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The cannabinoid type 1 (CB1 ) receptor and the dopamine type 2 (D2 ) receptor are co-localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB1 /D2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB1 /D2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB1 /D2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB1 /D2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB1 /D2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co-treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co-treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ-aminobutyric acid release in STHdhQ7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co-exposure to cannabinoids and different antipsychotics over acute and prolonged time periods.

Huntington’s disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB(1)) levels in the striatum, which is strongly correlated with HD pathogenesis. CB(1) positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and 2-arachidonoylglycerol. The goal of this study was to determine whether the recently characterized CB(1) allosteric modulators GAT211 (racemic), GAT228 (R-enantiomer), and GAT229 (S-enantiomer), affected the signs and symptoms of HD. GAT211, GAT228, and GAT229 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/2 mice, 10 mg/kg/d, 21 d, i.p.). GAT229 was a CB(1) PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/2 HD mice. GAT228 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/2 mice. GAT211 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB(1) PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/2 HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB(1) PAMs to treat the signs and symptoms of HD.

We apply the magic methyl effect to improve the potency/efficacy of GAT211, the prototypic 2-phenylindole-based cannabinoid type-1 receptor (CB1R) agonist-positive allosteric modulator (ago-PAM). Introducing a methyl group at the α-position of nitro group generated two diastereomers, the greater potency and efficacy of erythro, (±)-9 vs threo, (±)-10 constitutes the first demonstration of diastereoselective CB1R-allosteric modulator interaction. Of the (±)-9 enantiomers, (-)-(S,R)-13 evidenced improved potency over GAT211 as a CB1R ago-PAM, whereas (+)-(R,S)-14 was a CB1R allosteric agonist biased toward G protein- vs β-arrestin1/2-dependent signaling. (-)-(S,R)-13 and (+)-(R,S)-14 were devoid of undesirable side effects (triad test), and (+)-(R,S)-14 reduced intraocular pressure with an unprecedentedly long duration of action in a murine glaucoma model. (-)-(S,R)-13 docked into both a CB1R extracellular PAM and intracellular allosteric-agonist site(s), whereas (+)-(R,S)-14 preferentially engaged only the latter. Exploiting G-protein biased CB1R-allosteric modulation can offer safer therapeutic candidates for glaucoma and, potentially, other diseases.

Background and purpose We sought to understand why (-)-cannabidiol (CBD) and (-)-cannabidiol-dimethylheptyl (CBD-DMH) exhibit distinct pharmacology, despite near identical structures. Experimental approach HEK293A cells expressing either human type 1 cannabinoid (CB1 ) receptors or CB2 receptors were treated with CBD or CBD-DMH with or without the CB1 and CB2 receptor agonist CP55,940, CB1 receptor allosteric modulator Org27569 or CB2 receptor inverse agonist SR144528. Ligand binding, cAMP levels and βarrestin1 recruitment were measured. CBD and CBD-DMH binding was simulated with models of human CB1 or CB2 receptors, based on the recently published crystal structures of agonist-bound (5XRA) or antagonist-bound (5TGZ) human CB1 receptors. Key results At CB1 receptors, CBD was a negative allosteric modulator (NAM), and CBD-DMH was a mixed agonist/positive allosteric modulator. CBD and Org27569 shared multiple interacting residues in the antagonist-bound model of CB1 receptors (5TGZ) but shared a binding site with CP55,940 in the agonist-bound model of CB1 receptors (5XRA). The binding site for CBD-DMH in the CB1 receptor models overlapped with CP55,940 and Org27569. At CB2 receptors, CBD was a partial agonist, and CBD-DMH was a positive allosteric modulator of cAMP modulation but a NAM of βarrestin1 recruitment. CBD, CP55,940 and SR144528 shared a binding site in the CB2 receptor models that was separate from CBD-DMH. Conclusion and implications The pharmacological activity of CBD and CBD-DMH in HEK293A cells and their modelled binding sites at CB1 and CB2 receptors may explain their in vivo effects and illuminates the difficulties associated with the development of allosteric modulators for CB1 and CB2 receptors. Linked articles This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Type 1 cannabinoid (CB1) and dopamine 2 long form (D2L) receptors can physically interact to form heteromers that display unique pharmacology in vitro compared to homomeric complexes. Co-expression of CB1 and D2L and co-application of CB1 and D2 agonists increases cAMP levels while administration of either agonist alone decreases cAMP levels. To understand the observed co-agonist response, our first goal of the current study was to define the stoichiometry of CB1/D2L/Gα protein complexes. Using bioluminescence resonance energy transfer 2 (BRET2), we confirmed that, CB1 homodimers, D2L homodimers, and CB1/D2L heteromers are formed. By using sequential resonance energy transfer 2 (SRET2) combined with bimolecular fluorescence complementation (BiFC), we were able to demonstrate that CB1/D2L form heterotetramers consisting of CB1 and D2L homodimers. We demonstrated that CB1/D2L heterotetramers are coupled to at least two Gα proteins. The second aim of the study was to investigate allosteric effects of a D2L agonist (quinpirole) on CB1 receptor function and to investigate the effects of a CB1 agonist [arachidonyl-2-chloroethylamide (ACEA)] on D2L receptor function within CB1/D2L heterotetramers. Treating cells co-expressing CB1 and D2L with both ACEA and quinpirole switched CB1 and D2L receptor coupling and signaling from Gαi to Gαs proteins, enhanced β-arrestin1 recruitment and receptor co-internalization. The concept of bidirectional allosteric interaction within CB1/D2 heterotetramers has important implications for understanding the activity of receptor complexes in native tissues and under pathological conditions.

In addition to its known actions as a non-selective cyclooxygenase (COX) 1 and 2 inhibitor, we hypothesized that indomethacin can act as an allosteric modulator of the type 1 cannabinoid receptor (CB1R) because of its shared structural features with the known allosteric modulators of CB1R. Indomethacin enhanced the binding of [3H]CP55940 to hCB1R and enhanced AEA-dependent [35S]GTPγS binding to hCB1R in Chinese hamster ovary (CHO) cell membranes. Indomethacin (1 μM) also enhanced CP55940-dependent βarrestin1 recruitment, cAMP inhibition, ERK1/2 and PLCβ3 phosphorylation in HEK293A cells expressing hCB1R, but not in cells expressing hCB2R. Finally, indomethacin enhanced the magnitude and duration of CP55940-induced hypolocomotion, immobility, hypothermia, and anti-nociception in C57BL/6J mice. Together, these data support the hypothesis that indomethacin acted as a positive allosteric modulator of hCB1R. The identification of structural and functional features shared amongst allosteric modulators of CB1R may lead to the development of novel compounds designed for greater CB1R or COX selectivity and compounds designed to modulate both the prostaglandin and endocannabinoid systems.

(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (&szlig, arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R &gt, CB1R, with both ligands acting as full agonists in cAMP and &szlig, arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.

The cannabinoid 1 receptor (CB1R) is one of the most widely expressed metabotropic G protein-coupled receptors in brain, and its participation in various (patho)physiological processes has made CB1R activation a viable therapeutic modality. Adverse psychotropic effects limit the clinical utility of CB1R orthosteric agonists and have promoted the search for CB1R positive allosteric modulators (PAMs) with the promise of improved drug-like pharmacology and enhanced safety over typical CB1R agonists. In this study, we describe the synthesis and in vitro and ex vivo pharmacology of the novel allosteric CB1R modulator GAT211 (racemic) and its resolved enantiomers, GAT228 (R) and GAT229 (S). GAT211 engages CB1R allosteric site(s), enhances the binding of the orthosteric full agonist [3H]CP55,490, and reduces the binding of the orthosteric antagonist/inverse agonist [3H]SR141716A. GAT211 displayed both PAM and agonist activity in HEK293A and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in mouse-bra...

Proliferative vitreoretinopathy (PVR) can develop after ocular trauma or inflammation and is a common complication of surgery to correct retinal detachment. Currently, there are no pharmacological treatments for PVR. Cannabinoids acting at cannabinoid 2 receptor (CB2R) can decrease inflammation and fibrosis. The objective of this study was to examine the anti-inflammatory actions of CB2R as a candidate novel therapeutic target in experimental PVR. PVR was induced by intravitreal injection of dispase in wild-type (WT) and CB2R genetic knockout (CB2R −/− ) mice. Ocular pathology was studied at 24 h or one week after dispase injection. CB2R modulation was examined in WT mice, using the CB2R agonist, HU308, and the CB2R antagonist, AM630. Histopathological scoring and quantification of microglia was used to evaluate tissue pathology. Quantitative PCR and multiplex assays were used to assess changes in proinflammatory cytokines. Intravital microscopy (IVM) was used to visualize and quantify leukocyte-endothelial adhesion to the iridial microcirculation. Activation of CB2R with HU308 in WT mice with PVR decreased mean histopathological scores, the number of microglia, and leukocyte adhesion compared to vehicle-treated animals. Conversely, an increase in histopathological scores and activated microglia was observed in PVR animals after treatment with AM630. CB2R −/− mice with PVR exhibited exacerbated ocular histopathology, increased microglia numbers, and elevated protein levels of cytokines as compared to WT mice. In conclusion, our results indicate that intervention at early stage PVR with CB2R agonists reduces ocular inflammation and disease severity. CB2R may represent a therapeutic target to prevent PVR progression and vision loss. This article is part of the Special Issue entitled ‘Lipid Sensing G Protein-Coupled Receptors in the CNS’.

Central nervous system (CNS) injury, such as stroke or trauma, is known to increase susceptibility to various infections that adversely affect patient outcomes (CNS injury-induced immunodepression—CIDS). The endocannabinoid system (ECS) has been shown to have immunoregulatory properties. Therefore, the ECS might represent a druggable target to overcome CIDS. Evidence suggests that cannabinoid type 2 receptor (CB2R) activation can be protective during the early pro-inflammatory phase after CNS injury, as it limits neuro-inflammation and, therefore, attenuates CIDS severity. In the later phase post CNS injury, CB2R inhibition is suggested as a promising pharmacologic strategy to restore immune function in order to prevent infection.

The cannabinoid receptor CB2 plays a significant role in the regulation of immune function whereas neuronal expression remains a subject of contention. Multiple studies have described CB2 in retina and a recent study showed that CB2 deletion altered retinal visual processing. We revisited CB2 expression using immunohistochemistry and a recently developed CB2-eGFP reporter mouse. We examined the consequence of acute vs. prolonged CB2 deactivation on the electroretinogram (ERG) responses. We also examined lipidomics in CB2 knockout mice and potential changes in microglia using Scholl analysis. Consistent with a published report, in CB2 receptor knockout mice see an increased ERG scotopic a-wave, as well as stronger responses in dark adapted cone-driven ON bipolar cells and, to a lesser extent cone-driven ON bipolar cells early in light adaptation. Significantly, however, acute block with CB2 antagonist, AM630, did not mimic the results observed in the CB2 knockout mice whereas chronic (7 days) block did. Immunohistochemical studies show no CB2 in retina under non-pathological conditions, even with published antibodies. Retinal CB2–eGFP reporter signal is minimal under baseline conditions but upregulated by intraocular injection of either LPS or carrageenan. CB2 knockout mice see modest declines in a broad spectrum of cannabinoid-related lipids. The numbers and morphology of microglia were unaltered. In summary minimal CB2 expression is seen in healthy retina. CB2 appears to be upregulated under pathological conditions. Previously reported functional consequences of CB2 deletion are an adaptive response to prolonged blockade of these receptors. CB2 therefore impacts retinal signaling but perhaps in an indirect, potentially extra-ocular fashion. post-print 8285 KB

Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced.

Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gβγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.

Interstitial cystitis (IC) is a chronic bladder disorder with unclear etiology. The endocannabinoid system has been identified as a key regulator of immune function, with experimental evidence for the involvement of cannabinoid receptors in bladder inflammation. This study used intravital microscopy (IVM) and behavioral testing in lipopolysaccharide-induced IC, to investigate the anti-inflammatory analgesic effects of a natural dietary sesquiterpenoid, beta-caryophyllene (BCP), which is present in cannabis among other plants, and has reported agonist actions at the cannabinoid 2 receptor (CB2R). BCP&rsquo, s anti-inflammatory actions were compared to the synthetic CB2R-selective cannabinoid, HU308, and to an FDA-approved clinical treatment (dimethyl sulfoxide: DMSO). IVM data revealed that intravesical instillation of BCP and/or HU308 significantly reduces the number of adhering leukocytes in submucosal bladder venules and improves bladder capillary perfusion. The effects of BCP were found to be comparable to that of the selective CB2R synthetic cannabinoid, HU308, and superior to intravesical DMSO treatment. Oral treatment with BCP was also able to reduce bladder inflammation and significantly reduced mechanical allodynia in experimental IC. Based on our findings, we believe that CB2R activation may represent a viable therapeutic target for IC, and that drugs that activate CB2R, such as the generally regarded as safe (GRAS) dietary sesquiterpenoid, BCP, may serve as an adjunct and/or alternative treatment option for alleviating symptoms of inflammation and pain in the management of IC.

The human type 1 cannabinoid (hCB1) receptor is expressed at high levels in the central nervous system. mRNA variants of the coding region of this receptor, human cannabinoid hCB1a and hCB1b receptors, have been identified, their biological function remains unclear. The present study demonstrated that the three human cannabinoid hCB1 coding region variants are expressed in the human and monkey (Macaca fascicularis) brain. Western blot analyses of homogenates from different regions of the monkey brain demonstrated that proteins with the expected molecular weights of the cannabinoid CB1, CB1a and CB1b receptors were co-expressed throughout the brain. Given the co-localization of these receptors, we hypothesized that physical interactions between the three splice variants may affect cannabinoid pharmacology. The human cannabinoid hCB1, hCB1a, and hCB1b receptors formed homodimers and heterodimers, as determined by BRET in transiently transfected HEK 293A cells. We found that the co-expression of the human cannabinoid hCB1 and each of the splice variants increased cell surface expression of the human cannabinoid hCB1 receptor and increased Gi/o-dependent ERK phosphorylation in response to cannabinoid agonists. Therefore, the human cannabinoid hCB1 coding region splice variants play an important physiological role in the activity of the endocannabinoid system.

The type 1 cannabinoid receptor (CB1) is a G protein-coupled receptor that is expressed at high levels in the striatum. Activation of CB1 increases expression of neuronal trophic factors and inhibits neurotransmitter release from GABA-ergic striatal neurons. CB1 mRNA levels can be elevated by treatment with cannabinoids in non-neuronal cells. We wanted to determine whether cannabinoid treatment could induce CB1 expression in a cell culture model of striatal neurons and, if possible, determine the molecular mechanism by which this occurred. We found that treatment of STHdh(7/7) cells with the cannabinoids ACEA, mAEA, and AEA produced a CB1receptor-dependent increase in CB1 promoter activity, mRNA, and protein expression. This response was Akt- and NF-κB-dependent. Because decreased CB1 expression is thought to contribute to the pathogenesis of Huntington's disease (HD), we wanted to determine whether cannabinoids could increase CB1 expression in STHdh(7/111) and (111/111) cells expressing the mutant huntingtin protein. We observed that cannabinoid treatment increased CB1 mRNA levels approximately 10-fold in STHdh(7/111) and (111/111) cells, compared to vehicle treatment. Importantly, cannabinoid treatment improved ATP production, increased the expression of the trophic factor BDNF-2, and the mitochondrial regulator PGC1α, and reduced spontaneous GABA release, in HD cells. Therefore, cannabinoid-mediated increases in CB1 levels could reduce the severity of some molecular pathologies observed in HD.

The endocannabinoid, N-arachidonoylethanolamine (AEA), is degraded by the enzyme fatty acid amide hydrolase (FAAH). This study examined whether the FAAH inhibitor, URB597, increases retinal ganglion cell (RGC) survival following optic nerve axotomy in young and aged animals. URB597 alone, or together with either a CB1 or CB2 receptor antagonist, was administered daily for 1 or 2 weeks post-axotomy. Histological assessment of retinas indicated that URB597 increased RGC survival in young retina at 1 and 2 weeks post-axotomy. The increase in RGC survival at 2 weeks was accompanied by a reduction in phagocytic microglia. The CB1 antagonist, AM281, but not the CB2 antagonist, AM630, ablated URB597-mediated RGC neuroprotection. CB1 or CB2 antagonism increased phagocytic microglia in URB597 and vehicle-treated animals. In aged animals, URB597 increased RGC survival at 1 week, but not at 2 weeks post-axotomy and had no effect on microglia. Retinal Iba-1 positive microglia were also decreased in URB597-treated axotomized young animals and this decrease was mitigated by CB1 but not CB2 antagonism. As seen with phagocytotic microglia, the CB2 antagonist, AM630, increased Iba-1 positive microglia in the absence of URB597 treatment. Measurement of retinal endocannabinoid levels in URB597-treated animals at 2 weeks post-axotomy revealed a significant increase in AEA levels, accompanied by a decrease in the AEA metabolite, N-arachidonoyl glycine, in young animals but not aged animals. 2-arachidonoylglycerol levels were similar across all experimental groups. These data demonstrate that URB597-mediated retinal neuroprotective effects are mediated primarily through CB1 receptors and that URB597 neuroprotective efficacy declines with age.

Background and Purpose GPR18 is a recently deorphaned lipid receptor that is activated by the endogenous lipid N-arachidonoyl glycine (NAGly) as well the behaviourally inactive atypical cannabinoid, abnormal cannabidiol (Abn-CBD). The presence and/or function of any GPR18-based ocular signalling system remain essentially unstudied. The objectives of this research are: (i) to determine the disposition of GPR18 receptors and ligands in anterior murine eye, (ii) examine the effect of GPR18 activation on intraocular pressure (IOP) in a murine model, including knockout mice for CB1, CB2 and GPR55. Experimental Approach IOP was measured in mice following topical application of Abn-CBD, NAGly or the GPR55/GPR18 agonist O-1602, alone or with injection of the GPR18 antagonist, O-1918. GPR18 protein localization was assessed with immunohistochemistry. Endocannabinoids were measured using LC/MS-MS. Key Results GPR18 protein was expressed most prominently in the ciliary epithelium and the corneal epithelium and, interestingly, in the trabecular meshwork. The GPR18 ligand, NAGly, was also detected in mouse eye at a level comparable to that seen in the brain. Abn-CBD and NAGly, but not O-1602, significantly reduced IOP in all mice tested. The antagonist, O-1918, blocked the effects of Abn-CBD and NAGly. Conclusions and Implications We present evidence for a functional GPR18-based signalling system in the murine anterior eye, including receptors and ligands. GPR18 agonists, Abn-CBD and NAGly, reduce IOP independently of CB1, CB2 or GPR55. These findings suggest that GPR18 may serve as a desirable target for the development of novel ocular hypotensive medications. Linked Articles This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8

Cannabinoids are known to modulate cardiovascular functions including heart rate, vascular tone, and blood pressure in humans and animal models. Essential components of the endocannabinoid system, namely, the production, degradation, and signaling pathways of endocannabinoids have been described not only in the central and peripheral nervous system but also in myocardium, vasculature, platelets, and immune cells. The mechanisms of cardiovascular responses to endocannabinoids are often complex and may involve cannabinoid CB1 and CB2 receptors or non-CB1/2 receptor targets. Preclinical and some clinical studies have suggested that targeting the endocannabinoid system can improve cardiovascular functions in a number of pathophysiological conditions, including hypertension, metabolic syndrome, sepsis, and atherosclerosis. In this chapter, we summarize the local and systemic cardiovascular effects of cannabinoids and highlight our current knowledge regarding the therapeutic potential of endocannabinoid signaling and modulation.

Leukocyte-endothelial interactions within the microvasculature represent a hallmark of inflammation regardless of whether the inflammation results from non-infectious or infectious triggers. In this review, we highlight features of leukocyte recruitment in ocular disease and postulate mechanisms by which the infiltrating cells may lead to the progression of the ocular inflammatory response, including cytokine and chemokine production, T cell or non-T cell responses. Additionally, ex-vivo and in vivo methods used to study the general features of the immune response are discussed, with a specific focus on intravital imaging, which allows real-time non-invasive examination of leukocyte-endothelial interactions in the ocular microvasculature. At the present time there are still significant gaps in our understanding of the process of leukocyte recruitment in vivo in different microvascular beds. Further studies using non-invasive imaging approaches, such as intravital microscopy, provide an opportunity to study dynamic tissue-specific leukocyte-endothelial interactions in vivo and identify novel targets for early intervention in the inflammatory process. This knowledge is essential to the rational use of therapeutics to resolve inflammation in ocular disease.

UNLABELLED The type 1 cannabinoid receptor (CB(1) ) is an integral component of the endocannabinoid system that modulates several functions in the CNS and periphery. The majority of our knowledge of the endocannabinoid system involves ligand-receptor binding, mechanisms of signal transduction, and protein-protein interactions. In contrast, comparatively little is known about regulation of CB(1) gene expression. The levels and anatomical distribution of CB(1) mRNA and protein are developmental stage-specific and are dysregulated in several pathological conditions. Moreover, exposure to a variety of drugs, including cannabinoids themselves, alters CB(1) gene expression and mRNA levels. As such, alterations in CB(1) gene expression are likely to affect the optimal response to cannabinoid-based therapies, which are being developed to treat a growing number of conditions. Here, we will examine the regulation of CB(1) mRNA levels and the therapeutic potential inherent in manipulating expression of this gene. LINKED ARTICLES This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8.

Intraocular pressure (IOP) is the primary risk factor for glaucoma, a blinding eye disease. Cannabinoid agonists have long been known to decrease IOP, suggesting they may be useful in glaucoma treatment. However, the specific mechanism by which cannabinoids generate this ocular hypotensive effect remains unknown. The current evidence suggests the cannabinoids reduce IOP through actions at cannabinoid 1 (CB 1 ) receptors within the eye, and adrenergic receptors (ARs) may also contribute to this action of cannabinoids. Considering this, the present study aimed to elucidate the mechanism behind the ocular hypotensive properties of cannabinoids through the use of mice genetically lacking either cannabinoid receptors or βARs. Cannabinoid agonists, βAR antagonists, and βAR agonists decreased IOP in wild-type mice and CB 2 (−/−) mice. In contrast, none of these compounds were found to reduce IOP in βAR(−/−) or CB 1 (−/−) mice. Desensitization of the βARs and depletion of catecholamines in wild-type mice also eliminated the ability of the cannabinoid agonist ( R )-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3- de ]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) to reduce IOP, strongly implicating a role for both βARs and catecholamines in the ocular hypotensive properties of cannabinoids. Finally, CB 1 receptors were shown to colocalize with tyrosine hydroxylase, a marker for adrenergic neurons. Taken together, these findings suggest that βARs are required for the ocular hypotensive properties of cannabinoids, and cannabinoids reduce IOP by acting as indirect sympatholytics and inhibiting norepinephrine release within the eye.

Activation of dopamine receptor 2 long (D2L) switches the signaling of type 1 cannabinoid receptor (CB1) from Gαi to Gαs, a process thought to be mediated through CB1-D2L heteromerization. Given the clinical importance of D2 antagonists, the goal of this study was to determine if D2 antagonists could modulate CB1 signaling. Interactions between CB1 and D2L, Gαi, Gαs, and β-arrestin1 were studied using bioluminescence resonance energy transfer 2 (BRET(2)) in STHdh(Q7/Q7) cells. CB1-dependent extracellular regulated kinase (ERK)1/2, CREB phosphorylation, and CB1 internalization following cotreatment of CB1 agonist and D2 antagonist were quantified. Preassembled CB1-Gαi complexes were detected by BRET(2) Arachidonyl-2'-chloroethylamide (ACEA), a selective CB1 agonist, caused a rapid and transient increase in BRET efficiency (BRETEff) between Gαi-Rluc and CB1-green fluorescent protein 2 (GFP(2)), and a Gαi-dependent increase in ERK phosphorylation. Physical interactions between CB1 and D2L were observed using BRET(2) Cotreatment of STHdh(Q7/Q7) cells with ACEA and haloperidol, a D2 antagonist, inhibited BRETEff signals between Gαi-Rluc and CB1-GFP(2) and reduced the EMax and pEC50 of ACEA-mediated Gαi-dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRETEff between Gαs-Rluc and CB1-GFP(2) and increased the EMax and pEC50 of ACEA-induced Gαs-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB1 and D2L treated with ACEA, binding of haloperidol to D2 receptors switched CB1 coupling from Gαi to Gαs In addition, haloperidol treatment reduced ACEA-induced β-arrestin1 recruitment to CB1 and CB1 internalization. D2 antagonists allosterically modulate cannabinoid-induced CB1 coupling, signaling, and β-arrestin1 recruitment through binding to CB1-D2L heteromers. These findings indicate that D2 antagonism, like D2 agonists, change agonist-mediated CB1 coupling and signaling.

Background and purpose: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM). Experimental approach: CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis. Key results: WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10–100 μM) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+]i was pertussis toxin (PTX)-insensitive, therefore, independent of Gi/o coupling, but was attenuated by a dominant negative Gαq/11 subunit, implicating a Gq/11 signalling pathway. The increase in [Ca2+]i was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTX-sensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a Gi/o signalling pathway. CB1-Gq/11 coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+]i at concentrations ≥15 μM, and PTX-sensitive increases in ERK1/2 phosphorylation. Conclusions and implications: This study demonstrates that endogenous CB1 couples to both Gq/11 and Gi/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP. British Journal of Pharmacology (2007) 152, 1111–1120; doi:10.1038/sj.bjp.0707495; published online 8 October 2007

Glaucoma is an irreversible blinding eye disease which produces progressive retinal ganglion cell (RGC) loss. Intraocular pressure (IOP) is currently the only modifiable risk factor, and lowering IOP results in reduced risk of progression of the disorder. The endocannabinoid system (ECS) has attracted considerable attention as a potential target for the treatment of glaucoma, largely due to the observed IOP lowering effects seen after administration of exogenous cannabinoids. However, recent evidence has suggested that modulation of the ECS may also be neuroprotective. This paper will review the use of cannabinoids in glaucoma, presenting pertinent information regarding the pathophysiology of glaucoma and how alterations in cannabinoid signalling may contribute to glaucoma pathology. Additionally, the mechanisms and potential for the use of cannabinoids and other novel agents that target the endocannabinoid system in the treatment of glaucoma will be discussed.

In the present study, we compared the in vivo neuroprotective efficacy of intraperitoneally administered tetracycline and minocycline to enhance the survival of retinal ganglion cells (RGCs) following unilateral axotomy of the adult rat optic nerve. We also examined the effects of the tetracycline drugs on the activation of retinal microglia. RGCs in retinal whole-mounts were visualized by retrograde labeling with fluorogold. The presence of activated microglia was confirmed immunohistochemically using OX-42 monoclonal antibodies. Optic nerve axotomy produced RGC death and increased activation of microglia. No significant RGC loss was seen prior to 5 days and approximately 50% and 80-90% cell loss occurred at 7 and 14 days, respectively. Examination of the effects of tetracycline and minocycline on RGC survival at 7 days post-axotomy, revealed increased numbers of RGCs in minocycline-treated animals (75% of non-axotomized control) compared with vehicle-only (52% of control) and tetracycline-treated (58% of control) animals. The densities of RGCs (RGCs/mm2+/-S.D.) for control, vehicle-, tetracycline- and minocycline-treated axotomized animals were 1996+/-81, 1029+/-186, 1158+/-190 and 1497+/-312, respectively. The neuroprotective effect of minocycline seen at 7 days was transient, since RGCs present in minocycline-treated animals at 14 days post-axotomy (281+/-43, 14% of control) were not significantly different to vehicle-treated animals (225+/-47, 11% of control). OX-42 staining of activated retinal microglia was reduced in tetracycline- and minocycline-treated axotomized animals compared with axotomized animals receiving vehicle-only. These results demonstrate that systemic administration of the second-generation tetracycline derivative, minocycline, delays the death of axotomized RGCs by a mechanism that may be associated with inhibition of microglia activation. The neuroprotective efficacy of minocycline following optic nerve axotomy was superior to that of tetracycline.

We report on seizures during anesthesia induction in animals treated with a cannabinoid receptor 1 (CB1R) antagonist for experimental sepsis. Animals received surgery for colon ascendens stent peritonitis-induced sepsis or sham surgery followed by treatment of CB1R antagonist, CB1R agonist, or placebo. Fourteen hours later, animals received pentobarbital or ketamine for anesthesia induction and animal behavior was observed. Tonic-clonic seizures were observed in 5 of 12 septic animals (42%) treated with CB1R antagonist after induction of anesthesia with pentobarbital. The data suggest that CB1R inhibition in combination with pentobarbital may increase the incidence of anesthetic-induced seizures in the case of sepsis.

Purpose Topical beta-blockers, such as timolol, have been used extensively in the medical treatment of glaucoma to lower intraocular pressure (IOP). Recently, these drugs have been shown to have effects on the retinal and optic nerve circulation as well as potential neuroprotective properties. In the current study, the concentration of timolol attained in the cornea, iris-ciliary body, retina, vitreous, and plasma was measured after topical or intraperitoneal administration in rats to determine the relative contributions of each route to intraocular timolol concentrations. Materials and methods One group of rats received one drop of commercially available 0.5% timolol in the right eye and two drops in the left eye for 3 to 12 days. Another group of rats received one drop of 0.5% timolol in one eye only and concentrations were studied in the ocular tissues at 15, 30, 60, 120, and 240 minutes after instillation. The final group of rats received a single intraperitoneal injection of timolol ranging in concentration from 5 to 75 mg/kg after which tissue and plasma concentrations were measured 30 minutes after injection. All tissue and plasma concentrations were measured by high performance liquid chromatography. Results Rats that received topical timolol daily for 3 to 12 consecutive days accumulated timolol concentrations of 2.3 to 4.4 microg/g in cornea, 198 to 326 microg/g in iris, 0.05 to 0.11 microg/ml in vitreous, and 0.17 to 0.77 microg/g in retina. In rats that received a single drop of timolol in one eye, the tissue concentrations were higher in the treated eye than in the untreated eye in all cases except for vitreous. In these experiments, timolol levels in plasma were either low or not detectable. Increasing timolol doses administered intraperitoneally resulted in corresponding increased tissue and plasma concentrations. Conclusions Absorption of drug into the systemic circulation plays a significant role in delivering timolol to the retina and vitreous in addition to a local ocular route. A clear dose-response relationship exists in all ocular tissues studied after an intraperitoneal dose of timolol. High doses of timolol were required to achieve measurable concentrations of drug in the ocular tissues via our high performance liquid chromatography assay suggesting that a significant hepatic first-pass effect may be involved after an intraperitoneal injection of timolol.

Transcriptional dysregulation is a major pathological feature of Huntington's disease (HD). The goal of this study was to understand how p65/RelA co-regulated genes, specifically those of the cytokine and endocannabinoid systems, were affected in HD. p65/RelA levels were lower in human HD tissue and R6/2 HD mice, as were the levels of the type 1 cannabinoid receptor (CB1), IL-1β, IL-8, CCL5, GM-CSF, MIP-1β, and TNFα, all of which may be regulated by p65/RelA. Activation of p65/RelA restored CB1 and CCL5 expression in STHdh cell models of HD. Therefore, p65/RelA activation may normalize the expression of some genes in HD.

The endocannabinoid system plays a role in regulation of vasoactivity in the peripheral vasculature; however, little is known about its role in regulation of the CNS microvasculature. This study investigated the pharmacology of cannabinoids and cannabimimetic lipids in the retinal microvasculature, a CNS vascular bed that is autoregulated. Vessel diameter (edge detector) and calcium transients (fura-2) were recorded from segments of retinal microvasculature isolated from adult, male Fischer 344 rats. Results showed that abnormal cannabidiol (Abn-CBD), an agonist at the putative endothelial cannabinoid receptor, CBe, inhibited endothelin 1 (ET-1) induced vasoconstriction in retinal arterioles. These actions of Abn-CBD were independent of CB1/CB2 receptors and were not mediated by agonists for GPR55 or affected by nitric oxide synthase (NOS) inhibition. However, the vasorelaxant effects of Abn-CBD were abolished when the endothelium was removed and were inhibited by the small Ca(2+)-sensitive K channel (SKCa) blocker, apamin. The effects of the endogenous endocannabinoid metabolite, N-arachidonyl glycine (NAGly), a putative agonist for GPR18, were virtually identical to those of Abn-CBD. GPR18 mRNA and protein were present in the retina, and immunohistochemistry demonstrated that GPR18 was localized to the endothelium of retinal vessels. These findings demonstrate that Abn-CBD and NAGly inhibit ET-1 induced vasoconstriction in retinal arterioles by an endothelium-dependent signaling mechanism that involves SKCa channels. The endothelial localization of GPR18 suggests that GPR18 could contribute to cannabinoid and lipid-mediated retinal vasoactivity.

The endocannabinoid system represents a potential therapeutic target in sepsis due to the presence of cannabinoid receptors (CB2) on immune cells. In this review we discuss how various targets within the endocannabinoid system can be manipulated to treat the immune consequences of sepsis. One of the targets outlined are the endocannabinoid receptors and modulation of their activity through pharmacological agonists and antagonists. Another therapeutic target covered in this review is the modulation of the endocannabinoid degradative enzyme’s activity. Modulation of degradative enzyme activity can change the levels of endogenous cannabinoids thereby altering immune activity. Overall, activation of the CB2 receptors causes immunosuppression and can be beneficial during the hyperactivated immune state of sepsis, while suppression of the CB2 receptors may be beneficial during a hypoimmune septic state.

Mutations in genes that code for components of the Norrin-FZD4 ligand-receptor complex cause the inherited childhood blinding disorder familial exudative vitreoretinopathy (FEVR). Statistical evidence from studies of patients at risk for the acquired disease retinopathy of prematurity (ROP) suggest that rare polymorphisms in these same genes increase the risk of developing severe ROP, implying that decreased Norrin-FZD4 activity predisposes patients to more severe ROP. To test this hypothesis, we measured the development and recovery of retinopathy in wild type and Fzd4 heterozygous mice in the absence or presence of ocular ischemic retinopathy (OIR) treatment. Avascular and total retinal vascular areas and patterning were determined, and vessel number and caliber were quantified. In room air, there was a small delay in retinal vascularization in Fzd4 heterozygous mice that resolved as mice reached maturity suggestive of a slight defect in retinal vascular development. Subsequent to OIR treatment there was no difference between wild type and Fzd4 heterozygous mice in the vaso-obliterated area following exposure to high oxygen. Importantly, after return of Fzd4 heterozygous mice to room air subsequent to OIR treatment, there was a substantial delay in retinal revascularization of the avascular area surrounding the optic nerve, as well as delayed vascularization toward the periphery of the retina. Our study demonstrates that a small decrease in Norrin-Fzd4 dependent retinal vascular development lengthens the period during which complications from OIR could occur.

Seven-transmembrane G protein-coupled receptors (GPCRs) represent the single largest family of cell surface receptors. Signaling through these receptors is controlled by changes in the conformation of the receptor from inactive to active conformations, which in turn lead to the activation of multiple downstream signaling pathways. To facilitate greater diversity in signaling responses, many of these receptors are capable of adopting several distinct active conformations, in which each couples preferentially to its own set of downstream signaling partners. Because these unique signaling responses result from specific receptor active conformations, GPCR signaling may be directed toward these selective responses through either strength-of-signal effects resulting from partial agonism or through biased agonism and functional selectivity, resulting from the selective stabilization of one active conformation over the others. This review uses the CB 1 cannabinoid receptor as a specific example to highlight the contribution of two important aspects of GPCR function—orthosteric ligand binding and receptor heterodimerization—toward directed GPCR signaling.

PURPOSE Cannabinoids have been reported to mediate changes in vascular resistance through endothelial receptor targets. We examined involvement of the endothelium in cannabinoid-mediated vasoactive responses in resistance arterioles of the retina. METHODS Vascular responses to both intraluminal (IL) and extraluminal (EL) administration of the atypical cannabinoid, abnormal cannabidiol (abn-CBD), a prototypical agonist at the non-CB1/CB2 endothelial cannabinoid receptor (CBeR), were studied in endothelial intact and endothelial denuded, isolated perfused porcine retinal arterioles with and without endothelin-1 (ET-1) precontraction. The effects of AM251, a CB1 receptor antagonist, and O-1918, an analog of CBD reported to antagonize CBeR, were also studied. RESULTS Dose-dependent vasocontractile responses were induced by both IL and EL administration of abn-CBD in the absence of precontraction. Significantly greater vasoconstriction was induced by IL administration of abn-CBD than with EL administration. In contrast, only vasodilation to abn-CBD was observed in ET-1 precontracted retinal arterioles. Endothelium removal significantly reduced abn-CBD-induced vasoactivity when abn-CBD was used IL but not when applied EL. IL abn-CBD-induced vasoactivity was antagonized by O-1918 and AM251. CONCLUSIONS Cannabinoids show complex vasoactive actions in isolated perfused retinal arterioles. The fact that abn-CBD-mediated vasorelaxation was seen only in precontracted retinal vessels indicates that the abn-CBD-induced vasoactive response is highly dependent on vascular tone. Furthermore, IL and EL administration produced differential responses, and removal of endothelium blunted abn-CBD vasoactivity, highlighting the critical role of endothelium in abn-CBD vasoactivity. AM251 and O-1918 inhibition of abn-CBD-induced vasoactivity suggests the possibility of modulating abn-CBD-induced vasoactivity.

In systemic inflammation and sepsis, the endo-cannabinoid system is upregulated [1]. While it is known that neuronal cannabinoid signalling via cannabinoid receptor-1 (CB1) in the central nervous system represents an intrinsic neuroprotective response [2] and exerts anti-epileptic activity [3], inhibition of CB1 (CB1inh) has been suggested as an experimental target for sepsis therapy [4]. We studied the effects of CB1inh in rats with experimental sepsis during anesthesia induction with pentobarbital.

Regeneration of frog sympathetic neurons (B-cells) was found to be accompanied by sprouting of neurites within the ganglion. Neurons whose axons had been crushed and allowed to regenerate exhibited sprouts that arose mainly from the axon hillock and initial segment of the axon. Sprouting was apparent by 5–7 days and reached maximal values by 14–21 days, but had decreased to control levels by 42–49 days after injury. In contrast, neurons whose axons were prevented from regenerating (by cut and proximal ligation of nerves) exhibited sprouts which did not retract by 42–49 days. These results suggest that successful regeneration to targets may dictate the recovery of normal B-cell morphology in bullfrog sympathetic ganglia.

In bullfrog sympathetic B cells, axotomy decreases the amplitude and decay time of membrane afterhyperpolarization (AHP) and increases action potential (AP) duration. A second (test) axotomy, 7 days after an initial (conditioning) axotomy, did not amplify these changes. No recovery of AHP amplitude or AP duration occurred by 56 days post-axotomy, but AHP decay time recovered 21 days earlier than after test axotomy alone. Conditioning, previously shown to accelerate regeneration, speeds the return to normal of those membrane properties previously shown to recover after axotomy.

In bullfrog B-type sympathetic neurones axon injury produces substantial changes in somal membrane properties. These include a shortening of action potential afterhyperpolarization (AHP) and an increase in action potential (AP) duration. In the present experiments we compared two injury situations: nerve crush, which was followed by regeneration, and nerve cut, after which regeneration to the original target was prevented, to investigate whether these electrophysiological changes were related to axon regeneration. Both crush and cut injuries produced a similar maximum decrease in AHP duration (to 33 and 30%) by 14 days after axotomy. After nerve crush, AHP duration recovered to within control values by 42 days, while after cut it remained depressed. AHP amplitude decreased to the same extent after nerve crush or cut (to 62 and 58%), but the rate of decrease was slower following crush when compared with cut, and following both types of injury it still remained depressed at 42 and 49 days. Changes in AP duration also took longer to occur following nerve crush, reaching maximal values at 35-42 days, at which time AHP duration had returned to within the normal range. The early reduction in AHP duration and its rapid recovery in regenerating neurones suggests that the current underlying this membrane property is regulated by events associated with axon outgrowth and peripheral reconnection. In contrast, changes in AHP amplitude and AP repolarization appeared to be independent of the occurrence of axon regeneration and remained abnormal at 49 days despite the recovery of AHP duration. These results imply that the electrophysiological changes seen in B-cells following injury are differentially regulated during subsequent regeneration.