generating the REMs that characterize REM sleep, but most likely downstream from the REM sleep generator.

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Anaclet C, Pedersen NP, Lu J (2008) Medullary circuitry regulating trigeminal motor nucleus phasic activity during rapid eye movement sleep in the rat. Neuroscience 2008 Abstracts 784.16/RR67. Society for Neuroscience, Washington, DC.

Summary: Rapid Eye Movement (REM) sleep or paradoxical sleep is characterized by activation of the cortical and hippocampal EEG, atonia of postural muscles (neck and limbs), and phasic movements of cranial muscles (eyes, chin, ears and whiskers). We have previously established that glutamatergic neurons of the sublaterodorsal tegmentual nucleus (SLD) play a critical role in generating postural muscle atonia during REM sleep. It has been further proposed that the SLD produces REM motor atonia by stimulating spinal inhibitory neurons, which in turn inhibit spinal motor output neurons. It is not known however whether the SLD is also involved in the regulation of tonic and phasic events of cranial muscles during REM sleep (e.g., rapid eye movement, phasic masseter activation). Previous studies have shown that the supraolivary medulla (SOM, dorsal to the inferior olive) and parvocellular reticular (PCRt) nucleus in the medullary reticular formation project to relevant cranial motor nuclei, including: the trigeminal motor nucleus (Mo5), retroabducens region, facial nucleus (Mo7) and hypoglossal nucleus (Mo12). It is therefore possible that either the SOM or the PCRt (or both) may also be involved in regulating cranial muscle activity in REM sleep. To identify the cell groups responsible for REM phasic control of cranial motor nuclei, we examined masseter muscle EMG following cell-specific lesions (anti-orexin B IgG-saporin) of the SLD, SOM or PCRt. Following two weeks of surgical recovery, we recorded the EEG, EMG (neck and masseter muscles) and EOG continuously for two days. Control rats showed significant phasic activation of the masseter muscles, in particular during the second half of REM sleep episodes. This phasic bursting pattern was similar to eye movements during REM sleep. Neither SLD nor PCRt lesions altered the phasic activity of the masseter muscles during REM sleep, although, and as previously reported, SLD lesions did produce REM without atonia in postural muscles. By contrast, lesions in the SOM eliminated phasic activation of the masseter muscles during REM and produced myoclonic twitching of neck muscles. These results indicate that the SOM is involved in the induction of phasic REM activity of masseter muscles, likely via activation of Mo5, whereas SOM projections to the spinal cord are involved in suppression of myoclonic activity of postural muscles.

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Saenz CM, Borowski T, De Lacalle S (2008) Contrasting effects of estrogen on memory tasks in young female rats. Neuroscience 2008 Abstracts 794.17/UU7. Society for Neuroscience, Washington, DC.

Summary: Sleep deprivation may lead to behavioral alterations and it has been associated with a hyperalgesic state in human beings and animal models. The tricyclic antidepressant amitriptyline can be used as an analgesic drug in patients and in chronic pain animal models that are not improved with classical analgesics, such as spinal nerve injury induced model of peripheral neuropathy. The pain hypersensitivity following both paradoxical sleep deprivation (PSD) and peripheral nerve injury shares common spinal mechanisms, which involve at least the glutamate receptors and nitric oxide. In this way, we evaluated the effects of amitriptyline pretreatment in the thermal hyperalgesia observed in paradoxical sleep deprived rats. Amitriptyline (10 and 30 mg/Kg) or saline were administered i.p. during 11 days to male Wistar rats (n = 7/group, 250 – 350 g). In the last 3 or 4 days of treatment the animals were submitted to 72 or 96 hours of PSD, respectively, or remained in home cages, being subsequently evaluated for their thermal sensitivity on a hot plate test (52oC or 46oC), 1 or 24 hours after the last drug administration. In order to verify if the results observed in the highest withdrawal latencies were due to a reduction on the locomotor activity rather than an analgesic effect, the number of squares crossed in an open field arena during 5 minutes, subsequently to the hot plate test was counted. The results demonstrated that paw withdrawal latency response to 52oC was significantly lower in paradoxical sleep deprived rats than controls (-37%, p<0.05). This hyperalgesic effect was also detected in animals pre-treated with 10 mg/kg (-41%, p<0.05) or 30 mg/Kg (-53%, p<0.05) of amitriptyline. At the highest dose, both groups presented a higher withdrawal threshold when compared to their respective saline groups (+185%, p<0.05 and +112%, p<0.05; control and sleep deprived rats, respectively). However, in the open field test a decrease in the number of squares crossed in control animals was observed (-52%, p<0.05), but not in sleep deprived rats (-3%, p>0.05). When the animals were allowed to recover for 24h from sleep deprivation, the pre-treatment with amitriptyline (10 mg/Kg) was not able to prevent the hyperalgesic state (-60%, p<0.05). Even with lower thermal stimulus (46oC) and sleep deprivation period (72h), the difference between control and sleep deprived animals could still be detected (-40%, p<0.05), with no changes after an amitriptyline 10 mg/Kg pre-treatment (-43%, p<0.05). Overall, these findings highlight that thermal pain hypersensitivity induced by PSD was not prevented by amitriptyline pre-treatment, as observed in other models of inductive pain.

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

Savage ST, Oberg J, Pernold K, Mattsson A (2008) Enhanced sensitivity to phencyclidine following cortical cholinergic denervation. Neuroscience 2008 Abstracts 842.7/X2. Society for Neuroscience, Washington, DC.

Summary: Alterations in cholinergic signaling in the brain have been implicated as a contributing factor in the pathogenesis of schizophrenia. We have recently shown that cholinergic denervation of cortex cerebri by stereotaxic infusion of the immunotoxin 192 IgG-saporin in the nucleus basalis magnocellularis in adult rats, leads to an enhanced sensitivity to amphetamine. Thus, saporin lesioned rats show a marked increase in locomotor activity, as well as a potentiated dopamine release in nucleus accumbens when challenged with amphetamine. We hypothesize that the loss of cortical cholinergic input alters the activity of cortical glutamatergic neurons and in turn, their regulation of subcortical dopamine neurons. We have previously shown that this cortical cholinergic denervation leads to an increased locomotor response to the NMDA receptor antagonist phencyclidine (PCP), suggesting that disruption of cortical cholinergic activity can lead to disturbances of glutamatergic transmission. In current studies we are investigating attention and memory functions of rats with cholinergic denervation of neocortex using the novel object recognition task. Preliminary data from these investigations shows impairment in performance under PCP-challenge in saporin lesioned rats as compared to sham lesioned controls. These results indicate that cortical cholinergic deficits, in addition to leading to a dramatic potentiation of the locomotor response to PCP, can also lead to an enhanced sensitivity to PCP-induced cognitive impairments. Using pharmacological magnetic resonance imaging (MRI) we are investigating possible spatiotemporal differences in brain activation in rats with cortical cholinergic deficits following administration of PCP. Preliminary data have provided indications of a greater activation in cortical areas in saporin lesioned rats as compared to sham lesioned controls following PCP-challenge. Evaluations of possible alterations in social behavior following cortical cholinergic denervation are ongoing. Social interaction will be investigated under normal conditions, as well as after PCP-challenge. Preliminary results from these studies together with our previous results indicate that loss of cortical acetylcholine can lead to alterations in glutamatergic signaling. These observations are compatible with a possible role of cholinergic deficits in schizophrenia, and provide a possible link between different hypotheses of the disorder.

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

Khan AM, Rapp KL, Ponzio TA, Sanchez-Watts G, Watts AG (2008) Stimulus-, circuit- and intracellular-level determinants of MAP kinase and CREB activation in parvicellular hypothalamic paraventricular neurons. Neuroscience 2008 Abstracts 865.23/MM24. Society for Neuroscience, Washington, DC.

Summary: Systemic insulin or 2-deoxyglucose (2-DG) rapidly elevate phosphorylated MAP kinases (phospho-ERK1/2) and/or CRH hnRNA in PVHp neurons, and increase circulating ACTH and corticosterone. These neuroendocrine responses are likely driven by hindbrain-originating catecholaminergic (CA) neuron subpopulations, which richly innervate the PVHp and are activated by glycemic challenges. Supporting this, acute in vivo or in vitro PVH delivery of the prototypical catecholamine, norepinephrine (NE), recapitulates these responses (J Neurosci, 2007, 27:7344-7360). Here, we determined whether PVHp ERK/CREB phosphorylation responses require: 1) intact CA afferents, when triggered by three distinct in vivo challenges; and 2) upstream MEK kinase activity, when triggered by NE application in acute hypothalamic slices maintained in vitro. Methods. Rats given PVH microinjections of anti-dopamine-b-hydroxylase (DBH)-saporin antibody-toxin conjugate (DSAP) or mIgG-saporin control conjugate received either normal 0.9% saline vehicle or one of three systemic challenges: insulin (2 U/kg, i.v.); 2-DG (250 mg/kg, i.v.); or hypertonic saline (1.5 M, i.p.) and sacrificed 30 min later. Brains were processed for CRH mRNA/hnRNA hybridization, or DBH, phospho-ERK or phospho-CREB immunocytochemistry. Plasma was collected for hormone determinations at 0 and 30 min. In separate in vitro studies, acute hypothalamic slices received either no treatment (controls), or received bath-applied NE (100 mM) in the presence or absence of the MEK inhibitor, U0126 (10 mM), or the inactive MEK inhibitor analogue, U0124 (10 mM). Ten min later, slices were placed in fixative. Results. 1. Sham-lesioned animals: Relative to vehicle, all challenges elevated phospho-ERK1/2, phospho-CREB, and ACTH/corticosterone levels; and, except for insulin, also increased CRH hnRNA. 2. Lesioned animals: DSAP treatment selectively destroyed hindbrain-originating CA afferents. In insulin- and, to a lesser extent, 2-DG-treated animals, this loss was accompanied by markedly reduced PVH phospho-ERK1/2 and circulating ACTH/corticosterone. In contrast, these responses remained robust in CA-deafferented hypertonic saline-treated rats. Phospho-CREB levels were differentially reduced relative to phospho-ERK in lesioned rats. 3. Slices: NE-induced PVH elevations of phosphorylated ERK1/2 and CREB were reduced markedly by U0126, but not U0124, pre-treatment. Conclusions. PVHp phospho-ERK selectively couples to CA afferents during glycemic challenges and ERK/CREB recruitment appears to require MEK activity.

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Martin MM, Winter SS, Cheatwood JL, Carter LA, Jones JL, Weathered SL, Wagner SJ, Wallace DG (2008) Organization of food protection behavior is differentially influenced by 192 IgG-saporin lesions of either the medial septum or the nucleus basalis magnocellularis. Brain Res 1241:122-135. doi: 10.1016/j.brainres.2008.09.018

Summary: In this work the authors used a food-protection model to investigate the role of cholinergic neurons in the processing of information from internal and external sources. Rats received the following amounts of 192-IgG-SAP (Cat. #IT-01): 15 ng or 20 ng into the medial septum (MS), or 20 ng into the nucleus basalis magnocellularis (NB). While the NB lesions reduced the number of successful food protection behaviors, lesions in the MS disrupted the temporal organization of this behavior.

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Lecourtier L (2008) Involvement of the habenula in cognition through its regulatory role upon monoamine and acetylcholine transmissions. Neuroscience 2008 Abstracts 698.4. Society for Neuroscience, Washington, DC.

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