Annual ATS Poster of the Year Winners

Neuroscience 2019

Truckenbrod LM, Bumanglag AV, Chun E, Hernandez A, Federico QP, Maurer AP, Sloviter RS, Burke SN (2019) Targeted hippocampal GABA neuron ablation produces hippocampal sclerosis, epilepsy, and dissociable effects on the Morris water maze and object-place paired association tasks. Neuroscience 2019 Abstracts 158.03. Society for Neuroscience, Chicago, IL

An epileptogenic role for hippocampal GABAergic dysfunction has recently been reported (Chun et al., 2019). Specifically, selective ablation of hippocampal GABA neurons by Stable Substance P-saporin (SSP-saporin) conjugate caused dorsal hippocampal sclerosis and chronic epilepsy, without involving convulsive status epilepticus or widespread brain injury. The current study assessed cognitive function in chronically epileptic SSP-saporin-treated rats and their vehicle-injected controls ~8 months following injection. First, rats completed the Morris Water Maze test of spatial learning and memory (Morris et al., 1982). Animals then underwent testing with the object-place paired association (OPPA) task, which requires the hippocampus as well as functional connectivity between the hippocampus and cortical areas (Jo and Lee, 2010; Hernandez et al., 2017), and then a simple object discrimination task. Interestingly, both controls and rats with dorsal hippocampal sclerosis and chronic epilepsy were able to learn the location of the hidden platform in the Morris Water Maze task and could also acquire a simple pair-wise object discrimination. However, epileptic rats with dorsal hippocampal sclerosis were significantly impaired on the OPPA task, which requires animals to integrate spatial location memory with a correct object choice and is a more sensitive measure of cognitive dysfunction (Hernandez et al., 2015). These data indicate that, similar to humans with medial temporal lobe epilepsy, selective hippocampal sclerosis and epilepsy in this model do not result in global cognitive decline. Rather, cognitive functions that require functional connectivity between the hippocampus and cortical areas are selectively affected.

Featured Article in Targeting Trends: Bumanglag AV, Truckenbrod LM, Chun E, Hernandez AR, Federic QP, Maurer AP, Sloviter RS, Burke SN (2019) Targeted hippocampal GABA neuron ablation produces hippocampal sclerosis, epilepsy, and dissociable effects on the Morris Water Maze and Object-Place Paired Association tasks. Targeting Trends 20.

Neuroscience 2018

Abudukeyoumu N, Garcia-Munoz M, Nakano Y, Arbuthnott GW (2018) Impaired reach-to-grasp responses in mice depleted of striatal cholinergic interneurons. Neuroscience 2018 Abstracts 491.01 / MM13. Society for Neuroscience, San Diego, CA

Cholinergic interneurons (ChIs) are sparsely distributed within the striatum, a nucleus that plays important role in voluntary motor control, associated learning, procedural memory, action selection and planning and execution of movement. Sparsely distributed ChIs are 1-3% of all striatal neurons and the main source of striatal acetylcholine. Here we report the effect of depletion of ChIs in the dorsolateral striatum in a reach-to-grasp task. To selectively deplete ChIs, we used the saporin ribosome-inactivating-immunotoxin that targets choline acetyltransferase. C57BL/J male mice, 21 days old, received a stereotaxic unilateral infusion of the toxin (0.3µl/3min), and sham control group was injected with saline. Following one week postsurgery recovery, animals were food deprived for 12 h everyday and trained for 12 days at night during their active circadian cycle. The mean percentage ± SEM of successful performance in the reach-to-grasp task for the last 6 training sessions was 51.11 ± 4.09% (n = 25), 48.79 ± 7.7% (n = 9) and 26.28 ± 5.19% (n = 13) for intact control, sham control and ChIs-depleted mice, respectively. These results indicate that striatal depletion of ChIs impair reaching accuracy, whereas no significant differences were observed in control or sham operated mice. Moreover, a positive correlation between loss of ChIs and performance in the reach-to-grasp task was observed. Our results suggest that the participation of ChIs in striatal mediated motor learning impact on the function of interneurons and projection neurons of the whole striatal microcircuitry (Abudukeyoumu, N., Hernandez-Flores, T. et al. Eur. J. Neuroscience, in press).

Featured Article in Targeting Trends: Abudukeyoumu N, Garcia-Munoz M, Nakano Y, Arbuthnott GW (2018) Impaired reach-to-grasp responses in mice depleted of striatal cholinergic interneurons. Targeting Trends 19.

Neuroscience 2017

Falquetto B, Oliveira LM, Moreira TS, Takakura AC (2017) Role of orexinergic neurons in the chemosensory control of breathing in a Parkinson’s disease model. Neuroscience 2017 Abstracts 779.08 / HH1. Society for Neuroscience, Washington, DC

Parkinson´s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra compacta (SNpc). Non-motor symptoms such as neuropsychiatric, sleep and breathing disorders are also observed in PD. Previous study has already demonstrated that in 6-hydroxydopamine (6-OHDA)-model of PD there is a reduction in the number of phox2b neurons in the retrotrapezoid nucleus (RTN) and a decrease in the respiratory response to hypercapnia. Here, we tested the involvement of orexin cells from lateral hypothalamus/perifornical area (LH/PeF) on breathing in this model of PD. 6-OHDA (24 µg/µl) injections into the striatum reduced the number of catecholaminergic (40 days: 128 ± 10 and 60 days: 116 ± 13 vs. vehicle: 938 ± 15 neurons) and orexin-B-ir neurons (40 days: 310 ± 9 and 60 days: 258 ± 15 vs. vehicle: 412 ± 13 neurons). The injection of anti-Orexin-B saporin into the LH/PeF produces a further reduction in the number of orexinergic neurons in PD animals (79 ± 8 vs. control: 427 ± 14 neurons). The respiratory frequency (fR) at rest and in response to hypercapnia (7% CO2) was assessed 60 days after bilateral 6-OHDA or vehicle injections into the striatum and anti-Orexin-B saporin or IgG saporin into the LH/PeF during sleep and wakefulness in the dark and light phases of the diurnal cycle. Sixty days after 6-OHDA, we observed a reduction of fR at rest during sleep in the light phase only in PD animals (56 ± 2 vs. control: 66 ± 2 bpm). During the dark phase, there is a reduction in fR response to hypercapnia in PD animals with depletion of orexinergic neurons during wakefulness (119 ± 6 vs. control: 152 ± 3 bpm) and sleep (128 ± 7 vs. control: 147 ± 5 bpm). Our data suggest that orexinergic neurons are important to restore chemoreceptor function in a rat model of PD during sleep and wakefulness in rats.

Neuroscience 2016

Kelly SC, Nelson PT, Counts SE (2016) The locus coeruleus: a potential link between cerebrovascular and neuronal pathology in Alzheimer’s disease. Neuroscience 2016 Abstracts 786.11 / H7. Society for Neuroscience, San Diego, CA

Noradrenergic locus coeruleus (LC) neuron loss is a major feature of Alzheimer’s disease (AD). The LC is the primary source of norepinephrine (NE) in the forebrain, where it modulates attention and memory in vulnerable cognitive regions such as prefrontal cortex and hippocampus. Furthermore, LC-mediated NE signaling is thought to play a role in blood brain barrier maintenance and neurovascular coupling, suggesting that LC degeneration may impact the high comorbidity of cerebrovascular disease (CVD) and AD. However, the extent to which LC projection system degeneration occurs in the earliest stages of AD is not fully characterized to date. To address these issues, we analyzed LC tissue samples from University of Kentucky AD Center subjects who died with a premortem diagnosis of no cognitive impairment (NCI) and Braak stages 0-II at autopsy, NCI subjects with Braak stages III-V thought to be in a preclinical AD (PCAD) stage, and subjects with mild cognitive impairment (MCI) or mild AD (n = 5-6 cases/group). Paraffin-embedded pontine tissue blocks containing the LC were cut at 20µm, immunostained with tyrosine hydroxylase (TH, a marker for NE synthesis), and analyzed by stereology to estimate total LC neuron number (total number of neuromelanin-containing LC neurons) and the percentage of TH+ LC neurons. Preliminary analysis reveal a ~20% loss of both total and TH+ LC neurons in PCAD (p = 0.08), a ~30-35% loss of these neurons in MCI (p < 0.05), and a ~45-50% loss of total and TH+ neurons in AD (p < 0.01) compared to NCI. Studies were also performed to compare additional LC neuronal pathologies (phospho-tau, TDP-43, and 8dOHG) in the diagnostic groups. A substantial increase in 8dOHG and phospho-tau is observed in PCAD compared to NCI. The morphometric data will be correlated with postmortem neuropathologic and CVD variables (e.g., microinfarcts and cerebral amyloid angiopathy) to gauge the relationship between LC neurodegeneration and cerebral AD and vascular pathology. To model these relationships in vivo, we stereotactically lesioned LC projection neurons innervating the PFC, a major LC projection zone, in the TgF344-19 rat model of AD (6 months old) using the noradrenergic immunotoxin, dopamine-β-hydroxylase-saporin, or a control lesion (n = 8/group). Prior to sacrifice at 9 months, immunotoxin- and control-lesioned rats will be tested behaviorally on the Barnes maze task. Postmortem PFC will be analyzed for LC fiber innervation, NE and NE metabolite levels, CVD pathology and AD-like pathology. Taken together, these data will shed light on the multifactorial noradrenergic pathways contributing to neuronal and vascular pathologies during the onset of AD.

Featured Article in Targeting Trends: Kelly SC, Nelson PT, Counts SE (2017) The locus coeruleus: a potential link between cerebrovascular and neuronal pathology in Alzheimer’s disease. Targeting Trends 18.

Neuroscience 2015

Horner KA, Logan M, Murray RC (2015) Ablation of the patch compartment reduces cocaine-induced stereotypy. Neuroscience 2015 Abstracts 506.23/M12. Society for Neuroscience, Chicago IL

Repeated exposure to cocaine (COC) induces stereotypy, which is characterized as inflexible, repetitive behavior. Enhanced relative activation of the patch compartment of the striatum has been shown to positively correlate with the emergence of stereotypy following repeated COC treatment, suggesting that stereotypy may be related to preferential activation of this region. However, the specific contribution of the patch compartment to COC-induced stereotypy following repeated exposure is unknown. To elucidate the involvement of the patch compartment to the development of stereotypy in response to repeated COC exposure, we determined if destruction of this sub-region altered COC-induced behaviors. Animals were bilaterally infused in the striatum with the neurotoxin dermorphin-saporin (DERM-SAP; 17 ng/[[Unsupported Character – Symbol Font &#61549;]]l) to ablate the neurons of the patch compartment and allowed to recover for eight days. The animals were given daily injections of COC (25 mg/kg) or saline for one week, followed by a weeklong drug-free period. Animals were then given a challenge dose of COC, placed in activity chambers, observed for 2h and sacrificed. DERM-SAP pretreatment reduced the number of mu-labeled patches in the striatum. DERM-SAP pretreatment significantly reduced the intensity and spatial immobility of COC-induced stereotypy. In support of this observation, increased locomotor activity was seen in DERM-SAP pretreated, COC-treated animals. DERM-SAP pretreatment attenuated COC-induced c-Fos expression in the patch compartment, while enhancing COC-induced c-Fos expression in the matrix compartment. These data indicate that the patch compartment is necessary for repetitive behavior and suggests that alterations in activity in the patch vs matrix compartments may contribute to this phenomenon.

Featured Article in Targeting Trends: Horner KA, Murray R, Logan Merce MC (2016) Striatal patch compartment lesions reduce cocaine-induced repetitive behaviors. Targeting Trends 17(1).

Neuroscience 2014

Phillips K, Kucinski A, Albin R, Sarter M (2014) Impairments in gait, posture and complex movement control in rats modeling the multi-system, cholinergic-dopaminergic losses in PD. Neuroscience 2014 Abstracts 692.21. Society for Neuroscience, Washington, DC

In addition to striatal dopamine loss, degeneration of cholinergic neurons in the basal forebrain (BF) and the brainstem pedunculopontine nucleus (PPN) were documented in patients with Parkinson’s disease (PD). Loss of cholinergic projections to cortical, thalamic and midbrain regions have been associated with impairments in gait and postural control and a propensity for falls. We previously demonstrated that loss of cortical cholinergic inputs and the resulting impairments in attentional control ‘unmask’ gait and postural risk factors and thus yielded falls in rats with striatal dopamine loss (Kucinski et al., 2013). For this research we developed a new behavior task for the assessment of gait, postural control, and fall propensity (Michigan Complex Motor Control Task; MCMCT). Here, to determine the contributions of the PPN cholinergic projection system to complex movement control, we also lesioned the cholinergic pars compacta (posterior) division of the PPN by infusing anti-ChAT saporin-coupled immunotoxin. Rats received these lesions either in combination with BF cholinergic (192-IgG-saporin) or dorsomedial striatal dopamine loss (6-OHDA), or all three lesions together (“triples”). MCMCT performance by triples was characterized by more falls than in rats with just PPN lesions, PPN plus striatal dopamine loss, or rats with loss of both BF and PPN cholinergic neurons. High fall rates in triples persisted throughout the 20-day MCMCT testing sequence, indicating that daily practice did not improve the interactions between loss of attentional control and gait and postural deficits that underlie falls. Interestingly, combined loss of BF and PPN cholinergic neurons increased falls relative to controls and single lesions, suggesting that ascending cholinergic PPN loss sufficiently dysregulates striatal dopamine input for BF cholinergic cell loss to ‘unmask’ the impact of the former on striatal dysfunction. Finally, PPN cholinergic cell loss resulted in ballistic postural (recovery) movements and slip-triggered switches to asymmetrical gait. Such behavior was previously observed in rats after electrolytic lesions of the PPN region, considered a model of “Parkinsonian festination” (Cheng et al., 1981) and it may assist in maintaining balance by stabilizing the center of gravity. Collectively, our findings support the hypothesis that PPN cholinergic projections contribute to the mediation of gait symmetry and postural control, and when lesioned in combination with forebrain cholinergic and dopaminergic system, results in profound impairments in the control of complex movements. This research was supported by the Michael J. Fox Foundation.

Featured Article in Targeting Trends: Kucinski A (2015) Impairments in gait, posture and complex movement control in rats modeling the multi-system, cholinergic-dopaminergic losses in Parkinson’s Disease. Targeting Trends 16(1).

Neuroscience 2013

Khan D, Owens E, Zaben M, Dunnett SB, Gray WP (2013) CD4+ T lymphocytes interact with microglia to modulate hippocampal neurogenesis. Neuroscience 2013 Abstracts 699.04. Society for Neuroscience, San Diego, CA

Hippocampal neurogenesis occurs within the subgranular zone of the dentate gyrus and is important for learning and memory. Neurogenesis is impaired in patients with chronic temporal lobe epilepsy, an observation that may account for the learning and memory deficits that these patients commonly have. Emerging literature demonstrates that CD4+ T lymphocytes increase neurogenesis and enhance cognition; however, the exact mechanisms remain undetermined. Vasoactive Intestinal Peptide (VIP) receptors are expressed on T lymphocytes, microglia and hippocampal progenitor cells, hence this study was designed to investigate VIP’s role in mediating neuro-immune modulation. Hippocampal cultures (P7-10 Sprague Dawley rats) were generated and maintained for 3 days in vitro (DIV) and treated with 5% supernatant generated from C57/Bl6 mouse spleen using a CD4+ T lymphocyte isolation kit. BrdU and experimental conditions were added for the terminal 6 hours before fixation and then processed for BrdU and nestin. For phenotype analysis, experimental conditions were added at 3DIV and fixed at 6DIV to be processed for nestin and TuJ1. To deplete microglia, Mac-1-SAP was added at 2DIV for 24 hours before experimental conditions were added. 5% T lymphocytes supernatant increased proliferation of hippocampal nestin-expressing cells; an effect that is further enhanced under VIP treatment via VPAC1 receptor subtype. Examining potential cytokine mediators of this effect, PCR analysis showed 6-fold increase in IL-4 mRNA expression, and IL-4 antagonist abolished VIP proliferative effects. Using Mac-1-SAP to account for microglial involvement by depleting microglia, VIP proliferative effects were abolished. Our phenotyping studies also demonstrated an additional neurogenic effect under VIP treated supernatant compared to standard control conditions. Taken together, these results show VPAC1 receptor subtype expressed by CD4+ T lymphocytes mediates VIP proliferative effects on hippocampal cells via IL-4 cytokine release. Microglia mediates VIP proliferative effects. While we demonstrated before that VPAC2 mediates hippocampal progenitor cell survival, the findings of this study strongly implicate VPAC1 receptor as a neuro-immune mediator of hippocampal neurogenesis, and from a therapeutic perspective, shows that the effect can be pharmacologically manipulated.

Neuroscience 2012

Zushida K, Light K, Uchida S, Hevi C, Shumyatsky GP (2012) The GRP peptide and the GRPR-positive interneurons control fear acquisition and extinction. Neuroscience 2012 Abstracts 496.03. Society for Neuroscience, New Orleans, LA

The gastrin releasing peptide (GRP) is the marker of the neural circuits relaying fear-related conditioned stimulus (CS) information to the amygdala. The GRP is expressed by principal cells and the GRP-receptor (GRPR) is expressed by interneurons. The GRPR is expressed in the amygdala and hippocampus. To examine the role of the GRPR-positive interneurons in these two brain areas, we performed local injections of the bombesin-saporin (SAP)-toxin, which selectively eliminates the GRPR-expressing cells. The intra-BLA [lateral (LA) and basal nuclei (BA) of amygdala] injection of bombesin-SAP before fear conditioning significantly enhanced cued, but not contextual fear memory. We did not observe any significant effect of post-training intra-BLA injections of bombesin-SAP on fear memory recall. Also, there were no significant effects of bombesin-SAP on acquisition of contextual and cued fear memory in mice injected bombesin-SAP into LA, BA and central amygdala (CeA), respectively. Also, we examined cued fear memory in the GRP knockout mice and found significant enhancement in their cued fear memory. These results support the idea that GRPR-expressing interneurons play an inhibitory role in acquisition of fear memory and suggested inhibitory effect by the GRPR-expressing GABA interneurons on fear memory requires both LA and BA but not CeA.

Neuroscience 2011

Smith MA, Williams H, Krajewski SJ, Mcmullen NT, Rance NE (2011) Arcuate NK3 receptor-expressing KNDy neurons are essential for estrogen modulation of LH secretion and body weight in the female rat. Neuroscience 2011 Abstracts 712.07. Society for Neuroscience, Washington, DC

Arcuate kisspeptin, neurokinin B, and dynorphin (KNDy) neurons have been proposed to mediate estrogen negative feedback in multiple species. To determine if these neurons are essential for this feedback, we ablated KNDy neurons in the arcuate nucleus of female rats using [MePhe7]Neurokinin B, a selective NK3 receptor (NK3R) agonist, conjugated to Saporin ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). The specificity of this conjugate for NK3R-expressing KNDy neurons is described in a separate abstract (see Krajewski et al., Soc. Neurosci. Abstr. 2011). Twenty-four female rats were ovariectomized (OVX) and received bilateral arcuate microinjections of either [MePhe7]NKB-SAP or a scrambled peptide conjugated to Saporin (Blank-SAP controls). 20-23 days later, animals were implanted with s.c. silastic capsules containing 17β-estradiol (E2), and animals were sacrificed 11 days later. Blood samples for RIA of serum LH were taken at time of OVX and injections (baseline), 20-23 days post-OVX, and 11 days after E2-treatment. Because OVX and E2-treatment have well-described effects on body weight, animals were weighed at the same three time points. In control animals, OVX induced a 13-fold rise in serum LH, which returned to baseline 11 days after E2 replacement. In contrast, OVX had no effect on serum LH in [MePhe7]NKB-SAP animals. There was a small decrease in serum LH 11 days after E2 replacement in [MePhe7]NKB-SAP animals, but the magnitude of this change was much less than seen in control animals. Control animals also exhibited a 20% increase in body weight 20-23 days after OVX, followed by a significant reduction after E2 replacement. Surprisingly, neither OVX nor E2 replacement affected body weight in [MePhe7]NKB-SAP-treated animals. Rather, these animals showed a steady increase in body weight throughout the experiment, at rates comparable to intact female rats or OVX rats treated with E2 (Williams et al., Endocrinology, 2010). Immunohistochemical studies showed near-complete destruction of KNDy neurons in the arcuate nucleus of [MePhe7]NKB-SAP animals. There was preservation of proopiomelanocortin and neuropeptide Y immunoreactivity in the arcuate nucleus and GnRH-immunoreactive fibers in the median eminence. These data provide compelling evidence that arcuate KNDy neurons play an essential role in estrogen negative feedback on LH secretion as well as the estrogen modulation of body weight.

Krajewski SJ, Smith MA, Williams H, Ciofi P, Lai JY, Mcmullen NT, Rance NE (2011) Ablation of NK3 receptor-expressing KNDy neurons in the rat arcuate nucleus using [MePhe7]Neurokinin B-Saporin. Neuroscience 2011 Abstracts 712.09. Society for Neuroscience, Washington, DC

A subpopulation of neurons expressing kisspeptin, neurokinin B and dynorphin (KNDy neurons) has been shown to reside within the arcuate nucleus of many mammalian species. Although these peptides are critical for reproductive function, the precise role of the arcuate KNDy neurons is not fully understood. Here we describe a method to ablate KNDy neurons based on their co-expression of the Neurokinin 3 receptor (NK3R, Burke et al., J. Comp. Neurol, 2006). Saporin, a molecular neurotoxin, was conjugated to [MePhe7]Neurokinin B, a selective NK3R agonist ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). Binding studies revealed that the conjugation of saporin did not alter the affinity of [MePhe7]NKB to NK3R in rat cerebral cortex membranes. To investigate the specificity of this conjugate for ablation of NK3R neurons, stereotaxic surgery was used to bilaterally inject [MePhe7]NKB-SAP into the arcuate nucleus of female rats. Control rats were injected with saporin conjugated to a scrambled peptide (Blank-SAP, Advanced Targeting Systems). Rats were sacrificed 31-34 days later and the brains were processed for immunohistochemical studies. Nissl stained sections from [MePhe7]NKB-SAP-treated rats showed no signs of inflammation at the injection sites and no qualitative changes in cell density compared to Blank-SAP control rats. Immunohistochemistry revealed near-complete loss of NK3R-immunoreactive (ir) neurons throughout the arcuate nucleus of [MePhe7]NKB-SAP rats. When the injection site was dorsal to the arcuate nucleus, there was also variable loss of NK3R-ir cells in the lateral hypothalamus and zona incerta. In the arcuate nucleus, [MePhe7]NKB-SAP injections resulted in a 98% and 94% reduction in the number of kisspeptin and neurokinin B-ir neurons, respectively, compared to Blank-SAP controls. The number of dynorphin-ir neurons in the arcuate nucleus of [MePhe7]NKB-SAP-treated rats was reduced by 67%, a value consistent with the co-expression of NK3R on dynorphin neurons in our previous study (Burke et al., J. Comp. Neurol, 2006). In contrast, arcuate proopiomelanocortin and neuropeptide Y immunoreactivity were preserved in [MePhe7]NKB-SAP rats. Moreover, there was no difference in GnRH-ir fiber density in the median eminence between the two groups. These results document the utility of [MePhe7]NKB-SAP for selective ablation of NK3R-expressing KNDy neurons in rat hypothalamus. These rats were used to examine the role of KNDy neurons in the estrogen regulation of LH secretion and body weight in the female rat (see Smith et al., Soc. Neurosci. Abstr. 2011).

Featured Article in Targeting Trends: Rance NE, Mittelman-Smith MA, Krajewski-Hall SJ (2012) Use of a novel saporin conjugate (NK3-SAP) to study the function of neurokinin 3 receptor (NK3r)-expressing kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the rat arcuate nucleus. Targeting Trends 13(2).

Neuroscience 2010

Liu Y, Krencik R, Liu H, Ma L, Zhang X, Zhang S-C (2010) Functional cholinergic neurons from human embryonic stem cells. Neuroscience 2010 Abstracts 331.5/B19. Society for Neuroscience, San Diego, CA

Basal forebrain cholinergic neurons play a critical role in regulating memory and cognition. Degeneration or dysfunction of these neurons is associated with neurological conditions including Alzheimer’s disease and dementia. In this study, we aimed at generating cholinergic neurons from human embryonic stem cells (hESCs) for therapeutic development. hESCs were first differentiated to primitive neuroepithelial cells in a chemically defined medium. In the presence of sonic hedgehog, over 97% of the differentiated cells became Nkx2.1-expressing ventral forebrain progenitors. These ventral progenitors further differentiated to cholinergic neurons with basal forebrain characteristics by expressing ChAT, VAChAT, FoxG1, Nkx2.1, Islet1, ßIII-tubulin, MAP2, P75, Synapsin but not GABA, Glutamate, or Mash2. The hESC-generated cholinergic neurons were electrophysiologically active in vitro. Following transplantation into the hippocampus of mice, in which cholinergic neurons in the medial septum were destroyed by IgG-P75-saporin, the grafted human cells produced large cholinergic neurons. The animals transplanted with cholinergic neurons demonstrated an improvement in learning and memory deficit. These results indicate that the human stem cell-generated cholinergic neurons are functional, thus providing a new source for drug discovery and cell therapy for neurological disorders that affect cholinergic neurons.

Neuroscience 2009

Li A-J, Wang Q, Dinh TT, Ritter S (2009) Leptin-saporin injection into the arcuate nucleus lesions NPY/AGRP and POMC neurons and produces hyperphagia, obesity and changes in diurnal feeding patterns in rats. Neuroscience 2009 Abstracts 374.5/EE116. Society for Neuroscience, Chicago, IL

Leptin-saporin (Lep-SAP), a conjugate of leptin with a ribosomal inactivating toxin, saporin (Advanced Targeting Systems), is a novel toxin designed to destroy leptin receptor-expressing cells selectively in vitro. However, its lesioning properties in vivo are currently unknown. Here, we injected Lep-SAP into the arcuate nucleus (Arc), to examine its effects on feeding behavior and on leptin receptor-expressing NPY/AGRP and POMC neurons in this area. Immunohistochemical studies showed unilateral injection of Lep-SAP into the Arc dramatically reduced numbers of NPY-Y1- and α-MSH- positive neurons compared to the contralateral side injected with SAP control. Real-time PCR revealed only 11-21% of Agrp and Pomc expression remaining in the Arc after Lep-SAP injection into this region. Rats injected bilaterally with Lep-SAP were unresponsive to central leptin administration and showed dramatic increases in feeding, body weight and light-phase feeding, compared pre-injection baseline. Two weeks after injection, total daily feeding was increased by 75%, light phase feeding by 359% and dark phase feeding by 33%. Control SAP injections did not produce these changes. Clock gene expression in homogenates of whole hypothalamus and liver were quantified at ZT 5-7. Bmal1 expression in hypothalamus and liver of Lep-SAP rats was decreased, while hepatic Per1 expression was increased compared to control. Results demonstrate that Lep-SAP effectively lesions Arc leptin receptor-expressing NPY/AGRP and POMC neurons in vivo, and that rats with this lesion are hyperphagic and obese, possibly due to enhanced hunger drive, lack of responsiveness to leptin and/or changes in circadian control of feeding behavior.

Featured Article in Targeting Trends: Li AJ, Wang Q, Dinh TT, Ritter S (2010) Deletion of NPY/AGRP and POMC Neurons in the Arcuate Nucleus by Leptin-Saporin Produces Hyperphagia, Obesity and Changes in Diurnal Feeding Patterns in Rats. Targeting Trends 11(1).

Neuroscience 2008

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

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.

Featured Article in Targeting Trends: Khan AM, Rapp KL, Ponzio TA, Sanchez-Watts G, Watts AG (2009) Deletion of Catecholaminergic Neurons by Anti-DBH-Saporin Disrupts Hypothalamic MAP Kinase and CREB Activation. Targeting Trends 10(2).

Neuroscience 2007

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

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.

Featured Article in Targeting Trends: Pang KCH, Jiao X, Servatius RJ (2008) Role of medial septal GABAergic neurons in learning and extinction: Effects of the novel GABA immunotoxin GAT1-SAP. Targeting Trends 9(1).

Neuroscience 2006

Chauhan NB (2006) Cholinergic immunolesioning produced tangle-like inclusions in TgCRND8 brain. Neuroscience 2006 Abstracts 271.8. Society for Neuroscience, Atlanta, GA

Today’s Alzheimer’s disease (AD) research lacks a “complete” model that would represent both plaque and tangle pathology together with correlative memory deficits. Although currently developed transgenic model including APP/PS1/tau mutations do not “truly” represent AD because tangles observed in AD brain are independent of tau mutations. Subtly increased β-amyloid (Aβ) levels either due to familial mutations or sporadic causes, primarily targets pre-tangle cytopathology and degeneration of basal forebrain cholinergic neurons (BFCN) via deranged signaling of glygogen synthase kinase 3-beta (GSK3β)-, protein kinase A (PKA)-, and extracellular signal-regulated kinase (ERK2) of ERK-mitogen-activated protein kinase (MAPK) cascade, leading to reduced phosphorylation of cAMP responsive element binding protein (CREB) that results in synaptic and memory deficits much earlier than the emergence of classic AD-pathology. Thus, subtly elevated Aβ, together with BFCN deficits resulting from Aβ-induced deranged signaling, set up a vicious feedback loop to produce characteristic plaque- and tangle-pathology observed in AD. Based on these facts, we wished to test if selective lesioning of basal fore brain cholinergic neurons during the early stages of amyloid build-up will exacerbate tau phosphorylation and produce tangle-like inclusions in transgenic mice with APP mutations. We produced selective immunotoxic lesions of BFCN by injecting the BFCN-specific cholinergic immunotoxin, which is known to specifically target p75-expressing BFCN and spare p75-expressing cerebellar neurons (Mu-p75-Saporin, Advanced Targeting Systems, #IT-16), intracerebroventricularly (ICV) in TgCRND8 mice harboring Swedish (KM670/671NL) and Indiana (V717F) mutations. This model exhibited tangle-like inclusions, provoked already existing plaque pathology, and worsened already impaired behavioral deficits.

Featured Article in Targeting Trends: Chauhan N (2007) Cholinergic Immunolesioning Produced Tangle-like Inclusions in TgCRND8 Brain. Targeting Trends 8(1).

Neuroscience 2005

Zhang W, Gardell SE, Xie Y, Luo M, Rance NE, Vanderah TW, Porreca F, Lai J (2005) Pain facilitatory cells in the rostral ventromedial medulla coexpress opioid-μ receptors and cholecystokinin type 2 receptors. Neuroscience 2005 Abstracts 394.17. Society for Neuroscience, Washington, DC

Pain transmission can be modulated by descending input to the spinal dorsal horn from the rostral ventromedial medulla (RVM). RVM neurons that facilitate nociception are termed “ON-cells”, which are inhibited by mu-opioids, suggesting that they express opioid mu receptors (MOR). Focal application of cholecystokinin (CCK8(s)) into the RVM elicits acute thermal and tactile hypersensitivity and induces ON-cell activity. In situ hybridization using riboprobes for either rat MOR or rat cholecystokinin type-2 receptor (CCK-2) confirms the expression of these receptors in the RVM. Pretreatment with a toxin conjugate, CCK8(s)-saporin results in a significant loss of CCK-2 positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The pretreatment also significantly reduces the number of neurons labeled for MOR in the RVM, suggesting that MOR and CCK-2 may be co-localized in some RVM cells. Consistent with these data, similar pretreatment with the toxin conjugate, dermorphin-saporin, which selectively targets MOR expressing neurons, significantly reduces the number of MOR labeled cells in the RVM, blocks RVM CCK8(s) induced hyperalgesia and reduces the number of CCK-2 positive cells in the RVM. In situ hybridization using 35S-labeled CCK-2 riboprobes and Digoxigenin-labeled MOR riboprobes shows that over 80% of labeled RVM neurons co-express both MOR and CCK-2, ~15% express only CCK-2, and very few cells express only MOR. These findings represent the first direct demonstration of the phenotype of pain facilitatory neurons in the RVM. Together with previous studies showing that RVM CCK-2 antagonists reverse nerve injury-induced pain, this phenotype provides strong support for the view that endogenous CCK is a critical mediator of the descending pain facilitation, particularly in the maintenance of experimental neuropathic pain. Support Contributed By: NIDCR R01 DE016458

Featured Article in Targeting Trends: Lai J, Zhang W, Badghisi H, Hruby VJ, Porreca F (2006) The Biologically Active Cholecystokinin (26-33) peptide, [Tyr2-SO3]CCK-8, Retains High Affinity for CCK2 Receptors after Covalent Conjugation to Saporin. Targeting Trends 7(1).

Neuroscience 2004

Pearson MS, Woods M, Whiteside GT, Garrison AE, Pomonis JD, Walker K (2004) IB4-SAP reduces IB4 staining in the spinal cord and prevents axotomy induced sprouting of Aβ fibers. Neuroscience 2004 Abstracts 858.6. Society for Neuroscience, San Diego, CA

Peripheral nerve injury results in hyperalgesia and allodynia. It has been proposed that sprouting of myelinated touch responsive Aß-fibers into the innervation territory of pain sensitive C-fibers in the spinal cord contributes to these abnormal behaviors. The extent of sprouting has recently been challenged and it has been proposed that C-fibers rather than Aß-fibers are involved. We have investigated whether selectively ablating a population of small diameter nociceptors using isolectin B4 conjugated to saporin (IB4-SAP), reduces axotomy-induced sprouting. Male Sprague-Dawley rats received intraneural injections of either IB4-SAP or PBS (3 µl, 0.66 µg/µl) and two weeks later the sciatic nerve was axotomized at the mid-thigh level. Two weeks later, the sciatic nerve was injected with the retrograde tracer, cholera toxin-ß subunit (CTB) (2 µl, 2%) that selectively traces Aß-fibers. Three days post CTB the animals were perfused, the spinal cord harvested, sectioned and stained immunohistochemically for IB4 and CTB. IB4-SAP treatment resulted in a substantial reduction of IB4 staining in the spinal cord versus PBS injected controls. As previously described, axotomy resulted in considerable CTB immunostaining in laminae I, II and III compared to non-axotomized controls in which it was present only in laminae I and III. IB4-SAP treatment followed by axotomy resulted in a substantial reduction of CTB immunostaining in lamina II compared to PBS injected controls. These results suggest that intraneural IB4-SAP ablates a population of small diameter nociceptors and that axotomy induced CTB staining in lamina II is due to uptake of CTB by C-fibers.

Featured Article in Targeting Trends: Pearson M (2005) IB4-SAP Prevents Axotomy-Induced Sprouting of Aß Fibers. Targeting Trends 6(1).

Neuroscience 2003

McGaughy JA, Jindal M, Eichenbaum HB, Hasselmo ME (2003) Cholinergic deafferentation of the entorhinal cortex in rats impairs encoding of novel but not familiar stimuli in a delayed non-match to sample task (DNMS). Neuroscience 2003 Abstracts 425.4. Society for Neuroscience, New Orleans, LA

Muscarinic cholinergic receptor activation in entorhinal cortex (EC) activates intrinsic depolarizing membrane currents which cause self-sustained spiking activity in single neurons (Klink and Alonso, J. Neurophys. 77, 1997). This effect may underlie delay activity and match-dependent activity changes in delayed match to sample tasks (Fransen et al., J. Neurosci. 22, 2002) and could allow accurate maintenance of novel information without dependence on synaptic modification associated with previous exposure (familiarization). Consistent with this, research in human subjects suggest that the medial temporal lobes are specifically activated during working memory for novel but not familiar stimuli (Stern, et al. Hippocampus v. 11, 2001), and cholinergic deafferentation of the rhinal cortex in non-human primates has been shown to impair memory for trial-unique (novel) stimuli (Turchi et al., SFN abstracts v. 28). The current study tests the hypothesis that cholinergic deafferentation of the EC produces impairments in working memory for novel but not familiar stimuli. Prior to surgery rats were trained in an odor DNMS task with a brief delay. After reaching asymptotic performance, rats were infused with either 192-IgG-saporin (SAP) or its vehicle into the EC (0.01 µg/µl; 1.0 µl/injection; 6 infusions/hemisphere). Rats were not impaired at any delay when tested with familiar odors but showed significant, persistent impairments when tested with novel odors. An increase in task difficulty alone was insufficient to explain these effects. These data support the hypothesis that cholinergic afferents to the EC activate cellular mechanisms of sustained spiking activity necessary for maintenance of novel but not familiar stimuli in a working memory task. Support Contributed By: NIH MH61492, MH60013, DA16454.

Featured Article in Targeting Trends: McGaughy J (2004) Cholinergic Deafferentation of the Entorhinal Cortex Impairs Working Memory for Novel, but not Familiar Stimuli in a Delayed Non-Match to Sample (DNMS) Task. Targeting Trends 5(1).

Neuroscience 2002

Truitt WA, McKenna KE, Coolen LM (2002) Lesions Of Spinothalamic Neurons In Lumbar Spinal Cord Disrupt Ejaculatory Reflexes In Male Rats. Neuroscience 2002 Abstracts 69.2. Society for Neuroscience, Orlando, FL

Previously we tested the significance of a population of lumbar spinothalamic (LUST) cells for male sexual behavior in rats. Anatomically, LUST cells are positioned to relay ejaculation-related sensory signals from reproductive organs to the brain and express substance P receptors as well as several neuropeptides including galanin. Ablation of LUST neurons by the selective toxin SSP-saporin resulted in a complete disruption of ejaculatory behavior. These results suggested that LUST cells play a pivotal role in generation of ejaculatory behavior and may be part of a spinal ejaculation generator. To test this hypothesis, we investigated ejaculatory reflexes in male rats with LUST lesions, using the urethrogenital reflex model. SSP-saporin (4 ng/µl) was injected bilaterally into L3-L4 region in sexually experienced male Sprague Dawley rats. Ten days following surgery, animals were deeply anesthetized and spinal cords were transected at upper thoracic levels. Next, urethral stimulation was provided and muscle contractions were recorded in the bulbocavernous muscle (BCM). Following the experiment, animals were sacrificed and lesions were confirmed using immunostaining for galanin, a marker for LUST cells. In non-lesioned animals (n=5), urethral stimulation produced stereotypical reflex contraction of the BCM, and penile reflexes were observed. In contrast, in animals with complete lesions of LUST cells (n=5) the urogenital reflex was severely attenuated. These results indicate that LUST cells are involved in control of ejaculatory reflexes and are part of a spinal ejaculation generator. Supported by: NIH R01 MH60781(LMC)

Featured Article in Targeting Trends: Truitt W (2003) Deep Lumbar Neurons Control Ejaculation. Targeting Trends 4(1).

Neuroscience 2001

Blanco-Centurion CA, Salin-Pascual RJ, Gerashchenko D, Greco MA, Shiromani PJ (2001) Hypocretin B-Saporin Lesions Of The Brainstem Increase Rem Sleep At Night. Neuroscience 2001 Abstracts 410.9. Society for Neuroscience, San Diego, CA

Loss of hypocretin (Hcrt) neurons has been linked to narcolepsy. These neurons project widely throughout brain, but it is not known which projection to which target site produces what symptom of narcolepsy. We (Molec Brain Res, 88:176-182,2001) showed that Hcrt receptors are present in brainstem areas implicated in REM sleep. Since abnormal REM sleep triggering characterizes narcolepsy, we have used Hcrt-saporin, a toxin that selectively lesions Hcrt receptor bearing cells, to assess the effects of such lesions on sleep. In the present study, Sprague Dawley rats (n=21) were administered (under anesthesia) Hcrt-sap (100ng/1ul, vol=0.5 ul bilaterally) or saline to the locus subcoeruleus (LSC) or the medullary inhibitory area of Magoun and Rhines. Subsequently, continuous sleep recordings were made for 21 days. Sleep records were scored blind. In the medulla, Hcrt-sap (n=5) increased the length of REM sleep bouts (p<0.039), which produced a trend towards an increase in REM sleep at night. There were no significant changes in SWS or W. Lesions of the LSC (n=5) increased total sleep time at night (p<0.03) and produced a trend towards a REM sleep increase. Data from both target sites were combined and the Hcrt-sap lesioned rats (n=10 versus saline=11) had a significant increase in REM sleep (30%; p<0.015). At neither site, cataplectic attacks were evident. Our studies with Hcrt-sap indicate site-specific effects on sleep and EEG depending on which Hcrt-receptor bearing neurons are lesioned. All of the symptoms of narcolepsy are evident when the Hcrt-containing neurons are lost. Supported by: NS30140, AG09975, AG15853, MH55772, DVA Med Research.

Neuroscience 2000

Martin JL, Sloviter RS (2000) Focal Hippocampal Hyperexcitability After Focal Interneuron Ablation In The Rat By Substance P-Saporin. Neuroscience 2000 Abstracts 389.13. Society for Neuroscience, New Orleans, LA

Hyperexcitability after prolonged seizures or head trauma may result from interneuron malfunction or loss; but a causal relationship is in doubt because global insults produce widespread brain damage and other effects. We have therefore sought to destroy interneurons selectively using stable Substance P-saporin (SSPsap; ATS); a neurotoxin internalized by SP receptor (SPR)-expressing neurons. Improved immunofluorescent methods revealed that most GABA-; parvalbumin (PV)-; and somatostatin (SS)-positive (+) cells of all hippocampal regions (dentate gyrus and areas CA1-CA3) are SPR+; but that granule cells; mossy cells; and CA1-3 pyramidal cells are not. Intrahippocampal injections of SSPsap or vehicle were made under urethane anesthesia in 3 sites (20nl/site) of the dorsal hippocampus of 6 male Sprague-Dawley rats/group. After 5-90 days; rats were blindly evaluated in two sites for CA1 pyramidal cell and dentate granule cell responses to perforant path stimulation (PPS). SSPsap-treated rats exhibited relatively normal responses in some sites; but pathophysiology at other sites that was virtually identical to that seen after prolonged PPS or kainate (multiple population spikes and paired-pulse disinhibition in response to 0.1-2.0Hz perforant path (PP) stimuli). Abnormal responses were observed at the earliest time tested (5 days); and at 90 days. Anatomical analysis revealed selective loss of SPR+; PV+; SS+; and GABA+ neurons; and survival of principal cells and extrinsic afferents. Importantly; “epileptic” pathophysiology was observed exclusively in areas of interneuron loss. These data indicate that the pathophysiology produced by status epilepticus or head trauma can be replicated focally by selective interneuron loss alone; and provide the first direct evidence that highly focal interneuron loss per se is capable of replicating “epileptic” disinhibition and hyperexcitability. In addition; the pathophysiology is restricted to the region of the affected interneuron somata; suggesting a highly localized influence of inhibitory interneurons. Supported by: NIH grant NS18201.

Featured article in Targeting Trends: Sloviter R (2001) Immunolesioning Hippocampal Inhibitory Interneurons. Targeting Trends 2(4).

Neuroscience 1999

Schreihofer AM, Guyenet PG (1999) Abolition of cyanide-induced sympathoexcitation by selective lesion of bulbospinal catecholaminergic neurons. Neuroscience 1999 Abstracts 474.6. Society for Neuroscience, MIami, FL

The rostral ventrolateral medulla (RVLM) contains BS C1 adrenergic cells and non-CA neurons whose relative importance for the production of sympathetic vasomotor tone and cardiovascular reflexes remains unknown. In the present study we evaluate 2 sympathetic reflexes after selective lesions of BS CA neurons with the neurotoxin saporin-anti-dopamine beta hydroxylase (SAP-DBH). Rats received bilateral microinjections of SAP-DBH (42 ng/200nl/site) into the spinal cord centered at the intermediolateral cell column at T2 & T4 & T6 and were allowed to recover for 3-5 weeks. Arterial pressure (AP), heart rate (HR), and splanchnic nerve activity (SNA) were measured while the rats were chloralose-anesthetized, artificially ventilated, and paralyzed. Baseline AP and HR were comparable between control rats (n-7, 113 +/- 7 mmHg, 426 +/- 12 bpm) and lesioned rats (n=7, 123 +/- 4 mmHg, 448 +/- 7 bpm). In control rats stimulation of the carotid chemoreflex (100 micrograms/kg sodium cyanide, iv) produced a burst in SNA (266 +/- 26%) followed by inhibition (to 18 +/- 3% of base). In contrast, in the SAP-DBH-treated animals cyanide produced only an inhibition of SNA (to 33 +/- 7% of base). Mean AP responses mirrored the SNA responses. In contrast, the Bezold-Jarisch reflex was not diminished in the SAP-DBH-treated rats. Phenyl biguanide (5 micrograms/kg, iv) decreased AP (22 +/- 3 vs. 42 +/- 5 mmHg), SNA (85 +/- 4 vs. 67 +/- 6%), and HR (28 +/- 6 vs. 56 +/- 7 bpm) in control rats and lesioned rats respectively. Histological examination revealed that SAP-DBH depleted the vast majority of BS C1 cells (>70% of the rostral third of the C1 cell group), and >80% of the A5 cell group. These results indicate that sympathetic vasomotor tone persists and rats have a normal mean AP in the absence of most of BS C1 and A5 cells. Although the BS C1 cells may not be required for the Bezold-Jarisch reflex, BS CA cells are essential for the sympathoexcitatory response to cyanide. Work supported by NIH 28785.