Abstract The non-euphorigenic phytocannabinoid cannabidiol (CBD) has demonstrated therapeutic efficacy in childhood-onset epilepsies. Using a songbird preclinical model we have found that CBD promotes recovery of learned vocalizations following focal motor cortical injury. But questions about cellular mechanisms supporting this protection remained. Songbird vocal learning, like human speech, depends on development and maintenance of specialized neural circuits. Partial lesioning (microlesions) of the vocal pre-motor cortical-like song region HVC transiently disrupts song structure and triggers injury-associated cellular stress responses across interconnected song regions. Building on prior findings that CBD reduces neuroinflammation and synaptic loss in zebra finch song circuitry, we investigated potential astrocyte contributions. Here we report that HVC microlesions induce significant cell loss in HVC and its projection targets (vocal motor RA and striatal Area X), with a substantial fraction of apoptotic cells being astrocytes. CBD treatment reduces lesion-induced apoptosis and preserves astrocyte populations, indicating enhanced astrocyte viability as a major factor in CBD-mediated neuroprotection. Microlesions also elevate astrocyte stress, including increased lysosomal burden (LAMP1/LC3 expression) and astrocytic reactivity markers (C3, S100A10, aromatase). CBD attenuates these stress responses while enhancing neuroprotective metabolic and antioxidant mediators (glutamine synthetase [GS], glutamate-cysteine ligase modifier subunit [GCLM]), consistent with improved antioxidant and excitotoxicity resistance. Given that development-dependent sensorimotor skills (e.g. song in songbirds, language and many others in humans) depend on sensitive period establishment and ongoing post-learning maintenance of specialized neural circuits vulnerable to traumatic disruption, the zebra finch model provides a valuable preclinical platform for investigating glial-targeted interventions to promote circuit resilience and functional recovery after TBI.

The non-euphorigenic phytocannabinoid cannabidiol (CBD) has been used successfully to treat childhood-onset epilepsies. These conditions are associated with developmental delays that often include vocal learning. Zebra finch song, like language, is a complex behavior learned during a sensitive period of development. Song quality is maintained through continuous sensorimotor refinement involving circuits that control learning and production. Within the vocal motor circuit, HVC is a cortical-like region that when partially lesioned temporarily disrupts song structure. We previously found CBD (10 mg/kg/day) improves post-lesion vocal recovery. The present studies were done to begin to understand mechanisms possibly responsible for CBD vocal protection. We found CBD markedly reduced expression of inflammatory mediators and oxidative stress markers. These effects were associated with regionally-reduced expression of the microglial marker TMEM119. As microglia are key regulators of synaptic reorganization, we measured synapse densities, finding significant lesion-induced circuit-wide decreases that were largely reversed by CBD. Synaptic protection was accompanied by NRF2 activation and BDNF/ARC/ARG3.1/MSK1 expression implicating mechanisms important to song circuit node mitigation of oxidative stress and promotion of synaptic homeostasis. Our findings demonstrate that CBD promotes an array of neuroprotective processes consistent with modulation of multiple cell signaling systems, and suggest these mechanisms are important to post-lesion recovery of a complex learned behavior.

Using a songbird, zebra finches, as a developmental drug abuse model we found previously that cannabinoid agonists administered during the sensorimotor period of vocal learning (50-75 days of age) persistently alter song patterns and cocaine responsiveness in adulthood. However, these effects were not produced in adults exposed to similar treatment regimens. Currently, we have used the MAGL inhibitor, JZL184, to test whether enhanced endocannabinoid signaling may similarly alter cocaine responsiveness. We found that, as expected and consistent with prior results, repeated developmental (but not adult) treatments with Delta 9-tetrahy-drocannabinol (THC, 3 mg/kg QD IM) resulted in increased time spent in cocaine-paired chambers. Unexpectedly and in contrast, repeated developmental JZL184 (4 mg/kg QD IM) treatments decreased time spent in cocaine-conditioned chambers. That is, young finches repeatedly treated with JZL184 avoided cocaine-paired chambers later in adulthood, while similar development treatments with THC had the opposite effect. To begin to identify brain regions that may underly this differential responsiveness we used c-Fos expression as a marker of neuronal activity. Differences in c-Fos expression patterns following placement of cocaine-conditioned finches into vehicle-vs. cocaine-paired chambers suggest distinct involvement of circuits through striatal and amygdaloid regions in respective ef-fects of THC and JZL184. Results demonstrate that, like exogenous cannabinoid exposure, inhibition of MAGL activity during late post-natal development persistently alters behavior in adulthood. Contrasting effects of THC vs. MAGL inhibition with JZL184 suggests the latter alters development of brain regions to favor promotion of aversive rather than appetitive cocaine responsiveness later in adulthood.

Introduction: There are few vocal learning animals that are suitable for laboratory study, and so songbirds have unique utility for evaluating drug effects on behavior learned during a critical period of development. We previously found that purified botanically-derived cannabidiol (CBD, ≥98%) mitigates effects of partial ablation of zebra finch HVC, a pre-vocal motor cortical region. Here we expand prior work to determine ability of the euphorigenic cannabis constituent, Δ9-tetrahydrocannabinol (THC) to modulate CBD efficacy. Evidence suggests relative abundance of phytocannabinoids within cannabis extracts is an important determinant of activity, with CBD:THC of particular significance. As CBD-enriched extracts have become increasingly available both by prescription and over the counter, differential efficacy associated with distinct phytocannabinoid combinations and relative CBD:THC amounts is of increasing concern. Methods and Results: To evaluate THC modulation of CBD efficacy in mitigating the effects of partial ablation of zebra finch HVC, we have tested 3 mg/kg of purified botanically derived CBD (≥98%) containing 0.02, 0.08, 1, 3 and 5% THC. Results demonstrate differential efficacy on phonology and syntax, consistent with complex, hormetic dose-responses. On phonology, CBD with the lowest THC content (3% CBD + 0.02% THC) improved recovery while that with the highest THC content (3% CBD+5% THC) slowed it. In terms of syntax, all THC concentrations improved recovery time with the higher 3 mg/kg+3% THC being distinctly effective in returning behavior to pre-injury levels, and the highest 3 mg/kg CBD+5% THC for reducing the acute magnitude of syntax disruption. Differential phonology and syntax effects likely involve distinct neural circuits that control vocal learning and production. Understanding these systems-level effects will inform mechanisms underlying both phytocannabinoid action, and learning-dependent vocal recovery. Conclusions: Overall, we have found that efficacy of purified botanically derived CBD (≥98%) to influence vocal recovery varies with THC content in complex ways. This adds to evidence of differential efficacy with phytocannabinoid combinations and ratios thereof and underscores the importance of careful control over cannabis preparations used therapeutically.

Using a songbird, zebra finches, as a developmental drug abuse model we found previously that cannabinoid agonists administered during the sensorimotor period of vocal learning (50 – 75 days of age) persistently alter song patterns and positive cocaine reinforcement in adulthood. However, these effects were not produced in adults exposed to the same treatment regimens. Currently, we have used the indirect cannabinoid agonist, JZL184, to evaluate the ability of enhanced endocannabinoid signaling to similarly alter cocaine sensitivity. Although JZL184 increased sensitivity to cocaine-reinforced place preference, it did so in an aversive manner in contrast to Δ 9 -tetrahydrocannabinol (THC, 3 mg/kg QD IM). That is, animals developmentally treated with JZL184 (4 mg/kg QD IM) avoided cocaine-paired chambers later in adulthood. To begin to identify brain regions involved in this differential responsiveness we used c-Fos expression as a marker of neuronal activity. Differences in c-Fos expression patterns following placement of cocaine-conditioned animals into vehicle- vs. cocaine-paired chambers suggest distinct involvement of circuits through striatal and amygdaloid regions in respective effects of THC and JZL184. Results demonstrate that, like exogenous cannabinoid exposure, promoting endocannabinoid signaling during late post-natal development persistently alters behavior in adulthood. Contrasting effects of exogenous agonist vs. indirect agonist suggests the latter activates a discrete subpopulation of receptors that result in altered development of brain regions controlling negative rather than positive cocaine reinforcement.

The endocannabinoid system (ECS) plays a complex role in the development of neural circuitry during fetal brain development. The cannabinoid receptor type 1 (CB 1 ) controls synaptic strength at both excitatory and inhibitory synapses and thus contributes to the balance of excitatory and inhibitory signaling. Imbalances in the ratio of excitatory to inhibitory synapses have been implicated in various neuropsychiatric disorders associated with dysregulated central nervous system development including autism spectrum disorder, epilepsy, and schizophrenia. The role of CB 1 in human brain development has been difficult to study but advances in induced pluripotent stem cell technology have allowed us to model the fetal brain environment. Cortical spheroids resemble the cortex of the dorsal telencephalon during mid-fetal gestation and possess functional synapses, spontaneous activity, an astrocyte population, and pseudo-laminar organization. We first characterized the ECS using STORM microscopy and observed synaptic localization of components similar to that which is observed in the fetal brain. Next, using the CB 1 -selective antagonist SR141716A, we observed an increase in excitatory, and to a lesser extent, inhibitory synaptogenesis as measured by confocal image analysis. Further, CB 1 antagonism increased the variability of spontaneous activity within developing neural networks, as measured by microelectrode array. Overall, we have established that cortical spheroids express ECS components and are thus a useful model for exploring endocannabinoid mediation of childhood neuropsychiatric disease.

Cannabidiol (CBD), a non-euphorigenic compound derived from Cannabis, shows promise for improving recovery following cerebral ischemia and has recently been shown effective for the treatment of childhood seizures caused by Dravet and Lennox-Gastaut syndromes. Given evidence for activity to mitigate effects of CNS insult and dysfunction, we considered the possibility that CBD may also protect and improve functional recovery of a complex learned behavior. To test this hypothesis, we have applied a songbird, the adult male zebra finch, as a novel pre-clinical animal model. Their learned vocalizations were temporarily disrupted with bilateral microlesions of HVC (used as a proper name) a pre-vocal motor cortical-like brain region that drives song. These microlesions destroy about 10% of HVC, and temporarily impair song production, syntax and phonology for about seven days. Recovery requires sensorimotor learning as it depends upon auditory feedback. Four CBD doses (0, 1, 10 and 100 mg/kg) within three surgery conditions (microlesion, no-microlesion, sham-microlesion) were evaluated (n = 5-6). Birds were recorded over 20 days: three baseline; six pre-microlesion drug treatment days and; 11 post-microlesion treatment and recovery days. Results indicate 10 and 100 mg/kg CBD effectively reduced the time required to recover vocal phonology and syntax. In the case of phonology, the magnitude of microlesion-related disruptions were also reduced. These results suggest CBD holds promise to improve functional recovery of complex learned behaviors following brain injury, and represent establishment of an important new animal model to screen drugs for efficacy to improve vocal recovery.

Zebra finches are songbirds that learn vocal patterns during a sensitive period of development that approximates adolescence. Exposure of these animals to a cannabinoid agonist during their period of sensorimotor vocal learning alters song patterns produced in adulthood. Thus, songbirds have unique value in studying developmental effects of drug exposure on a naturally learned behavior. A missing feature of this animal model has been a method to study drug reinforcement of behavior. To address this gap we have adapted place conditioning methods, used previously to determine that singing behavior is rewarding, to study cocaine reinforcement of behavior. We have found that cocaine dose-dependently reinforces both place conditioning and aversion at potencies consistent with those observed in mammalian species. Use of this place conditioning method has allowed us to determine that, when administered during periods of sensorimotor vocal learning, delta-9-THC, but not nicotine persistently increases sensitivity to cocaine through adulthood. Establishment of this method significantly expands the songbird drug exposure model, and holds promise for better appreciation of mechanisms important to sensorimotor learning that is dependent upon successful progress through sensitive periods of CNS development.

KLFromRecordingDays allows measurement of Kullback–Leibler (KL) distances between 2D probability distributions of vocal acoustic features. Greater KL distance measures reflect increased phonological divergence across the vocalizations compared. The software has been used to compare *.wav file recordings made by Sound Analysis Recorder 2011 of songbird vocalizations pre- and post-drug and surgical manipulations. Recordings from individual animals in *.wav format are first organized into subdirectories by recording day and then segmented into individual syllables uttered and acoustic features of these syllables using Sound Analysis Pro 2011 (SAP). KLFromRecordingDays uses syllable acoustic feature data output by SAP to a MySQL table to generate and compare “template” (typically pre-treatment) and “target” (typically post-treatment) probability distributions. These distributions are a series of virtual 2D plots of the duration of each syllable (as x-axis) to each of 13 other acoustic features measured by SAP for that syllable (as y-axes). Differences between “template” and “target” probability distributions for each acoustic feature are determined by calculating KL distance, a measure of divergence of the target 2D distribution pattern from that of the template. KL distances and the mean KL distance across all acoustic features are calculated for each recording day and output to an Excel spreadsheet. Resulting data for individual subjects may then be pooled across treatment groups and graphically summarized and used for statistical comparisons. Because SAP-generated MySQL files are accessed directly, data limits associated with spreadsheet output are avoided, and the totality of vocal output over weeks may be objectively analyzed all at once. The software has been useful for measuring drug effects on songbird vocalizations and assessing recovery from damage to regions of vocal motor cortex. It may be useful in studies employing other species, and as part of speech therapies tracking progress in producing distinct speech sounds in isolation.

Cannabinoids include the active constituents of Cannabis or are molecules that mimic the structure and/or function of these Cannabis-derived molecules. Cannabinoids produce many of their cellular and organ system effects by interacting with the well-characterized CB1 and CB2 receptors. However, it has become clear that not all effects of cannabinoid drugs are attributable to their interaction with CB1 and CB2 receptors. Evidence now demonstrates that cannabinoid agents produce effects by modulating activity of the entire array of cellular macromolecules targeted by other drug classes, including: other receptor types; ion channels; transporters; enzymes, and protein- and non-protein cellular structures. This review summarizes evidence for these interactions in the CNS and in cancer, and is organized according to the cellular targets involved. The CNS represents a well-studied area and cancer is emerging in terms of understanding mechanisms by which cannabinoids modulate their activity. Considering the CNS and cancer together allow identification of non-cannabinoid receptor targets that are shared and divergent in both systems. This comparative approach allows the identified targets to be compared and contrasted, suggesting potential new areas of investigation. It also provides insight into the diverse sources of efficacy employed by this interesting class of drugs. Obtaining a comprehensive understanding of the diverse mechanisms of cannabinoid action may lead to the design and development of therapeutic agents with greater efficacy and specificity for their cellular targets.

During typical late-postnatal CNS development, net reductions in dendritic spine densities are associated with activity-dependent learning. Prior results showed agonist exposure in young animals increased spine densities in a subset of song regions while adult exposures did not, suggesting endocannabinoid signaling regulates dendritic spine dynamics important to vocal development. Here we addressed this question using the CB1 receptor-selective antagonist SR141716A (SR) to disrupt endocannabinoid signaling both during and after vocal learning. We hypothesized antagonist exposure during vocal development, but not adulthood, would alter spine densities. Following 25days of exposure and a 25day maturation period, 3D reconstructions of Golgi-Cox stained neurons were used to measure spine densities. We found antagonist treatments during both age periods increased densities within Area X (basal ganglia) and following adult treatments within HVC (premotor cortical-like). Results suggest both inappropriate cannabinoid receptor stimulation and inhibition are capable of similar disregulatory effects during establishment of circuits important to vocal learning, with antagonism extending these effects through adulthood. Given clinical evidence of depressant effects of SR, we tested the ability of the antidepressant monoamine oxidase inhibitor (MAOI) phenelzine to mitigate SR-induced spine density increases. This was confirmed implicating interaction between monoamine and endocannabinoid systems. Finally, we evaluated acute effects of these drugs to alter ability of novel song exposure to increase spine densities in auditory NCM and other regions, finding when combined, SR and phenelzine increased densities within Area X. These results contribute to understanding relevance of dendritic spine dynamics in neuronal development, drug abuse, and depression.

Prior work shows developmental cannabinoid exposure alters zebra finch vocal development in a manner associated with altered CNS physiology, including changes in patterns of CB1 receptor immunoreactivity, endocannabinoid concentrations and dendritic spine densities. These results raise questions about the selectivity of developmental cannabinoid effects: are they a consequence of a generalized developmental disruption, or are effects produced through more selective and distinct interactions with biochemical pathways that control receptor, endogenous ligand and dendritic spine dynamics? To begin to address this question we have examined effects of developmental cannabinoid exposure on the pattern and density of expression of proteins critical to dendritic (MAP2) and axonal (Nf-200) structure to determine the extent to which dendritic vs. axonal neuronal morphology may be altered. Results demonstrate developmental, but not adult cannabinoid treatments produce generalized changes in expression of both dendritic and axonal cytoskeletal proteins within brain regions and cells known to express CB1 cannabinoid receptors. Results clearly demonstrate that cannabinoid exposure during a period of sensorimotor development, but not adulthood, produce profound effects upon both dendritic and axonal morphology that persist through at least early adulthood. These findings suggest an ability of exogenous cannabinoids to alter general processes responsible for normal brain development. Results also further implicate the importance of endocannabinoid signaling to peri-pubertal periods of adolescence, and underscore potential consequences of cannabinoid abuse during periods of late-postnatal CNS development.

Zebra finch song is a learned behavior dependent upon successful progress through a sensitive period of late-postnatal development. This learning is associated with maturation of distinct brain nuclei and the fiber tract interconnections between them. We have previously found remarkably distinct and dense CB1 cannabinoid receptor expression within many of these song control brain regions, implying a normal role for endocannabinoid signaling in vocal learning. Activation of CB1 receptors via daily treatments with exogenous agonist during sensorimotor stages of song learning (but not in adulthood) results in persistent alteration of song patterns. Now we are working to understand physiological changes responsible for this cannabinoid-altered vocal learning. We have found that song-altering developmental treatments are associated with changes in expression of endocannabinoid signaling elements, including CB1 receptors and the principal CNS endogenous agonist, 2-AG. Within CNS, 2-AG is produced largely through activity of the α isoform of the enzyme diacylglycerol lipase (DAGLα). To better appreciate the role of 2-AG production in normal vocal development we have determined the spatial distribution of DAGLα expression within zebra finch CNS during vocal development. Early during vocal development at 25 days, DAGLα staining is typically light and of fibroid processes. Staining peaks late in the sensorimotor stage of song learning at 75 days and is characterized by fiber, neuropil and some staining of both small and large cell somata. Results provide insight to the normal role for endocannabinoid signaling in the maturation of brain regions responsible for song learning and vocal-motor output, and suggest mechanisms by which exogenous cannabinoid exposure alters acquisition of this form of vocal communication.

Cannabinoids are well-established to alter processes of sensory perception; however neurophysiological mechanisms responsible remain unclear. Arc, an immediate-early gene (IEG) product involved in dendritic spine dynamics and necessary for plasticity changes such as long-term potentiation, is rapidly induced within zebra finch caudal medial nidopallium (NCM) following novel song exposure, a response that habituates after repeated stimuli. Arc appears unique in its rapid postsynaptic dendritic expression following excitatory input. Previously, we found that vocal development-altering cannabinoid treatments are associated with elevated dendritic spine densities in motor-(HVC) and learning-related (Area X) song regions of zebra finch telencephalon. Given Arc's dendritic morphological role, we hypothesized that cannabinoid-altered spine densities may involve Arc-related signaling. To test this, we examined the ability of the cannabinoid agonist WIN55212-2 (WIN) to (1) acutely disrupt song-induced Arc expression, (2) interfere with habituation to auditory stimuli, and (3) alter dendritic spine densities in auditory regions. We found that WIN (3 mg/kg) acutely reduced Arc expression within both NCM and Field L2 in an antagonist-reversible manner. WIN did not alter Arc expression in thalamic auditory relay nucleus ovoidalis (Ov), suggesting that cannabinoid signaling selectively alters responses to auditory stimulation. Novel song stimulation rapidly increased dendritic spine densities within auditory telencephalon, an effect blocked by WIN pretreatments. Taken together, cannabinoid inhibition of both Arc induction and its habituation to repeated stimuli, combined with prevention of rapid increases in dendritic spine densities, implicates cannabinoid signaling in modulation of physiological processes important to auditory responsiveness and memory.

Normal CNS development proceeds through late-postnatal stages of adolescent development. The activity-dependence of this development underscores the significance of CNS-active drug exposure prior to completion of brain maturation. Exogenous modulation of signaling important in regulating normal development is of particular concern. This mini-review presents a summary of the accumulated behavioral, physiological and biochemical evidence supporting such a key regulatory role for endocannabinoid signaling during late-postnatal CNS development. Our focus is on the data obtained using a unique zebra finch model of developmental psychopharmacology. This animal has allowed investigation of neuronal morphological effects essential to establishment and maintenance of neural circuitry, including processes related to synaptogenesis and dendritic spine dynamics. Altered neurophysiology that follows exogenous cannabinoid exposure during adolescent development has the potential to persistently alter cognition, learning and memory.

Recent findings indicate that cannabinoid-altered vocal development involves elevated densities of dendritic spines in a subset of brain regions involved in zebra finch song learning and production suggesting that cannabinoid receptor activation may regulate cell structure. Here we report that activation of zebra finch CB1 receptors (zfCB(1), delivered by a lentivector to CHO cells) produces dose-dependent biphasic effects on the mean length of filopodia expressed: Low agonist concentrations (3 nM WIN55212-2) increase lengths while higher concentrations reduce them. In contrast, treatment of zfCB(1)-expressing cells with the antagonist/inverse agonist SR141716A causes increases in both mean filopodia length and number at 30 and 100 nM. These results demonstrate that CB1 receptor activation can differentially influence filiopodia elongation depending on dose, and demonstrate that manipulation of cannabinoid receptor activity is capable of modulating cell morphology.

Centrally acting cannabinoids are well known for their ability to impair functions associated with both learning and memory but appreciation of the physiological mechanisms underlying these actions, particularly those that persist, remains incomplete. Our earlier studies have shown that song stereotypy is persistently reduced in male zebra finches that have been developmentally exposed to cannabinoids. In the present work, we examined the extent to which changes in neuronal morphology (dendritic spine densities and soma size) within brain regions associated with zebra finch vocal learning are affected by late-postnatal cannabinoid agonist exposure. We found that daily treatment with the cannabinoid agonist WIN55212-2 (WIN, 1 mg/kg IM) is associated with 27% and 31% elevations in dendritic spine densities in the song regions Area X and HVC, respectively. We also found an overall increase in cell diameter within HVC. Changes in dendritic spine densities were only produced following developmental exposure; treatments given to adults that had completed vocal learning were not effective. These findings have important implications for understanding how repeated cannabinoid exposure can produce significant, lasting alteration of brain morphology, which may contribute to altered development and behavior.

BACKGROUND:Previously we have found that cannabinoid treatment of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. Such persistently altered behavior must be attributable to changes in physiological substrates responsible for song. We are currently working to identify the nature of such physiological changes, and to understand how they contribute to altered vocal learning. One possibility is that developmental agonist exposure results in altered expression of elements of endocannabinoid signaling systems. To test this hypothesis we have studied effects of the potent cannabinoid receptor agonist WIN55212-2 (WIN) on endocannabinoid levels and densities of CB1 immunostaining in zebra finch brain.RESULTS:We found that late postnatal WIN treatment caused a long-term global disregulation of both levels of the endocannabinoid, 2-arachidonyl glycerol (2-AG) and densities of CB1 immunostaining across brain regions, while repeated cannabinoid treatment in adults produced few long-term changes in the endogenous cannabinoid system.CONCLUSIONS:Our findings indicate that the zebra finch endocannabinoid system is particularly sensitive to exogenous agonist exposure during the critical period of song learning and provide insight into susceptible brain areas.

We have demonstrated that nicotine attenuated ethanol-induced ataxia via nicotinic-acetylcholine-receptor (nAChR) subtypes α4β2 and α7. In the present study, ethanol (2g/kg; i.p.)-induced ataxia was assessed by Rotorod performance following repeated intracerebellar infusion of α4β2- and α7-selective agonists. Localization of α4β2 and α7 nAChRs was confirmed immunohistochemically. Cerebellar NOx (nitrite+nitrate) was determined flurometrically. Repeated intracerebellar microinfusion of the α4β2-selective agonist, RJR-2403 (for 1, 2, 3, 5 or 7 days) or the α7-selective agonist, PNU-282987 (1, 2, 3 or 5 days), dose-dependently attenuated ethanol-induced ataxia. These results suggest the development of cross-tolerance between ethanol-induced ataxia and α4β2 and α7 nAChR agonists. With RJR-2403, the cross-tolerance was maximal after a 5-day treatment and lasted 48h. Cross-tolerance was maximal after a 1-day treatment with PNU-282987 and lasted 72h. Pretreatment with α4β2- and α7-selective antagonists, dihydro-β-erythroidine and methyllycaconitine, respectively, prevented the development of cross-tolerance confirming α4β2 and α7 involvement. Repeated agonist infusions elevated cerebellar NOx 16h after the last treatment while acute ethanol exposure decreased it. Pretreatment with repeated RJR-2403 or PNU-282987 reversed ethanol-induced decrease in NOx. The NOx data suggests the involvement of the nitric oxide (NO)–cGMP signaling pathway in the cross-tolerance that develops between α4β2- and α7-selective agonists and ethanol ataxia. Both α4β2 and α7 subtypes exhibited high immunoreactivity in Purkinje but sparse expression in molecular and granular cell layers. Our results support a role for α4β2 and α7 nAChR subtypes in the development of cross-tolerance between nicotine and ethanol with the NO signaling pathway as a potential mechanism.