Neuroscience Needs to Test Both Statistical and Scientific Hypotheses.

Experimental neuroscience typically uses "p-valued" statistical testing procedures (null hypothesis significance testing; NHST) in evaluating its results. The rote, often misguided, application of NHST (Gigerenzer, 2008) has led to errors and "questionable research practices." Although the problems could be avoided with better statistics training (Lakens, 2021), there have been calls to abandon NHST altogether. One suggestion is to replace NHST with "estimation statistics" (Cumming and Calin-Jageman, 2017; Calin-Jageman and Cumming, 2019). Estimation statistics emphasizes the uncertainty inherent in scientific investigations and uses metrics, e.g., confidence intervals (CIs), that draw attention to uncertainty. Besides procedural steps and methods, the Estimation Approach prefers expressing "quantitative," rather than "qualitative" conclusions and making generalizations, rather than testing scientific hypotheses. The Estimation Approach embodies a philosophy of science-its ultimate goals, experimental mindset, and specific aims-that diverges unhelpfully from what laboratory-based neuroscience needs. The Estimation Approach meshes naturally with, e.g., clinical neuroscience, drug development, human psychology, and social sciences. It fits less well with much of the neuroscience published in the Journal of Neuroscience, for example. In contrast, the philosophy behind NHST fits naturally with traditional, evaluative testing of scientific hypotheses. Finally, some Estimation Approach remedies, e.g., replication, ideally with "preregistration," are incompatible with much experimental neuroscience. This Dual Perspective essay argues that, while neuroscience can benefit from practical aspects of estimation statistics, entirely replacing conventional methods with the Estimation Approach would be a mistake. NHST testing should be retained and improved.SIGNIFICANCE STATEMENT Experimental neuroscience relies on statistical procedures to assess the meaning and importance of its research findings. Optimal scientific communication demands a common set of assumptions for expressing and evaluating results. Problems arising from misuse of conventional significance testing methods have led to a proposal to replace significance testing with an Estimation Statistics Approach. Practical elements of the Estimation Approach can usefully be incorporated into conventional methods. However, the prevailing philosophy of the Estimation Approach does not address certain important needs of much experimental neuroscience. Neuroscience should adopt beneficial elements of the Estimation Approach without giving up the advantages of significance testing.

Weeding out bad waves: towards selective cannabinoid circuit control in epilepsy.

Endocannabinoids are lipid-derived messengers, and both their synthesis and breakdown are under tight spatiotemporal regulation. As retrograde signalling molecules, endocannabinoids are synthesized postsynaptically but activate presynaptic cannabinoid receptor 1 (CB1) receptors to inhibit neurotransmitter release. In turn, CB1-expressing inhibitory and excitatory synapses act as strategically placed control points for activity-dependent regulation of dynamically changing normal and pathological oscillatory network activity. Here, we highlight emerging principles of cannabinoid circuit control and plasticity, and discuss their relevance for epilepsy and related comorbidities. New insights into cannabinoid signalling may facilitate the translation of the recent interest in cannabis-related substances as antiseizure medications to evidence-based treatment strategies.

Interlamellar CA1 network in the hippocampus.

To understand the cellular basis of learning and memory, the neurophysiology of the hippocampus has been largely examined in thin transverse slice preparations. However, the synaptic architecture along the longitudinal septo-temporal axis perpendicular to the transverse projections in CA1 is largely unknown, despite its potential significance for understanding the information processing carried out by the hippocampus. Here, using a battery of powerful techniques, including 3D digital holography and focal glutamate uncaging, voltage-sensitive dye, two-photon imaging, electrophysiology, and immunohistochemistry, we show that CA1 pyramidal neurons are connected to one another in an associational and well-organized fashion along the longitudinal axis of the hippocampus. Such CA1 longitudinal connections mediate reliable signal transfer among the pyramidal cells and express significant synaptic plasticity. These results illustrate a need to reconceptualize hippocampal CA1 network function to include not only processing in the transverse plane, but also operations made possible by the longitudinal network. Our data will thus provide an essential basis for future computational modeling studies on information processing operations carried out in the full 3D hippocampal network that underlies its complex cognitive functions.

Homer protein-metabotropic glutamate receptor binding regulates endocannabinoid signaling and affects hyperexcitability in a mouse model of fragile X syndrome.

The Fmr1 knock-out mouse model of fragile X syndrome (Fmr1(-/y)) has an epileptogenic phenotype that is triggered by group I metabotropic glutamate receptor (mGluR) activation. We found that a membrane-permeable peptide that disrupts mGluR5 interactions with long-form Homers enhanced mGluR-induced epileptiform burst firing in wild-type (WT) animals, replicating the early stages of hyperexcitability in Fmr1(-/y). The peptide enhanced mGluR-evoked endocannabinoid (eCB)-mediated suppression of inhibitory synapses, decreased it at excitatory synapses in WTs, but had no effect on eCB actions in Fmr1(-/y). At a low concentration, the mGluR agonist did not generate eCBs at excitatory synapses but nevertheless induced burst firing in both Fmr1(-/y) and peptide-treated WT slices. This burst firing was suppressed by a cannabinoid receptor antagonist. We suggest that integrity of Homer scaffolds is essential for normal mGluR-eCB functioning and that aberrant eCB signaling resulting from disturbances of this molecular structure contributes to the epileptic phenotype of Fmr1(-/y).

Optogenetic identification of an intrinsic cholinergically driven inhibitory oscillator sensitive to cannabinoids and opioids in hippocampal CA1.

Neuronal electrical oscillations in the theta (4-14 Hz) and gamma (30-80 Hz) ranges are necessary for the performance of certain animal behaviours and cognitive processes. Perisomatic GABAergic inhibition is prominently involved in cortical oscillations driven by ACh release from septal cholinergic afferents. In neocortex and hippocampal CA3 regions, parvalbumin (PV)-expressing basket cells, activated by ACh and glutamatergic agonists, largely mediate oscillations. However, in CA1 hippocampus in vitro, cholinergic agonists or the optogenetic release of endogenous ACh from septal afferents induces rhythmic, theta-frequency inhibitory postsynaptic currents (IPSCs) in pyramidal cells, even with glutamatergic transmission blocked. The IPSCs are regulated by exogenous and endogenous cannabinoids, suggesting that they arise from type 1 cannabinoid receptor-expressing (CB1R+) interneurons - mainly cholecystokinin (CCK)-expressing cells. Nevertheless, an occult contribution of PV-expressing interneurons to these rhythms remained conceivable. Here, we directly test this hypothesis by selectively silencing CA1 PV-expressing cells optogenetically with halorhodopsin or archaerhodopsin. However, this had no effect on theta-frequency IPSC rhythms induced by carbachol (CCh). In contrast, the silencing of glutamic acid decarboxylase 2-positive interneurons, which include the CCK-expressing basket cells, strongly suppressed inhibitory oscillations; PV-expressing interneurons appear to play no role. The low-frequency IPSC oscillations induced by CCh or optogenetically stimulated ACh release were also inhibited by a µ-opioid receptor (MOR) agonist, which was unexpected because MORs in CA1 are not usually associated with CCK-expressing cells. Our results reveal novel properties of an inhibitory oscillator circuit within CA1 that is activated by muscarinic agonists. The oscillations could contribute to behaviourally relevant, atropine-sensitive, theta rhythms and link cannabinoid and opioid actions functionally.

Developmental increase in hippocampal endocannabinoid mobilization: role of metabotropic glutamate receptor subtype 5 and phospholipase C.

Endocannabinoids (eCBs) released from postsynaptic neurons mediate retrograde suppression of neurotransmitter release at central synapses. eCBs are crucial for establishing proper synaptic connectivity in the developing nervous system. Mobilization of eCBs is driven either by a rise in intracellular Ca(2+) (depolarization-induced suppression of inhibition, DSI) or postsynaptic G protein-coupled receptors (GPCRs) that activate phospholipase C beta (PLCß). To determine whether eCB mobilization changes between neonatal and juvenile ages, we used whole cell voltage-clamp recordings of CA1 neurons from rat hippocampal slices at postnatal days 1-18 (neonatal) and 19-43 (juvenile), because many neurophysiological parameters change dramatically between approximately postnatal days 18-20. We found that DSI was slightly greater in juveniles than in neonates, while eCB mobilization stimulated by GPCRs was unchanged. However, when DSI was elicited during GPCR activation, its increase was much greater in juveniles, suggesting that eCB mobilization caused by the synergy between the Ca(2+) and GPCR pathways is developmentally upregulated. Western blotting revealed significant increases in both metabotropic type glutamate receptor 5 (mGluR5) and PLCß1 proteins in juveniles compared with neonates. Responses to pharmacological activation or inhibition of PLC implied that eCB upregulation is associated with a functional increase in PLC activity. We conclude that synergistic eCB mobilization in hippocampal CA1 neurons is greater in juveniles than in neonates, and that this may result from increases in the mGluR5-PLCß1 eCB pathway. The data enhance our understanding of the developmental regulation of the eCB system and may provide insight into diseases caused by improper cortical wiring, or the impact of cannabis exposure during development.

Evidence of calcium-permeable AMPA receptors in dendritic spines of CA1 pyramidal neurons.

GluA2-lacking, calcium-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) have unique properties, but their presence at excitatory synapses in pyramidal cells is controversial. We have tested certain predictions of the model that such receptors are present in CA1 cells and show here that the polyamine spermine, but not philanthotoxin, causes use-dependent inhibition of synaptically evoked excitatory responses in stratum radiatum, but not s. oriens, in cultured and acute hippocampal slices. Stimulation of single dendritic spines by photolytic release of caged glutamate induced an N-methyl-d-aspartate receptor-independent, use- and spermine-sensitive calcium influx only at apical spines in cultured slices. Bath application of glutamate also triggered a spermine-sensitive influx of cobalt into CA1 cell dendrites in s. radiatum. Responses of single apical, but not basal, spines to photostimulation displayed prominent paired-pulse facilitation (PPF) consistent with use-dependent relief of cytoplasmic polyamine block. Responses at apical dendrites were diminished, and PPF was increased, by spermine. Intracellular application of pep2m, which inhibits recycling of GluA2-containing AMPARs, reduced apical spine responses and increased PPF. We conclude that some calcium-permeable, polyamine-sensitive AMPARs, perhaps lacking GluA2 subunits, are present at synapses on apical dendrites of CA1 pyramidal cells, which may allow distinct forms of synaptic plasticity and computation at different sets of excitatory inputs.

Nerve terminal nicotinic acetylcholine receptors initiate quantal GABA release from perisomatic interneurons by activating axonal T-type (Cav3) Ca²âº channels and Ca²âº release from stores.

Release of conventional neurotransmitters is mainly controlled by calcium (Ca²âº) influx via high-voltage-activated (HVA), Ca(v)2, channels ("N-, P/Q-, or R-types") that are opened by action potentials. Regulation of transmission by subthreshold depolarizations does occur, but there is little evidence that low-voltage-activated, Ca(v)3 ("T-type"), channels take part. GABA release from cortical perisomatic-targeting interneurons affects numerous physiological processes, and yet its underlying control mechanisms are not fully understood. We investigated whether T-type Ca²âº channels are involved in regulating GABA transmission from these cells in rat hippocampal CA1 using a combination of whole-cell voltage-clamp, multiple-fluorescence confocal microscopy, dual-immunolabeling electron-microscopy, and optogenetic methods. We show that Ca(v)3.1, T-type Ca²âº channels can be activated by α3ß4 nicotinic acetylcholine receptors (nAChRs) that are located on the synaptic regions of the GABAergic perisomatic-targeting interneuronal axons, including the parvalbumin-expressing cells. Asynchronous, quantal GABA release can be triggered by Ca²âº influx through presynaptic T-type Ca²âº channels, augmented by Ca²âº from internal stores, following focal microiontophoretic activation of the α3ß4 nAChRs. The resulting GABA release can inhibit pyramidal cells. The T-type Ca²âº channel-dependent mechanism is not dependent on, or accompanied by, HVA channel Ca²âº influx, and is insensitive to agonists of cannabinoid, µ-opioid, or GABA(B) receptors. It may therefore operate in parallel with the normal HVA-dependent processes. The results reveal new aspects of the regulation of GABA transmission and contribute to a deeper understanding of ACh and nicotine actions in CNS.

Endocannabinoids at the synapse a decade after the dies mirabilis (29 March 2001): what we still do not know.

Endogenous cannabinoids (endocannabinoids, eCBs) are ubiquitous regulators of synaptic transmission in the brain, mediating numerous forms of short- and long-term plasticity, and having strong influences on synapse formation and neurogenesis. Their roles as retrograde messengers that suppress both excitatory and inhibitory transmission are well-established. Yet, despite intensive investigation, many basic aspects of the eCB system are not understood. This brief review highlights recent advances, problems that remain unresolved, and avenues for future exploration. While 2-arachidonoylglycerol (2-AG) is probably the major eCB for intercellular CB1R-dependent signalling, anandamide (AEA) has come to the forefront in several novel contexts, both as a dual endovanilloid/endocannabinoid that regulates synaptic transmission acutely and as the source of a steady eCB tone in hippocampus. Complexities in the cellular processing of 2-AG are receiving renewed attention, as they are increasingly recognized as major determinants of how 2-AG affects cells. Long-standing fundamental issues such as the synthesis pathway for AEA and the molecular mechanism(s) underlying cellular uptake and release of eCBs remain problematical.

Enhanced endocannabinoid signaling elevates neuronal excitability in fragile X syndrome.

Fragile X syndrome (FXS) results from deficiency of fragile X mental retardation protein (FMRP). FXS is the most common heritable form of mental retardation, and is associated with the occurrence of seizures. Factors responsible for initiating FXS-related hyperexcitability are poorly understood. Many protein-synthesis-dependent functions of group I metabotropic glutamate receptors (Gp1 mGluRs) are exaggerated in FXS. Gp1 mGluR activation can mobilize endocannabinoids (eCBs) in the hippocampus and thereby increase excitability, but whether FMRP affects eCBs is unknown. We studied Fmr1 knock-out (KO) mice lacking FMRP to test the hypothesis that eCB function is altered in FXS. Whole-cell evoked IPSCs (eIPSCs) and field potentials were recorded in the CA1 region of acute hippocampal slices. Three eCB-mediated responses were examined: depolarization-induced suppression of inhibition (DSI), mGluR-initiated eCB-dependent inhibitory short-term depression (eCB-iSTD), and eCB-dependent inhibitory long-term depression (eCB-iLTD). Low concentrations of a Gp1 mGluR agonist produced larger eCB-mediated responses in Fmr1 KO mice than in wild-type (WT) mice, without affecting DSI. Western blots revealed that levels of mGluR1, mGluR5, or cannabinoid receptor (CB1R) were unchanged in Fmr1 KO animals, suggesting that the coupling between mGluR activation and eCB mobilization was enhanced by FMRP deletion. The increased susceptibility of Fmr1 KO slices to eCB-iLTD was physiologically relevant, since long-term potentiation of EPSP-spike (E-S) coupling induced by the mGluR agonist was markedly larger in Fmr1 KO mice than in WT animals. Alterations in eCB signaling could contribute to the cognitive dysfunction associated with FXS.

Metaplastic control of the endocannabinoid system at inhibitory synapses in hippocampus.

The modifiability of neuronal response plasticity is called "metaplasticity." In suppressing synaptic inhibition and facilitating induction of long-term excitatory synaptic plasticity, endocannabinoids (eCBs) act as agents of metaplasticity. We now report the discovery of a calcium-dependent mechanism that regulates eCB mobilization by metabotropic glutamate receptor (mGluR) activation. The switch-like mechanism primes cells to release eCBs and requires a transient rise in intracellular Ca2+ concentration ([Ca2+]i) but not concurrent activation of mGluRs. Conversely, short-term, [Ca2+]i-dependent eCB release can be persistently enhanced by mGluR activation. Hence, eCBs are also objects of metaplasticity, subject to higher levels of physiological control.

Not too excited? Thank your endocannabinoids.

Endocannabinoids can mediate neuroprotection, but it is not known how. In this issue of Neuron, Monory et al. use mutant mice and localized viral targeting to produce conditional knockouts of the cannabinoid CB1 receptor. They show that protection against kainic acid-induced seizures and cell death is conferred by CB1Rs on hippocampal glutamatergic nerve terminals.

Synaptic cross talk between perisomatic-targeting interneuron classes expressing cholecystokinin and parvalbumin in hippocampus.

Cholescystokinin (CCK)- or parvalbumin (PV)-containing interneurons are the major perisomatic-targeting interneurons in the cerebral cortex, including hippocampus, and are thought to form mutually exclusive networks. We used several techniques to test the alternative hypothesis that CCK and PV cells are coupled by chemical synapses. Triple immunofluorescence confocal microscopy revealed numerous axosomatic, axodendritic, and axoaxonic contacts stained for CCK, PV, and the presynaptic marker synaptophysin. The existence of mutual CCK and PV synapses was supported by dual EM immunolabeling. Paired whole-cell recordings detected unitary GABA(A)ergic synaptic transmission between identified CCK and PV cells, and single CCK cells could transiently inhibit action potential firing of synaptically coupled PV cells. We conclude that the major hippocampal perisomatic-targeting interneurons communicate synaptically. This communication should affect neuronal network activity, including neuronal oscillations, in which the CCK and PV cells have well established roles. The prevalence of CCK and PV networks in other brain regions suggests that internetwork interactions could be generally important.

Scientific Hypothesis-Testing Strengthens Neuroscience Research.

Science needs to understand the strength of its findings. This essay considers the evaluation of studies that test scientific (not statistical) hypotheses. A scientific hypothesis is a putative explanation for an observation or phenomenon; it makes (or "entails") testable predictions that must be true if the hypothesis is true and that lead to its rejection if they are false. The question is, "how should we judge the strength of a hypothesis that passes a series of experimental tests?" This question is especially relevant in view of the "reproducibility crisis" that is the cause of great unease. Reproducibility is said to be a dire problem because major neuroscience conclusions supposedly rest entirely on the outcomes of single, p valued statistical tests. To investigate this concern, I propose to (1) ask whether neuroscience typically does base major conclusions on single tests; (2) discuss the advantages of testing multiple predictions to evaluate a hypothesis; and (3) review ways in which multiple outcomes can be combined to assess the overall strength of a project that tests multiple predictions of one hypothesis. I argue that scientific hypothesis testing in general, and combining the results of several experiments in particular, may justify placing greater confidence in multiple-testing procedures than in other ways of conducting science.

Cholecystokinin inhibits endocannabinoid-sensitive hippocampal IPSPs and stimulates others.

Cholecystokinin (CCK) is the most abundant neuropeptide in the central nervous system. In the hippocampal CA1 region, CCK is co-localized with GABA in a subset of interneurons that synapse on pyramidal cell somata and apical dendrites. CCK-containing interneurons also uniquely express a high level of the cannabinoid receptor, CB(1), and mediate the retrograde signaling process called DSI. Reported effects of CCK on inhibitory post-synaptic potentials (IPSPs) in hippocampus are inconsistent, and include both increases and decreases in activity. Hippocampal interneurons are very heterogeneous, and these results could be reconciled if CCK affected different interneurons in different ways. To test this prediction, we used sharp microelectrode recordings from pyramidal cells with ionotropic glutamate receptors blocked, and investigated the effects of CCK on pharmacologically distinct groups of IPSPs during long-term recordings. We find that CCK, acting via the CCK(2) receptor, increases some IPSPs and decreases others, and most significantly, that the affected IPSPs can be classified into two groups by their pharmacological properties. IPSPs that are increased by carbachol (CCh-sIPSPs), are depressed by CCK, omega-conotoxin GVIA, and endocannabinoids. IPSPs that are enhanced by CCK (CCK-sIPSPs) are blocked by omega-agatoxin IVA, and are unaffected by carbachol or endocannabinoids. Interestingly, a CCK(2) antagonist enhances CCh-sIPSPs, suggesting normally they may be partially suppressed by endogenous CCK. In summary, our data are compatible with the hypothesis that CCK has opposite actions on sIPSPs that originate from functionally distinct interneurons.

Distinctions among GABAA and GABAB responses revealed by calcium channel antagonists, cannabinoids, opioids, and synaptic plasticity in rat hippocampus.

RATIONALE: Hippocampal interneurons release gamma-aminobutyric acid (GABA) and produce fast GABA(A)- and slow GABA(B)-inhibitory postsynaptic potentials (IPSPs). The regulation of GABA(B) eIPSPs or the interneurons that produce them are not well understood. In addition, while both micro-opioid receptors (microORs) and cannabinoid CB1R receptors (CB1Rs) are present on hippocampal interneurons, it is not clear how these two systems interact. OBJECTIVES: This study tests the hypotheses that: (1) all interneurons can initiate both GABA(A) and GABA(B) inhibitory postsynaptic potentials; (2) GABA(B) responses are insensitive to mGluR-triggered, endocannabinoid (eCB)-mediated inhibitory long-term depression (iLTD); (3) GABA(B) responses are produced by interneurons that express microOR; and (4) CB1R-dependent and microOR-dependent response interact. MATERIALS AND METHODS: Pharmacological and electrophysiological approaches were used in acute rat hippocampal slices. High resistance microelectrode recordings were made from pyramidal cells, while interneurons were stimulated extracellularly. RESULTS: GABA(B) responses were found to be produced by interneurons that release GABA via either presynaptic N-type or P/Q-type calcium channels but that they are insensitive to suppression by eCBs or eCB-mediated iLTD. GABA(B) IPSPs were sensitive to suppression by a microOR agonist, suggesting a major source of GABA(B) responses is the microOR-expressing interneuron population. A small eCB-iLTD (10% eIPSP reduction) persisted in conotoxin. eCB-iLTD was blocked by a microOR agonist in 6/13 slices. CONCLUSIONS: GABA(B) responses cannot be produced by all interneurons. CB1R or microOR agonists will differentially alter the balance of activity in hippocampal circuits. CB1R- and microOR-mediated responses can interact.

Inhibition of cyclooxygenase-2 potentiates retrograde endocannabinoid effects in hippocampus.

In hippocampal pyramidal cells, a rise in Ca(2+) releases endocannabinoids that activate the presynaptic cannabinoid receptor (CB1R) and transiently reduce GABAergic transmission-a process called depolarization-induced suppression of inhibition (DSI). The mechanism that limits the duration of endocannabinoid action in intact cells is unknown. Here we show that inhibition of cyclooxygenase-2 (COX-2), not fatty acid amide hydrolase (FAAH), prolongs DSI, suggesting that COX-2 limits endocannabinoid action.

Endocannabinoids facilitate the induction of LTP in the hippocampus.

Exogenous cannabinoids disrupt behavioral learning and impede induction of long-term potentiation (LTP) in the hippocampus, yet endogenous cannabinoids (endocannabinoids) transiently suppress inhibitory post-synaptic currents (IPSCs) by activating cannabinoid CB1 receptors on GABAergic interneurons. We found that release of endocannabinoids by a rat CA1 pyramidal cell during this depolarization-induced suppression of inhibition (DSI) enabled a normally ineffective train of excitatory post-synaptic currents (EPSCs) to induce LTP in that cell, but not in neighboring cells. By showing that endocannabinoids facilitate LTP induction and help target LTP to single cells, these data shed new light on the physiological roles of endocannabinoids and may lead to a greater understanding of their effects on behavior and potential clinical use.

Endocannabinoid signaling dynamics probed with optical tools.

Intercellular signaling dynamics critically influence the functional roles that the signals can play. Small lipids are synthesized and released from neurons, acting as intercellular signals in regulating neurotransmitter release, modulating ion channels on target cells, and modifying synaptic plasticity. The repertoire of biological effects of lipids such as endocannabinoids (eCBs) is rapidly expanding, yet lipid signaling dynamics have not been studied. The eCB system constitutes a powerful tool for bioassaying the dynamics of lipid signaling. The eCBs are synthesized in, and released from, postsynaptic somatodendritic domains that are readily accessible to whole-cell patch electrodes. The dramatic effects of these lipid signals are detected electrophysiologically as CB1-dependent alterations in conventional synaptic transmission, which therefore serve as a sensitive reporter of eCB actions. We used electrophysiological recording, photolytic release of caged glutamate and a newly developed caged AEA (anandamide), together with rapid [Ca2+]i measurements, to investigate the dynamics of retrograde eCB signaling between CA1 pyramidal cells and GABAergic synapses in rat hippocampus in vitro. We show that, at 22 degrees C, eCB synthesis and release must occur within 75-190 ms after the initiating stimulus, almost an order of magnitude faster than previously thought. At 37 degrees C, the time could be < 50 ms. Activation of CB1 and downstream processes constitute a significant fraction of the total delay and are identified as major rate-limiting steps in retrograde signaling. Our findings imply that lipid messenger dynamics are comparable with those of metabotropic neurotransmitters and can modulate neuronal interactions on a similarly fast time scale.

Endocannabinoid identification in the brain: studies of breakdown lead to breakthrough, and there may be NO hope.

Endocannabinoids are a class of fatty acid derivatives defined by their ability to interact with the specific cannabinoid receptors that were originally identified as the targets of Delta9-tetrahydocannabinol (Delta9-THC), the psychoactive component of cannabis. Endocannabinoids have been implicated in a growing number of important physiological and behavioral events. A full understanding of the functions of endocannabinoids will involve knowing which ones are active, and how they are produced, during any given physical event. However, studying these small lipids in the brain presents many technical challenges. New selective pharmacological tools promise to be very useful in unraveling the complexities of endocannabinoid signaling, but parallel developments from the investigation of the cellular neurophysiology of the endocannabinoid systems highlight the difficulties remaining.