NIH K99 Application (Non-funded, 2014)
Specific Aims. Fetal alcohol spectrum disorders (FASD) include a wide-range of abnormal neuroanatomical, neurochemical, and neurobehavioral outcomes resulting from the teratogenic effects of alcohol on the developing embryo or fetus [1]. One of the key aims of FASD research is the development of successful treatments to improve cognition in affected individuals. Utilizing an animal model of FASD, Thomas and colleagues have demonstrated significant cognitive improvement in alcohol-exposed rats following developmental choline supplementation [2-4]. Choline is a constituent of acetylcholine, but also acts as a full agonist on α7 nicotinic acetylcholine receptors [5]. Our preliminary data demonstrate significant alterations in nicotinic acetylcholine receptors in the hippocampus of alcohol-exposed rats (see Figure 1) [6]. Specifically, we show that alcohol exposure over postnatal days (PD) 4-9 in the rat, a period of brain development equivalent to the third trimester in humans, causes significant reductions in α7 nicotinic acetylcholine receptors in the hippocampus, an effect partially mitigated by choline supplementation. The cholinergic system acting on nicotinic acetylcholine receptors in the hippocampus significantly modulates synaptic plasticity and hippocampus-dependent behaviors [7, 8]. Alterations in hippocampus nicotinic acetylcholine receptors, especially reductions in α7 subunits in alcohol-exposed rats, may contribute to impaired induction of long-term potentiation (LTP) within the CA1 region of the hippocampus [9, 10] and deficits in hippocampus-mediated contextual fear conditioning [11-14]. In this proposal, we hypothesize that application of the selective α7 nicotinic acetylcholine receptor agonist DMXB will facilitate induction of CA1 LTP and significantly improve contextual fear conditioning in rats exposed to a high binge-like dose of alcohol over postnatal days (PD) 4-9 (a model of late gestational alcohol exposure). Drugs that target the α7 nicotinic acetylcholine receptor have been utilized to improve cognitive outcomes in a number of disorders associated with cholinergic dysfunction, such as Alzheimer’s disease, Parkinson’s disease, schizophrenia, and ADHD [15, 16]. The results from these studies will highlight a potential therapeutic target to improve cognitive outcomes in FASD.
Aim 1: Determine the role of α7 nicotinic acetylcholine receptors in the induction of LTP in the hippocampus in a rat model of FASD.
The induction of LTP at the Schaffer collateral (SC)-CA1 pathway is a well-established cellular model of synaptic plasticity, learning, and memory. Timing-dependent potentiation of the SC-CA1 synapse is mediated by α7 nicotinic acetylcholine receptors, blocked by the α7 antagonist MLA, and is absent in α7 KO mice [17, 18]. We hypothesize that reductions in synaptic efficacy [19] and impaired induction of CA1 LTP [9, 10] in rats exposed to alcohol during the neonatal period arise, in part, from reduction of α7 nicotinic receptors in the hippocampus. The goal of Aim 1 is to examine timing-dependent potentiation of the SC-CA1 synapse in vitro in alcohol-exposed and control rats. We expect alcohol-exposed rats to show impaired induction of LTP under basal conditions. We hypothesize that application of DMXB, a selective α7 agonist, will facilitate the induction of CA1 LTP in control and alcohol-exposed rats in a dose-dependent manner. We further hypothesize a rightward shift in the bell-shaped dose-response curve in alcohol-exposed rats – i.e., the peak facilitation will occur with a higher concentration of DMXB compared to controls. Co-administration of the α4β2 antagonist DHβE or the α7 antagonist MLA will demonstrate that DMXB facilitates LTP via α7 nicotinic receptors. This aim will determine if pharmacologically targeting the α7 nicotinic receptor can mitigate LTP induction-deficits in the hippocampus in alcohol-exposed rats.
Aim 2: Determine the role of hippocampal α7 nicotinic acetylcholine receptors in contextual fear conditioning in a rat model of FASD.
The α7 nicotinic receptor mediates the protein synthesis-dependent phase of CA1 LTP [20]. Behaviorally, blockade of α7 nicotinic receptors in the dorsal hippocampus following, but not prior to fear acquisition impairs context conditioning [21, 22], further suggesting a roll for α7 nicotinic receptors in long-term memory formation. Rats exposed to alcohol over the neonatal period show significant impairments in contextual fear conditioning [11-14], but control-level measures of short-term retention (e.g., context acquisition and/or post-shock freezing; [14, 23]. We hypothesize that reductions in α7 nicotinic receptors in the dorsal hippocampus mediate, in part, the contextual conditioning impairments in alcohol-exposed rats. The goal of Aim 2 is examine the role of dorsal hippocampus α7 nicotinic receptors in the consolidation of contextual fear memories in alcohol-exposed and control rats. Rats will undergo standard delay fear conditioning starting on PD 30. One minute following acquisition (PD 30), we will infuse DMXB or MLA into the dorsal hippocampus. We do expect impaired context conditioning in alcohol-exposed rats treated with saline relative to controls. We hypothesize that DMXB will facilitate contextual conditioning in both control and alcohol-exposed rats following a dose-response curve, with maximal facilitation occurring at a higher-dose in alcohol-exposed rats. Application of DHβE will not affect conditioning in either group. However, MLA will disrupt conditioning in both groups. This aim will determine if pharmacologically targeting the α7 nicotinic receptor can mitigate contextual fear conditioning impairments in alcohol-exposed rats.
CANDIDATE’S BACKGROUND
I have studied the learning and memory impairments resulting from developmental alcohol exposure for the past seven years. My focus is on brain-behavior relationships and I have utilized behavioral, anatomical, and molecular strategies to better understand the neural abnormalities underlying cognitive impairments in models of developmental disorders [e.g., 24, 25], with a particular emphasis on animal models of Fetal Alcohol Spectrum Disorders (FASD) [6, 11-14, 26]. A secondary focus of my work examines the ontogeny of learning and memory [27, 28]. My current work examines potential treatments for cognitive impairments in FASD. My findings that developmental alcohol exposure significantly alters nicotinic receptor densities in the hippocampus [6] form the basis of this K99 application to examine nicotinic receptor function in a rat model of FASD. An examination of the cholinergic system could answer significant questions concerning the neural basis of cognitive impairment in FASD and facilitate the development of effective treatments that target this system. Training in cellular physiology techniques will supplement and extend my training in behavioral, anatomical, and molecular techniques.
Together, I can apply these techniques to answer the fundamental questions that drive my research.
My research experience in behavioral neuroscience began during my undergraduate studies at California State University, Sacramento. Under the mentorship of Dr. Jeffrey Calton, I used in vivo single cell recording methods to examine how cholinergic and serotonergic drugs modulate the firing of head direction cells in the dorsal thalamus [29]. My graduate training in behavioral neuroscience occurred under the mentorship of Dr. Mark E. Stanton at the University of Delaware. Under Dr. Stanton, my focus was examining how the timing and dose of alcohol during development affects subsequent hippocampus function in a rat model of FASD. My National Research Service Award (F31; NIAAA) supported my dissertation research. These studies addressed a number of key questions concerning developmental alcohol exposure and hippocampus function. I combined contextual fear conditioning and experience-dependent induction of immediate early genes in the hippocampus to explore brain-behavior relationships in FASD. I received training on fear conditioning paradigms from Dr. Jeffrey Rosen and training in molecular and anatomical techniques (e.g., immunohistochemistry and unbiased stereology) from Dr. Anna Klintsova. These techniques allowed me to examine both behavioral and functional aspects of hippocampus-mediated behaviors in rats developmentally exposed to alcohol [11, 12]. These studies demonstrated relationships between the amount and timing of developmental alcohol exposure and subsequent contextual learning [12, 13]. Further, I report that the contextual learning impairments in alcohol-exposed rats primarily result from the action of alcohol on the developing hippocampus during a limited neonatal period, and that these impairments likely reflect contextual learning deficits related to CA1 pyramidal cell number/activation [12]. In addition to highlighting important variables affecting hippocampus vulnerability to alcohol, my studies also highlighted effective ways to assess brain-behavior relationships by combining behavioral and molecular techniques. My graduate studies resulted in nine (of ten) peer-review publications and were recognized by the Fetal Alcohol Spectrum Disorders Study Group, who awarded me the 2012 Merit Award in honor of my scientific contributions to the study of FASD.
Following graduate school, I was awarded the Kaplan Postdoctoral Fellowship in Developmental Issues (San Diego State University; SDSU) to study the effects of supplemental choline on brain and behavior in a rat model of FASD under the mentorship of Dr. Jennifer Thomas (Center for Behavioral Teratology [CBT], SDSU). My postdoctoral studies are a direct extension of my doctoral work where I utilize my behavioral, anatomical, and immunohistochemical background to examine potential treatments to improve cognitive outcomes in FASD. Dr. Thomas’s work has focused primarily on cognitive treatments for FASD [2-4, 30-37]. For example, rats exposed to alcohol developmentally and subsequently administered choline show marked improvements in a number of hippocampus-mediated behaviors. My postdoctoral studies have examined the neural mechanisms that underlie these improvements, with a particular emphasis on the cholinergic systems. My postdoctoral studies aim to understand the impact of developmental alcohol exposure on both prefrontal and hippocampus development and how the timing of choline supplementation affects both neural and behavioral outcomes. My behavioral data demonstrate impaired fear extinction in alcohol-exposed rats. Choline is able to mitigate some effect only in adults supplemented during juvenile development, suggesting that choline may be affecting hippocampal and prefrontal function. I next examined muscarinic and nicotinic receptor densities in the hippocampus and prefrontal cortex through quantitative autoradiography. My findings demonstrate that developmental alcohol exposure significantly alters acetylcholine receptors, that the alterations are receptor sub-type and brain region specific, and that choline can mitigate some, but not all of these effects [6]. I am currently writing up these results for publication.
CAREER GOALS AND OBJECTIVES
My postdoctoral studies have identified a potential target for improving cognitive outcomes in FASD. Rats exposed to alcohol over the neonatal period, a period of brain development in the rat equivalent to the third trimester in humans, show significant reductions in α7 nicotinic acetylcholine receptor (nAChR) densities in the hippocampus. The α7 nAChR is a significant modulator of both hippocampus function and hippocampus-mediated behavior. Reductions in this receptor may underlie the impairments in synaptic plasticity and cognition that are present in animal models of FASD. Further, pharmacologically targeting this receptor with selective agonists may prove efficacious at mitigating cellular and behavioral impairments in FASD, as targeting this receptor improves outcomes in other disorders of cholinergic function (e.g., Alzheimer’s disease, schizophrenia, and ADHD). This important preclinical work may aid in the development of pharmacological treatments to improve cognitive outcomes in individuals with FASD.
I approached Dr. Marisa Roberto at The Scripps Research Institute (TSRI) to receive training in in vitro cell physiology techniques [38-40]. Cell physiology techniques will complement and extend the behavioral and molecular methods that I currently employ. Specifically, cell physiology techniques will allow me to address how pharmacologically targeting α7 nAChR modulates hippocampus function in a cellular model of synaptic plasticity, learning, and memory. This proposal will examine how modulation of the cholinergic system (by targeting specific acetylcholine receptor subtypes) affects both synaptic plasticity within the hippocampus (K99 phase) and behaviors that rely on such plasticity (R00 phase). The results of these studies will provide insights into the neural basis of cognitive impairment in FASD, may form a foundation for the development of new FASD treatments, and provide necessary training in electrophysiology techniques that will allow me to continue to address questions of brain-behavior relationships in an animal model of FASD. The results of the studies outlined in the proposal will provide valuable information concerning the relationship between cholinergic function, hippocampal synaptic plasticity, and behavioral outcomes in alcohol-exposed rats. Future grant applications will augment these data by examining the impact of developmental alcohol exposure across the life span. An understanding of how alcohol exposure during early development affects age-related cognitive decline and overall health is an important and little-explored topic with significant public health implications.
I will also greatly benefit from additional training in lab/personnel management, effective communication, writing, and career development form Drs. Roberto and Thomas as I transition to the independent phase of my research career. I will also receive additional consultation from Dr. Donna Gruol, an expert in alcohol-exposure and hippocampus plasticity [41-43]. During the second year of the mentoring phase, I will apply for faculty positions in behavioral neuroscience. My goal is to obtain an assistant professorship at a major research university with a core alcohol-research program. SDSU has provided a guarantee for a research faculty position following successful completion of the first two years of this award. The funding provided during the independent phase of the award will allow me to set up an independent lab and continue the studies outlined in Aim 2.
I am particularly interested in the effects that developmental alcohol exposure has on 1) specific hippocampus-mediated learning processes, 2) the cellular and molecular substrates that support such processes, 3) the effects that potential FASD treatments have on brain and behavior, and 4) the effects that early developmental insults have across the life span. My goal is to strengthen my skills and productivity as a researcher, and to develop the independence necessary for a successful career at an academic research institution. The current proposal will provide the necessary skills to make me a unique and productive researcher in the future. Ultimately, a greater understanding of the neural basis of cognitive impairment in FASD will significantly contribute to the development of effective treatments to improve cognitive outcomes in those affected by FASD.
CAREER DEVELOPMENT/TRAINING ACTIVITIES DURING THE AWARD PERIOD
Ongoing Training Activities:
- Quarterly meetings with Drs. Roberto, Thomas, Gruol to discuss training and research progress.
- Weekly lab meetings (Dr. Roberto)
- Monthly CBT Meetings (Dr. Thomas)
- Weekly lab meetings (Dr. Thomas)
- Readings of relevant research in FASD research, hippocampus cell physiology, cholinergic systems, and fear conditioning
- Attendance and presentations at scientific conferences (Fetal Alcohol Spectrum Disorders Study Group, Research Society on Alcoholism, Society for Neuroscience)
- Responsible conduct of research training activities: IACUC training, discussions, and related readings
Activities under the Award: K99 (2014-16)
Research Activities (75%)
- Complete Aim 1 of the current proposal, including data collection and analysis
- Write and submit associated manuscripts from Aim 1
- Presentation at RSA, FASDG, SfN
Education/Training Activities (20%)
- Continue ‘ongoing training activities’ as described previously
- Take courses: TSRI Kellogg School of Science and Technology course Ethics in Science, as well as courses relevant to behavioral neuroscience and molecular pharmacology (Structural Biology in Cell Signaling and Drug Discovery, Intracellular Signal Transduction)
- Bi-weekly one-on-one meetings with Dr. Roberto during the electrophysiological training and initiation of experiments in Specific Aim #1
- Weekly TSRI departmental seminars with all PIs, staff scientists, and pre- and postdoctoral fellows
- Bi-weekly seminars hosted by outside top-tier neuroscience and addictions experts hosted by CNAD and Neuroscience Affinity groups
- Receive training in in vitro electrophysiology techniques (Drs. Roberto and Gruol)
- Detailed training in electrophysiological data management and analysis (Drs. Roberto and Gruol)
Career Development/Job Market Preparation (5%)
- TSRI Career Services Office career workshops on laboratory management, time management, effective communication in the research lab, grantsmanship and funding, effective research manuscript preparation and approaches for reviewing grants and research manuscripts
- Year 2 of K99: Apply and interview for tenure-track research positions at institutions with strong record of alcohol research
- Practice and presentation of job talks at CBT and TSRI meetings and relevant seminars
- Consultations with Dr. Thomas and Dr. Roberto regarding career goals
- Participation in local career development activities
Activities under the Award: R00 (2016-19)
Research (at least 75%)
- Completion of Aim 2 of the current proposal, including data collection/analysis
- Manuscript preparation of associated manuscripts
- Presentation of findings at relevant scientific meetings including RSA, FASDG, SfN
- Grant Preparation (R01)
Continued Career Development/Training Activities
- Continued education in responsible conduct of research
- Consultations with senior scientists regarding career progress
- Participation in professional development conferences/seminars
BACKGROUND AND SIGNIFICANCE
Alcohol is a known teratogen that can cause significant damage to the developing embryo and fetus [44]. Fetal Alcohol Syndrome (FAS) refers to a pattern of pre- and postnatal growth deficiencies, facial dysmorphology, and central nervous system (CNS) dysfunction in offspring exposed to alcohol during gestational development [45-47]. Research over the previous 40 years has revealed that FAS is only one of a spectrum of possible adverse outcomes resulting from developmental alcohol exposure. Fetal alcohol spectrum disorders (FASD) refer to the range of neurobehavioral and structural abnormalities resulting from the actions of alcohol on the developing embryo and fetus [44, 48]. The prevalence of FAS is estimated to affect 2-7 per 1000 live births [49], with significantly higher rates of FASD reported: FASD affect 2-5% of live births in the US [49] and as many as 20.9% of live births in South Africa [50]. FASD substantially affect the lives of those affected and their families [51] and are associated with significant costs to society. The life-time costs of caring for an individual with an FASD are estimated to be around one million dollars [52]. Altogether, FASD are associated with billions of dollars in annual economic costs [44, 52]. The impact to the individuals affected by FASD, their families, and society make prenatal alcohol exposure a significant public health concern [1, 52].
The most detrimental effects of developmental alcohol exposure are those it has on the developing brain and its subsequent impact on neurobehavioral outcomes [44]. Individuals with FASD demonstrate significant impairments in a number of cognitive domains, including deficits in attention [53, 54], goal-directed behavior, motor control, and learning and memory [1, 55]. Cognitive impairments in FASD likely reflect underlying structural damage and/or functional aberrations in brain regions important for cognitive function. Individuals with FASD show structural and/or functional deficits in the prefrontal cortex, caudate-putamen, cerebellum, and the hippocampus [1, 56-58].
Efforts to educate the public about the dangers of prenatal alcohol exposure have shown some success [59, 60], yet some women continue to consume alcohol during pregnancy. The Center for Disease Control and Prevention (CDC) recently estimated that 7.6% of pregnant women in the United States consume alcohol (CDC, 2012). There are regional differences too, with an estimated 52.6% of Italian women reporting alcohol use during pregnancy [61]. There is a significant need, therefore, for the development of targeted treatments and interventions to improve outcomes in children and adults prenatally exposed to alcohol. Research in animal models is helping to identify effective treatments to mitigate neurobehavioral impairments in FASD [3, 4, 62, 63]. Treatments that target a known neural pathology are likely to result in significant neurobehavioral improvement.
Research has identified the cholinergic system as a potential target for FASD treatments. The cholinergic system significantly modulates hippocampus function [7, 64], hippocampus-mediated behaviors [8, 21, 65], and is affected by developmental alcohol exposure [62, 66-71]. The nutrient choline is a precursor to the neurotransmitter acetylcholine, in addition to being an agonist at the α7 nicotinic acetylcholine receptor (nAChR). Choline supplementation over early development mitigates many of the hippocampus-dependent learning impairments present in developmentally alcohol-exposed rats [3, 4, 72]. We recently demonstrated that developmental alcohol exposure causes significant reductions in the densities of α7 nAChR in multiple brain regions, including reductions in the dorsal hippocampus, an effect that is partially mitigated by choline supplementation (see Figure 1) [6]. If reductions in α7 nAChR in the hippocampus mediate, in part, the cognitive impairments present in FASD, than interventions and treatments that target these receptors may mitigate neurobehavioral effects. In this proposal, we hypothesize that pharmacologically targeting the α7 nAChR will lead to significant improvements in hippocampus neural plasticity (i.e., CA1 LTP induction) and hippocampus-mediated behavior (i.e., contextual fear conditioning) in rats exposed to alcohol developmentally.
The Cholinergic System in FASD: The cholinergic system has a significant neuromodulatory role in neural plasticity and cognition [7, 8, 73]. Acetylcholine (ACh) acts upon two broad classes of receptor types, muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors. mAChR are slower-acting G-protein coupled receptors composed of five subtypes (M1-M5) [For review of mAChR, see 7] whereas nAChR are fast-acting ionotropic receptors comprised of α (α2-α10) and/or β (β2-β4) subunits. The α7 nAChR has a number of properties that make it a significant modulator of neural transmission and a potential target for improving outcomes in cognitive disorders. First, the α7 nAChR is densely located pre- and postsynaptically on both glutamatergic pyramidal cells and GABAergic interneurons in the hippocampus [74-76], where it modulates the release of other neurotransmitters [77, 78]; reductions in this receptor, then, can have wide ranging effects. Second, the α7 nAChR is highly permeable to Ca2+ ions, more so than other nAChR subtypes or NMDA receptors [79]. Ca2+ ions can activate a number of second-messenger cascades (e.g., ERK1/2, CREB) that are involved in long-lasting changes in synaptic plasticity and long-term memory formation [16]. Finally, a number of highly selective agonists (e.g., DMXB), antagonists (e.g., MLA), and allosteric modulators have been developed that target the α7 nAChR [5]. Researchers utilizing these selective agents have demonstrated that α7 nAChR significantly modulate 1) long-term potentiation of the CA1 region of the hippocampus [17, 18, 20, 80], 2) hippocampus-dependent behaviors [21, 81], and 3) that targeting this receptor leads to improved hippocampal synaptic plasticity and hippocampus-mediated behavior in a number of animal models of disorders of cholinergic function, including models of Alzheimer’s, schizophrenia, and ADHD [16, 82, 83].
Developmental alcohol exposure is associated with impaired cholinergic function [62, 66, 71]. In animal models of FASD, developmental alcohol exposure causes reductions in cholinergic neurons in the medial septum and striatum [68, 71] and alterations in muscarinic receptor densities in the hippocampus [62, 67-70]. Our preliminary data demonstrate that rats exposed to a high binge-like dose of alcohol over postnatal days (PD) 4-9, a period of time equivalent to the third trimester in humans, show significant reductions in α7 nAChR densities in each of the major subfields of the hippocampus (CA1, CA3, and DG) relative to controls. Supplemental choline partly attenuated this effect in developmentally alcohol-exposed rats (Figure 1). Given the significant modulatory role that these receptors play in both hippocampus function and behavior, reductions in these receptors are likely to have a significant negative impact on hippocampus function and hippocampus-mediated behavior.
Developmental Alcohol Exposure and LTP induction in the Hippocampus: Long-term potentiation (LTP) refers to long-lasting changes in hippocampus synaptic efficacy following high frequency stimulation. LTP is a model of cellular plasticity, learning, and memory. Learning induces LTP-like changes in the hippocampus [84], suggesting that impairments in hippocampal LTP induction may underlie learning and memory impairments in models of FASD. Rats exposed to alcohol over the prenatal and early postnatal period demonstrate significant impairments in LTP induction in multiple pathways in the hippocampus, including the Perforant Path – Dentate Gyrus (PP-DG) [85-87] and the Schaffer Collateral – Cornu Ammonis 1 (SC-CA1) pathways [9, 10]. Developmental alcohol exposure also causes significant alterations in hippocampus excitatory and inhibitory neurotransmission [19, 88-90], which may underlie the LTP induction impairments. LTP induction in the SC-CA1 pathway involves activation of α7 nAChRs [17, 18, 20, 80]. Application of the α7 nAChR agonist DMXB can reverse the SC-CA1 LTP induction impairments in a mouse model of Alzheimer’s [82, 83], a disease associated with significant reductions in α7 nAChRs [91]. Our preliminary data demonstrates significant reductions in hippocampus α7 nAChRs in adolescent rats exposed to alcohol over the neonatal period (Figure 1), which we hypothesize contributes to SC-CA1 LTP induction impairments following this alcohol-exposure paradigm [9, 10]. If our hypothesis is correct, application of DMXB will facilitate SC-CA1 LTP induction in alcohol-exposed rats, where we expect to observe a reduced LTP magnitude. Other researchers have utilized other potential treatments that demonstrate facilitated PP-DG LTP in rats prenatally exposed to alcohol [87, 92]. In this proposal, we will examine LTP in the SC-CA1 pathway. Output from the CA1 neurons provides input to many brain regions that are critical for proper cognitive function. Thus, alterations in the function of this pathway by alcohol could have a significant impact on brain function. Our studies will utilize a combination of electrophysiological and behavioral techniques to gain an understanding of the role of α7 nAChR in the physiological and behavioral impairments observed following developmental alcohol exposure.
Contextual Fear Conditioning as a Behavioral Assay of Hippocampus Function. Fear conditioning to discrete conditioning stimuli (e.g., tones and lights) engages a well-described neural circuit centered on the amygdala [93, 94]. In addition to this basic fear circuit, contextual fear conditioning further recruits the hippocampus. For example, 1) context conditioning is disrupted by lesions, inactivation, or NMDA receptor blockade of the dorsal hippocampus [28, 93] and 2) contextual fear conditioning increases the expression of learning-related markers (e.g., ERK1/2, CREB, Arc) in the CA1 regions of the hippocampus [95, 96]. Prenatal or early postnatal alcohol exposure significantly disrupts contextual fear conditioning in both juvenile and adult rats [11, 14, 97, 98]. Alcohol-exposed rats show normal fear conditioning to discrete stimuli, suggesting that contextual fear impairments likely reflect hippocampus dysfunction and not dysfunction of the underlying basic fear circuit. My previous studies demonstrate that rats exposed to alcohol over the early neonatal period show significant contextual conditioning impairments. These impairments are evident even following alcohol exposure limited to a short window in the neonatal period (PD7-9), and are associated with CA1 pyramidal cell loss, reductions in CA1 c-Fos expression, and alterations in adult hippocampal neurogenesis [11-14]. The cholinergic system significantly modulates contextual fear conditioning [8, 21, 99]. Our preliminary data (Figure 2) demonstrate significant reductions in contextual fear conditioning in alcohol-exposed rats. If a drug applied immediately before conditioning impairs behavior, that drug is thought to affect memory acquisition. If a drug applied immediately following conditioning impairs behavior, that drug is thought to affect memory consolidation [27]. The acquisition of contextual fear conditioning can be disrupted by pre-conditioning blockade of mAChR and non-α7 nAChR subtypes (via scopolamine and mecamylamine, respectively [21, 99], whereas the consolidation of contextual memory can be blocked by the post-conditioning infusion of the α7 nAChR antagonist MLA [21]. A recent study demonstrated that injections of the acetylcholinesterase inhibitor physostigmine can mitigate contextual fear conditioning impairments in rats exposed to alcohol over the neonatal period [100]. Because physostigmine will increase cholinergic transmission non-selectively, it is difficult to distinguish the particular pathway (muscarinic vs. nicotinic receptors) by which it has its beneficial effects. However, measures of short-term contextual memory, like post-shock freezing, are not different in alcohol-exposed rats compared to controls [14, 23]. This suggests that the contextual fear conditioning impairments in alcohol-exposed rats likely result from a failure to consolidate long-term context memory formation. Because blockade of α7 nAChR disrupts the consolidation of context memories [21], we hypothesize that reductions in these receptors in the hippocampus of alcohol-exposed rats contributes to contextual memory consolidation impairments. We further hypothesize that hippocampus infusion of the α7 nAChR agonist DMXB following contextual fear acquisition will significantly mitigate learning impairments in alcohol-exposed rats.
Taken together, the studies presented in this proposal will investigate the therapeutic potential of targeting the acetylcholine system for the treatment of cognitive impairment in FASD.
INNOVATION
The present proposal hypothesizes that alterations in hippocampal α7 nAChR underlie impaired neural plasticity and memory deficits associated with developmental alcohol exposure. Although it has been previously shown that early alcohol exposure disrupts cholinergic development, this is the first study to target specific cholinergic receptors in an effort to improve neural function and cognitive performance. In addition, previous work examining the effects of neonatal alcohol exposure on synaptic plasticity has had great success utilizing complementary behavioral measures and ex vivo electrophysiology techniques, yet few laboratories effectively utilize both capabilities in tandem. Thus, the technical innovation of this proposal will be to integrate slice electrophysiology and behavioral assessment to identify the mechanism driving LTP-impairment in CA1 that occur following neonatal alcohol exposure.
APPROACH
Specific Aim 1: The role of α7 nicotinic acetylcholine receptors in the induction of LTP in the hippocampus in a rat model of FASD. The goal of Aim 1 is to demonstrate that pharmacologically targeting the α7 nicotinic receptor can mitigate LTP induction-deficits in the hippocampus in alcohol-exposed rats. High-frequency stimulation (HFS) of CA3 Schaffer collateral (SC) fibers can lead to a long-lasting enhancement of CA1 efficacy known as long-term potentiation (LTP). In animal models of FASD, both prenatal and early postnatal alcohol exposure result in impaired HFS induced LTP in the SC-CA1 pathway [9, 10, 101]. Neuronal nicotinic acetylcholine receptors (nAChR) significantly modulate the induction of LTP in the CA1 [7]. Specifically, the selective α7 nAChR agonist DMXB facilitates CA1 LTP in a dose-dependent fashion [80, 83]. Further, CA1 LTP induction is blocked by application of the α7 nAChR antagonist methyllycaconite (MLA), but not the non-α7 nAChR antagonist DHβE, and is absent in α7 knock-out mice [17, 18, 83]. Given our preliminary data showing reductions in CA1 α7 nAChR in adolescent rats (PD 30) exposed to alcohol over PD 4-9, we hypothesize that impaired HFS-induced LTP in the SC-CA1 pathway of alcohol-exposed rats results, in part, from reductions in α7 nAChR and that this impairment can be mitigated by the application of the selective α7 nAChR agonist DMXB.
To test this hypothesis, studies of synaptic function will be carried out in hippocampal slices from rats that receive 5.25 g/kg/day of alcohol (EtOH) or sham intubations (SI) over postnatal days (PD) 4-9 (which serves as a model of late gestational alcohol exposure) as previously described [12]. At PD 30, rats will be anesthetized, rapidly decapitated, and their brains will be removed and placed in oxygenated (95% O2/%5 CO2) artificial cerebral spinal fluid (ACSF). Transverse slices (350 µm) will be cut from the dorsal hippocampus and maintained in ACSF at 30°C with a continuous perfusion rate. Electrophysiological recordings will follow methods outlined in Roberto et al. [38, 39]. Briefly, synaptic responses will be evoked by a concentric bipolar stimulating electrode placed into the SC, while recording electrodes (1–3 MΩ tipped microelectrodes filled with ACSF) will be placed into the CA1 stratum pyramidale (somatic region; population spikes) and the stratum radiatum (dendritic region; field excitatory postsynaptic potentials [fEPSPs]). Thus, extracellular field potentials in area CA1 will be recorded simultaneously from the somatic and dendritic regions. Synaptic responses will be elicited by electrical stimulation of the SC pathway and response parameters for each slice will be determined by an input/output (I/O) protocol. I/O curves for the slope of the dendritic fEPSP and amplitude of the somatic population spike will be generated starting at the threshold stimulus intensity required to elicit a dendritic fEPSP, up to the intensity required to elicit the maximum somatic population spike amplitude. The I/O curves will be generated immediately before and 60 or 120 minutes after HFS, when LTP will be evident. Baseline measurements of fEPSPs for LTP experiments will involve fEPSPs evoked by SC stimulation every 30 s. The stimulus strength will be adjusted for each slice such that the fEPSP is equal to ∼40-50% of the maximal amplitude determined in the I/O relationship. LTP will be induced by a single train of HFS (100 Hz for 1s). The slice will be considered to exhibit LTP if the slope of the dendritic fEPSP response remains at an elevated level of >125% of baseline for >60 min following the HFS. Posttetanic potentiation (PTP) and paired-pulse facilitation (PPF) (transient increases in synaptic transmission induced by pathway activation) will also be examined. PTP and PPF are forms of short-term potentiation that result from increased transmitter release caused by a transient elevation of intracellular Ca2+ in repetitively activated synaptic terminals [102]. PTP is induced by the HFS stimulation used to induce LTP and occurs immediately after the HFS. PPF is induced by low frequency stimulation (e.g., every 30 sec) at an inter-stimulus interval of 50 ms. Synaptic function will be studied in hippocampal slices under control conditions (ACSF perfusion) and in the presence of α7 nAChR drugs. The following drugs will be added to the ACSF for different experimental conditions: 3-(2,4-Dimethoxybenzylidene)-anabaseine (DMXB, a selective α7 nAChR agonist; 1.0 or 10 µM), methyllycaconitine (MLA, α7 nAChR antagonist; 0.1 µM), or dihydro-beta-erythroidine (DHβE, α4β2 nAChR antagonist; 10 µM). These drugs will be bath-applied in the ACSF following the protocol of Chen et al [51], or if shorter exposure times are needed, by a localized perfusion system. The experiments will be divided into 4 phases: 1) assess synaptic function (i.e., I/O relationship, PFF, LTP) in both SI and EtOH rats (Exp. 1A); 2) assess DMXB effects on synaptic function in both groups at both doses (Exp. 1B); 3) assess the effects of DMXB in the presence of MLA (DMXB + MLA; Exp. 1C); and 4) assess the effects of DMXB in the presence of DHβE (DMXB + DHβE; Exp. 1D).
Predicted Outcomes and Interpretation. Previous studies examining excitatory synaptic transmission and presynaptic transmitter release (i.e., I/O curves and PPF, respectively) in control and alcohol-exposed rats under basal conditions in the SC-CA1 pathway report no group differences in these measures [10, 103]. Neuronal α7 and non-α7 nAChR are located at both pre-synaptic and post-synaptic sites, where presynaptic receptors can modulate the release of other neurotransmitters, including glutamate. [7, 16]. Our working hypothesis is that reductions in α7 nAChR contribute to hippocampal dysfunction in EtOH rats and should therefore result in LTP impairment due to increased PPF and decreased PTP compared to SI. Agonists of nAChRs show a bell-shaped dose-response curve [104]. Based on previous studies on effects of DMXB on fEPSPs [83], we expect that 1.0 µM DMXB will enhance (relative to ACSF) fEPSP amplitudes in controls, but not 10 µM concentration [83]. We also hypothesize that in EtOH rats, application of 10 µM DMXB (but not 1.0 µM DMXB) will enhance baseline fEPSP amplitudes, facilitating PTP and consequently the induction of LTP likely via both pre- and postsynaptic mechanisms. This shift in the dose-response of DMXB in EtOH rats is expected because of the lower densities of hippocampal α7 nAChR [6] (and Fig. 1) and thus a higher concentration of the drug will be needed to facilitate LTP induction compared to SI rats. The effects of DMXB (in both groups) are expected to be blocked by MLA, but not DHβE. LTP in the SC-CA1. A number of studies have examined the effects of both prenatal and early postnatal alcohol-exposure on LTP induction in the SC-CA1 pathway [9, 10, 19, 105, 106]. Those studies utilizing a third-trimester model have demonstrated significant SC-CA1 LTP impairment in alcohol-exposed rats [9, 10], although these findings are not consistently found [19, 107]. Numerous differences between studies could account for discrepant findings (e.g., route of alcohol administration, peak BACs, strain differences, age, sex, and LTP induction protocols). Puglia and Valenzuela (2010) report that the LTP impairments observed in alcohol-exposed rats were only present during the second half of their 60-minute recording session [see Figure 3B in 9]. That is, the impairment in LTP was not in the induction or short-term potentiation of CA1, but in the maintenance of LTP (defined as late LTP) in alcohol-exposed rats [9]. The α7 nAChR participates in both protein synthesis independent LTP (early LTP; < 60 min.) and protein synthesis-dependent LTP (late LTP; > 60 min) within the CA1 [20]. It is possible that developmental alcohol exposure affects late LTP, but not early LTP. To examine this possibility, we will record fEPSPs for 120 minutes to cover both early and late LTP. Due to reduction in α7 nAChR density, we hypothesize that EtOH rats will show reductions in HFS-induced LTP relative to SI rats (i.e., a smaller magnitude or failure of LTP). DMXB is expected to further enhance both early and late LTP in SI rats at 1.0 μM, but not the 10 μM concentration [83]. In contrast, DMXB will facilitate the induction of both early and late LTP in EtOH rats at the 10 μM, but not the 1.0 μM concentration, indicative of a rightward shift in the dose-response curve. These changes in the DMXB dose-response curve between SI and EtOH rats is predicted to follow the same pattern observed between control and β-amyloid infused rats [82]. DMXB + MLA is expected to block induction of LTP in both groups, which will demonstrate that DMXB acts upon α7 nAChR and that these receptors are necessary for LTP induction [17, 18, 83]. DMXB + DHβE is expected to have a similar result to those demonstrated by DMXB-alone in both groups. These results are outlined in Table 1.
Alternative outcomes. Neuronal α7 nAChR are located on both glutamatergic pyramidal cells and GABAergic interneurons in the hippocampus [7]. In addition to their stimulatory effect on pyramidal cells, activation of α7 nAChR on GABAergic interneurons may facilitate CA1 LTP induction by activating interneurons that project to other interneurons resulting in disinhibition of CA1 pyramidal cells [108]. However, there are reports that blockade of α7 nAChR with MLA facilitates HFS-induced CA1 LTP [109, 110]. These results might be interpreted in terms of the actions of α7 nAChR activation of GABAergic interneurons that directly inhibit CA1 pyramidal cells [108]. If this latter interpretation were the case, we would expect DMXB and MLA to have opposite effects to those we hypothesize. Further, we would expect EtOH rats to show facilitated CA1 LTP due to increased non-α7 and decreased α7 nAChR [see 109]. However, studies examining CA1 LTP in rats exposed to alcohol over the neonatal period do not report facilitated LTP [9, 10, 19]. Given the recent work showing that α7 nAChR activation facilitates while inactivation of these receptors impairs CA1 LTP [17, 18, 20, 83] and that CA1 LTP induction is impaired in alcohol-exposed rats [9, 10], we believe that our hypothesis that DMXB will facilitate CA1 LTP induction under our proposed protocols will stand. There is also the possibility that DMXB will have no effect in EtOH rats. This may result from qualitative, not quantitative group differences in α7 nAChR (i.e., there could be functional abnormalities in these receptors that reduce the effectiveness of direct agonist action). A failure to observe LTP modulation by DMXB, MLA, or DHβE in EtOH rats may necessitate an investigation into nicotinic receptor dynamics (via patch-clamp methods). Additional experiments that examine additional α7 nAChR drug doses or non-α7 nAChR drugs on HFS induction of CA1 LTP in EtOH and SI rats may be undertaken.
Future directions. Additional studies could build upon the findings of Aim 1. First, one of the goals of Aim 1 is to examine the therapeutic potential of using an α7 nAChR agonist (DMXB) to mitigate the impairments in hippocampal neural plasticity in a pre-clinical model of FASD. If our hypothesis is correct, then targeting this receptor could lead to improved cognitive outcomes in FASD affected individuals. α7 nAChR are characterized by rapid (<100 ms) desensitization after agonist binding [111] which might limit the therapeutic potential of full agonists at this receptor. An alternative would be to examine positive allosteric modulators (ligands that bind to receptors without desensitizing them), which have been used to treat cognitive deficits in other disorders of cholinergic function [5, 16]. An extension of the current Aim would be to examine if positive allosteric modulators of α7 nAChR could act to facilitate CA1 LTP in EtOH rats. Second, activation of α7 nAChR activates a number of signaling cascades associated with long-term synaptic plasticity and memory, including extracellular-signal regulated kinase 1/2 (ERK1/2) and cAMP response element binding (CREB) protein [16]. ERK1/2 and CREB may likely underlie the enhancement of late LTP by activation of α7 nAChR [20]. An examination of ERK1/2 and CREB following HFS induced CA1 LTP in alcohol-exposed rats, under ACSF and DMXB, would be the next step in understanding the molecular aspects of hippocampal α7 nAChRs. This will be accomplished following methods for examining ERK1/2 as previously described by Roberto et al. [38]. The modulation of HFS induced LTP by nAChRs could also be examined in additional hippocampal pathways, such as the PP-DG and the DG-CA3 mossy fiber pathways. Multiple studies have examined the effects of developmental alcohol exposure on HFS induction of LTP in the PP-DG pathway [85, 86, 88], but the modulation of LTP in this pathway by nAChRs. The modulation of LTP in the PP-DG pathway by non-α7 and α7 nAChRs is distinct from their modulation of LTP in the SC-CA1 pathway [7]. An examination of nAChR modulation of LTP in the PP-DG pathway of EtOH rats could provide additional insights into the functional consequences of altered nicotinic acetylcholine receptors.
Specific Aim 2: The role of hippocampal α7 nicotinic acetylcholine receptors in contextual fear conditioning in a rat model of FASD. Specific Aim 2 will complement and extend the findings of Aim 1 by testing the hypothesis that DMXB will significantly mitigate behavioral impairments in rats exposed to alcohol over the neonatal period. Contextual fear conditioning is associated with a significant increase of acetylcholine release in the hippocampus [112]. Nicotinic acetylcholine receptors in the hippocampus significantly modulate contextual fear conditioning [8]. For example, hippocampal blockade of α7 nAChRs with MLA following, but not before conditioning significantly disrupts the consolidation of contextual fear memories [21]. Rats exposed to alcohol over the neonatal period show control-level post-shock freezing [14, 23], suggesting intact short-term contextual memory. However, when tested 24 h following conditioning, alcohol-exposed rats show significant contextual learning impairments compared to controls over a variety of training parameters [11-14, 100, 113]. Together these data suggest that the contextual fear conditioning impairments in alcohol-exposed rats likely arise from a failure of contextual memory consolidation. Given the role of hippocampal α7 nAChR in contextual fear memory consolidation [21] and our preliminary data showing reductions in hippocampal α7 nAChR in alcohol-exposed rats [6], we hypothesize that infusion of the α7 nAChR agonist DMXB into the dorsal hippocampus following contextual fear conditioning will significantly mitigate contextual memory impairments seen in alcohol-exposed rats.
A full description of methods can be found under General Procedures. Briefly, EtOH and SI rats will be treated over PD 4-9 following the design of Aim 1. Experiments 2A and 2B will include undisturbed (UD) controls. Cannula surgeries will follow modified procedures outlined in Schiffino et al. 2011 [28]. Following a two-day recovery, all rats will undergo standard delay fear conditioning starting on PD 30. Freezing behavior during each session will be used as a measure of fear conditioning. Rats will be tested for contextual fear in the training context on PD 31 and tested for CS conditioning in an alternate context on PD 32. Rats will receive bilateral injections of phosphate buffered saline (PBS), DMXB, or MLA into the dorsal hippocampus one minute following conditioning on PD 30, a time these drugs are likely to influence the consolidation of contextual fear memories [21]. The concentrations of DMXB (0.38, 1.9, or 3.80 mg/side) are derived from concentrations used in Aim 1. The concentrations of MLA (13.5 or 35 mg/side) follow previous reports [21, 22].
We will perform four separate experiments under this Aim. Vago and Kesner [21] concluded that α7 nAChRs mediate contextual memory consolidation due to the ability of MLA to disrupt context conditioning when infused into the dorsal hippocampus one minute following conditioning. However, experiments testing the hypothesis that stimulating hippocampus α7 nAChRs (via DMXB) will facilitate contextual memory consolidation have yet to be examined. A 2 (Sex: male vs. female) x 3 (PBS vs. MLA [35 mg/side]-before vs. MLA [35 mg/side]-after) design will test the hypothesis that MLA infused into the hippocampus one minute following, but not 15 minutes before conditioning will disrupt contextual fear conditioning in UD PD 30 rats (Exp. 2A). A 2 (Sex) X 4 (DMXB: 0.0, 0.38, 1.90, 3.80 mg/side) design will test the hypothesis that hippocampus infusions of DMXB administered one minute following conditioning will significantly facilitate contextual fear conditioning UD PD 30 rats (Exp. 2B). These studies in normally developing adolescent rats will confirm that blockade of hippocampal α7 nAChRs disrupts contextual fear consolidation and that stimulation of these receptors will facilitate contextual fear conditioning. A 2 (Sex) X 2 (dosing condition: EtOH vs. SI) X 3 (DMXB: 0.0, 0.38, 3.80 mg/side) design will test the hypothesis that DMXB infused into the hippocampus one minute following training can mitigate contextual fear conditioning impairments in EtOH rats (Exp. 3A). Finally, a 2 (sex) X 2 (dosing condition) X 3 (MLA: 0.0, 13.5, or 35 mg/side) design will test the hypothesis that infusion of MLA into the hippocampus one minute following conditioning disrupts contextual fear conditioning in both EtOH and SI rats (Exp. 3B).
Predicted Outcomes and Interpretation. Experiment 2A will demonstrate that MLA infused into the dorsal hippocampus following, but not before training will disrupt contextual conditioning relative to PBS-infused UD rats. This study will confirm the conclusions of Vago & Kesner [27] that α7 nAChRs are involved in contextual memory consolidation and extend their findings to adolescent male and female rats. Experiment 1B will demonstrate that DMXB infused into the dorsal hippocampus following contextual fear training will dose-dependently facilitate context conditioning relative to PBS-infused UD rats. We expect DMXB to facilitate context conditioning following a bell-shaped dose-response curve commonly seen with nicotinic drugs [8, 104]; that is, DMXB will facilitate contextual conditioning at the 0.38 and 1.90 mg/side dose, with no enhancement seen in the 3.80 mg/kg dose. We do not anticipate any significant main effects or interactions with sex at this developmental age [13, 14, 26]. In Experiments 2 and 3, EtOH and SI rats are not expected to differ in their levels of freezing during Training or during CS Testing, following previous reports [14, 72, 113, 114]. These results will indicate that developmental alcohol exposure does not significantly disrupt discrete (e.g., tone) CS-US (e.g., foot shock) associations, negatively affect the basic fear circuit (centered on the amygdala), or disrupt freezing responses (ruling out a potential performance effect). PBS-treated EtOH rats are expected to show significant reductions in contextual freezing during Context Testing relative to PBS-treated SI rats [11-14].
Previous studies have concluded that α7 nAChR do not participate in the acquisition of contextual fear conditioning because pre-conditioning treatment with MLA in normally developing rodents failed to impair context conditioning [for review see 8, 22]. In a separate experiment however, MLA administered one minute or six hours following conditioning resulted in significant contextual learning impairments, suggesting that α7 nAChR participate in the consolidation of context memories [21]. Given these data, we anticipate significant facilitation of context conditioning in SI rats administered the DMXB at the 0.38, but not the 3.80 mg/side dose (replicating the result of Exp. 1B). This dose-response effect would follow our prediction for HFS-induced CA1 LTP in SI rats from Aim 1, where the lower, but not higher concentration of DMXB is expected to facilitate LTP induction in SI rats. In EtOH rats, the higher, but not lower concentration will mitigate the contextual fear conditioning impairment. This shift in the dose-response curve of DMXB in EtOH rats is expected because of lower densities of the α7 nAChR in the hippocampus [6] and thus a higher concentration of the drug will be needed in EtOH rats to mitigate contextual conditioning impairments. Infusion of MLA into the dorsal hippocampus is expected to impair conditioning in both SI and EtOH rats [21], however the effective concentrations are expected to differ between groups. MLA at the lower dose (13.5 mg/side) is expected to further disrupt context conditioning in EtOH rats, while both EtOH and SI rats infused with the higher dose (35 mg/side) will show contextual fear impairments relative to PBS-treated SI rats.
Alternative Outcomes and Future Directions. It is possible that local hippocampal infusions of DMXB in EtOH rats will fail to mitigate contextual fear conditioning impairments. Potential causes may relate to alterations in 1) α7 nAChR properties, 2) activation of downstream messengers, or 3) potential drug dose issues. If the results of Aim 1 show that DMXB can mitigate LTP induction impairments in EtOH rats, then we can assume that receptor properties and some downstream effects are functional in the EtOH rat. Work in normally developing (UD) rats should help to select the appropriate effective doses in controls (Exps. 2A and 2B), which are hypothesized to differ from the effective doses in EtOH rats. We will examine additional drug doses if no effects are seen. Alternatively, although acute effects of DMXB are reported to improve behaviors (e.g., water maze, inhibitory avoidance [81, 82]), chronic exposure to DMXB can also facilitate behaviors [115, 116]. An alternative method to our approach would be to examine a more chronic developmental exposure to DMXB, following models of developmental choline supplementation (another α7 nAChR agonist) which can improve cognitive performance in alcohol-exposed rats [3, 4, 6].
Additionally, although we do not expect any sex effects, significant sex effects in behavior will necessitate an examination of sex effects in LTP induction. We hypothesize that the results of Aim 1 and those of Aim 2 will complement each other (i.e., that DMXB will mitigate both LTP impairments and behavioral deficits in EtOH rats). However, our findings may reveal different effects. Aim 1 examines SC-CA1 LTP, but local infusion of DMXB into the dorsal hippocampus (Aim 2) will affect α7 nAChR in all of the subfields of the hippocampus, not just CA1. Activation of α7 nAChR in the DG also facilitates PP-DG LTP [7, 117]. Application of drugs throughout the hippocampus may have differential effects than drugs applied directly to CA1. Our preliminary data demonstrate that reductions in α7 nAChR in EtOH rats occur in each of the hippocampal subfields. Dorsal hippocampus infusions of DMXB could potentially facilitate neurotransmission in all subfields. In addition, the timing of drug application (5 minutes prior to HFS [Aim 1] or one minute following training [Aim 2]) may result in differential effects. Timing plays a significant role in α7 nAChR dependent CA1 LTP, where stimulation of the CA1 stratum oriens at 300 ms prior to SC stimulation results in LTP, whereas stimulation at 50 ms prior to SC stimulation results in short term depression [18]. The timing of our drug application (one minute following training) is demonstrated to affect context memory consolidation [21]. However, this effect is seen with blockade, but not activation of α7 nAChR. Experiment 2B will address this issue. However, failure for DMXB to facilitate contextual conditioning in normally developing controls may require an examination of additional times of drug application. Finally, histological examination of cannula placement will verify that drugs were infused into the dorsal hippocampus. Any animals with improper placement will be excluded. We may also examine drug infusion into the overlying cortex to assess generalized drug effects outside of the hippocampus. In addition, infusion of a radiolabeled drug may be warranted to examine the spread of the drug.
Future directions will be to examine the effectiveness of DMXB and other nicotinic acetylcholine drugs targeted at the hippocampus, medial prefrontal cortex, and/or amygdala to facilitate additional fear conditioning paradigms including context-preexposure-dependent contextual fear, trace fear, and fear extinction. These variants of fear conditioning utilize different brain regions and neural processes [93, 118, 119]. By examining how other nicotinic receptor drugs modulate behavior on a variety of behavioral tasks, we are likely to better understand the neural impairments that result from developmental alcohol exposure and neural targets that may improve different aspects of cognition.
GENERAL PROCEDURES
Subjects. Offspring of Sprague-Dawley rats mated at The Scripps Research Institute rat breeding colony (Aim 1) or at the Center for Behavioral Teratology, San Diego State University (Aim 2) will serve as subjects. The 22nd day of gestation (GD 22) is designated as the day of birth (postnatal day (PD) 0). On PD 2, litters will be culled to eight pups (4 males and 4 females). Pups will remain housed with their dams until PD 21 when they will be weaned and housed together in groups of 3-5 with same-sex litter mates for the remainder of the studies. Physiology experiments under Aim 1 will involve male-only subjects. An examination of only male subjects will make the studies of a more manageable size and limit potential sex differences in hippocampus LTP induction [120, 121]. Studies in Aim 2 will include both male and female rats (with a minimum of 8 animals per sex per group to examine potential sex effects). Rats will have access to standard rat chow and water ad libitum throughout the study. The animal facility and testing rooms are maintained on a 12:12-hour light-dark cycle (lights on at 0600 hr.). Only one rat per sex per treatment condition per litter will be assigned to any given experimental group to control for litter effects.
Developmental Alcohol Exposure. On PD 2, rats will be randomly assigned into one of two developmental alcohol exposure groups. Administration of alcohol via gastric intubations will follow procedures extensively use by the applicant, sponsor, and others [14, 31, 122]. Alcohol-exposed (AE) rats will receive alcohol at a dose of 5.25 g/kg/day (11.96% v/v in a milk formula) via intragastric intubations occurring over PD 4-9. Over PD 4-9, AE rats will receive two feedings of an ethanol-milk mixture and two feedings of a milk-only mixture, with each feeding separated by 2 hr. Sham-intubated (SI) control rats will receive the same intubation procedure without any infusion of liquid. Sham-intubated rats do not receive additional milk feedings to avoid excessive weight gain during the neonatal period [122]. On PD 6, blood will be collected from a small tail clip 1.5 hours following the second feeding and blood alcohol concentrations (BACs) determined using an Analox GL5 Analyzer (Analox Instruments, Luneburg, MA).
nAChR Drugs. All drugs are available through Sigma (St. Luis, MO). Dimethoxybenzylidene)-anabaseine (DMXB [also known as GTS-21]) is a selective α7 nAChR agonist with mild antagonism on α4β2 nAChRs [123]. Pharmacokinetic studies demonstrate that DMXB (administered i.v. in adult male rats) has a plasma half-life of 3-4 hours [124] Alpha-methyllycaconitine (MLA) is a selective α7 nAChR antagonist [5]. Dihydro-beta-erythroidine (DHβE) is a selective antagonist at the α4β2 nAChR [125]. For physiology experiments, drugs will be bath applied 20 (MLA or DHβE) or five (DMXB) minutes prior to HFS. For drug infusions, injection cannula attached with polyethylene tubing to a 10 µL Hamilton syringe mounted to a syringe pump will be placed into each guide cannula (extending 1 mm below the guide cannula). Drugs will be dissolved in sterile phosphate-buffered saline (PBS) and infused at a rate of 0.5 μL per minute. Injection cannula will be left in place for an additional minute to allow for drug diffusion before removal.
Brain slices and electrophysiology. Standard hippocampus slice preparation and methods (e.g., extracellular recording of fEPSPs and population spikes, PPF, LTP induction etc.) will be as described in Dr. Roberto’s papers [38, 39]. All experiments have been designed to minimize the number of animals required within the constraint of maintaining sufficient statistical power for proper interpretation of the results. The sample size for individual experimental groups in Specific Aim 1 is n = 12-14. We estimate that each experiment on a different drug will require at least 10-12 recordings (at approximately 1-2 fEPSPs/population spikes a day, about 8-12 animals) to attain statistical significance, provided drug responses are fairly uniform and consistent. Otherwise, 12-24 recordings (12-20 rats) may be required.
Fear Conditioning. Animals will be briefly handled (3 minutes/day) on two consecutive days prior to behavioral testing. Fear conditioning will occur in one of two distinct contexts. Context A is one of four rat testing chambers (Coulbourn Instruments, Whitehall, PA) measuring 12x10x12 situated within a sound-attenuated isolation cubicle. The front and back walls are made out of clear Plexiglas while the two side walls consisted of stainless steel plates. A stainless steel celling with a central hole allows a ceiling-mounted USB video camera to record activity. A house light mounted on an interior wall provides illumination and a fan mounted onto the isolation cubicle provides constant background noise (60 dB). The conditioning stimulus (CS) is a 10 s 80-dB, 1600 Hz white noise delivered through a speaker mounted to the interior chamber wall. A grid floor consisting of 26 stainless steel rods (diameter of each rod is 0.185 inches and the grid spacing (center to center) is 0.563 inches) delivers the foot shock unconditioned stimulus (US; 0.6 mA) from a precision animal shocker (Coulbourn Instruments, Whitehall, PA). Training will include three CS-US pairings with a 120s interstimulus interval following a 120 s baseline period. Testing will occur over a 5-minute stimulus-free session in Context A. CS Testing will follow the Training protocol without foot shocks in Context B. FreezeFrame3 software (Actimetrics, Wilmette, IL) controls the presentation of stimuli and received video input. Animal movement is determined by measuring changes in pixel luminance, where freezing is defined as a bout of 0.75 s or longer without changes in pixel luminance. Context B involved slight manipulations to Context A. The interior walls of the testing chamber consisted of black and white vertical stripes (~1 inch apart) and a mesh wire insert flooring. Our previous studies demonstrate little generalization between Context A and B.
Cannula Surgeries. Cannula surgeriesfollow methods previously described [28]. On PD 28, rats will be anesthetized with a ketamine/xylazine cocktail (1 mg/kg) and s.c. injection of buprenorphine (0.03 mg/kg) to alleviate pain prior to surgical implantation of stainless-steel guide cannulas (Plastic One, Roanoke, VA). Rats are mounted into a stereotaxic frame. Small holes are drilled with a 1/16 inch drill bit through the skull and guide cannulas are implanted bilaterally, terminating in the dorsal hippocampus following coordinates [126] modified for developing (PD 28) rats: anteroposterior (AP), +2.6 mm relative to interaural midline, mediolateral (ML), ±2.3 mm and dorsoventral (DV), -2.0 mm relative to Bergman. A subset of animals will receive cannula placement into the overlying cortex (missing the hippocampus with a DV -1.0) to assess the behavioral effects of drugs outside of the hippocampus. Two stainless steel wires are inserted into the skull surface and dental acrylic is used to secure the cannula assembly at the end of the surgery. Rats are placed onto a heating pad for 45 min following surgery and allowed 24 h to recover before behavioral testing.
Histology. Methods used to confirm cannula placement have been previously described [28]. Following behavioral testing, rats are deeply anesthetized with an i.p. injection of a ketamine/xylazine cocktail. 2% pontamine skyblue dye (0.5 µL) is injected through each guide cannula to stain cannula placement. Brains are removed and frozen in -20-ͦC methylbutane. Brain sections (40 µm) through the dorsal hippocampus are collected using a cryostat and are stained with 0.1% Cresyl Violet as previously described [12]. Sections are examined to confirm cannula placement within the dorsal hippocampus. Data obtained from rats with cannula placement not within the dorsal hippocampus are removed.
Data Analysis. Body weights are analyzed during the ethanol treatment period (PD 4-9), during each day of behavioral training, and prior to brain collection. For in vitro studies, measurements are made of the somatic population spike amplitude as well as the slope of the dendritic fEPSP in all protocols. Population spike amplitudes are measured from a line extrapolated between the peaks of the two rising components to the peak of the intervening downward deflection (i.e., the spike [38, 39]). The stimulation voltages of the I/O data were normalized such that the voltage required to produce threshold responses of the dendritic fEPSP and population spike are assigned a value of 0 V for each slice, and all stimuli are normalized to this value. For paired-pulse data, the relative amount of facilitation for each slice is expressed as the ratio of the second response with respect to the first response. Compiled data are expressed at the means +/- SE. Statistical analyses are done using ANOVA (factorial) and the Fisher’s protected least-significant difference (PLSD) post hoc test. Fear conditioning data are collected using FreezeFrame3 software (Actimetrics, Wilmette IL) with the bout length set at 0.75 s and the freezing threshold initially set as described in the program instructions. Freezing is defined as changes in pixels luminance falling below a threshold for at least 0.75 s. An experimenter blind to treatment groups will adjust the threshold so that freezing behavior involves the absence of all movements except those needed for respiration [127]. Freezing behavior is scored as the percentage of time spent freezing during a given bout of time. Training and CS Testing will use a repeated-measures ANOVA to examine changes in freezing over the training/CS testing session with ethanol treatment, sex, and drug treatment as between factors and training/testing bout (baseline, CS 1, CS 2, CS 3) as repeated factors. A factorial ANOVA is used to assess freezing behavior during the 5 min context test.
TIMELINE AND FEASIBILITY OF RESEARCH PLAN
The research plan and information about the total number of rats needed for each experiment is summarized in Table 2. Table 3 provides an experimental time-line. Aim 1 will be conducted under the K99 portion of the grant. The applicant will receive extensive training in in-vitro physiology by the co-sponsor Dr. Marissa Roberto. The initial procedural training will occur during the first six months of the grant. Aim 1 experiments (Exps. 1A-1D) are each expected to require six-twelve months to complete. This will leave the applicant an additional six months during the K99 phase to overcome unforeseen circumstances, write up manuscripts for publication, and test alternative results and future directions. Time estimates for behavioral experiments (Aim 2; R00) are based on processing up to 16 rats (2 litters) per month. Experiments 2A and 2B are expected to require six months to complete. Experiments 3A will take three months to complete while Exp.3B will require 4.5 months to complete. This will allow 1.25 years under the R00 to set up a lab, trouble shoot, write up manuscripts, and test alternative results and future directions.
References
94. LeDoux, J., The amygdala. Current biology : CB, 2007. 17(20): p. R868-74.
Nathen Murawski 