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.

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.

David Casarett's Stoned: A Doctor's Case for Medical Marijuana.

With legal cannabis sales at $5.4 billion in 2015 and expected to rise by another billion this year in the United States, legalization and marijuana's impact on health is a hot topic of national debate. Casarett, a physician at the University of Pennsylvania, immerses himself in the culture, science, and smoke of medical marijuana in order to sort out the truth behind the buzz. Our reviewer, who has authored more than 120 research papers and reviews on the regulation of synaptic inhibition and endocannabinoids, tell us what the author got right, but also overlooked on his journey to learn more about a complex and controversial subject.

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.

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).

Seizing an opportunity for the endocannabinoid system.

Exogenous cannabinoids can limit seizures and neurodegeneration, and their actions are largely mimicked by endogenous cannabinoids (endocannabinoids). Endocannabinoids are mobilized by epileptiform activity and in turn influence this activity by inhibiting synaptic transmission; both excitatory and some inhibitory synapses can be suppressed, leading to potentially complex outcomes. Moreover, the endocannabinoid system is not a fixed entity, and its strength can be enhanced or reduced. Endocannabinoids and their receptors are altered by epileptic seizures in ways that can reduce the efficacy of both exogenous and endogenous cannabinoids in sometimes unexpected ways.

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.

Muscarinic cholinergic receptors modulate inhibitory synaptic rhythms in hippocampus and neocortex.

Activation of muscarinic acetylcholine (ACh) receptors (mAChRs) powerfully affects many neuronal properties as well as numerous cognitive behaviors. Small neuronal circuits constitute an intermediate level of organization between neurons and behaviors, and mAChRs affect interactions among cells that compose these circuits. Circuit activity is often assessed by extracellular recordings of the local field potentials (LFPs), which are analogous to in vivo EEGs, generated by coordinated neuronal interactions. Coherent forms of physiologically relevant circuit activity manifest themselves as rhythmic oscillations in the LFPs. Frequencies of rhythmic oscillations that are most closely associated with animal behavior are in the range of 4-80 Hz, which is subdivided into theta (4-14 Hz), beta (15-29 Hz) and gamma (30-80 Hz) bands. Activation of mAChRs triggers rhythmic oscillations in these bands in the hippocampus and neocortex. Inhibitory responses mediated by GABAergic interneurons constitute a prominent feature of these oscillations, and indeed, appear to be their major underlying factor in many cases. An important issue is which interneurons are involved in rhythm generation. Besides affecting cellular and network properties directly, mAChRs can cause the mobilization of endogenous cannabinoids (endocannabinoids, eCBs) that, by acting on the principal cannabinoid receptor of the brain, CB1R, regulate the release of certain neurotransmitters, including GABA. CB1Rs are heavily expressed on only a subset of interneurons and, at lower density, on glutamatergic neurons. Exogenous cannabinoids typically disrupt oscillations in the theta (θ) and gamma (γ) ranges, which probably contributes to the behavioral effects of these drugs. It is important to understand how neuronal circuit activity is affected by mAChR-driven eCBs, as this information will provide deeper insight into the actions of ACh itself, as well as into the effects of eCBs and exogenous cannabinoids in animal behavior. After covering some basic aspects of the mAChR system, this review will focus on recent findings concerning the mechanisms and circuitry that generate θ and γ rhythms in hippocampus and neocortex. The ability of optogenetic methods to probe the many roles of ACh in rhythm generation is highlighted.

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.

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.

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.

Getting high on the endocannabinoid system.

The endogenous cannabinoid system-named for the plant that led to its discovery-is one of the most important physiologic systems involved in establishing and maintaining human health. Endocannabinoids and their receptors are found throughout the body: in the brain, organs, connective tissues, glands, and immune cells. With its complex actions in our immune system, nervous system, and virtually all of the body's organs, the endocannabinoids are literally a bridge between body and mind. By understanding this system, we begin to see a mechanism that could connect brain activity and states of physical health and disease.

Prostaglandin E2 stimulates estradiol synthesis in the cerebellum postnatally with associated effects on Purkinje neuron dendritic arbor and electrophysiological properties.

Prostaglandins (PGs) are ubiquitous membrane-derived, lipid-signaling molecules with wide ranging effects throughout the body. In the brain, PGE(2) is the key regulator of fever after inflammation but is also implicated in neural development and synaptic plasticity. The steroid hormone estradiol is also a key regulator of neural development and synaptic plasticity. Recently, we showed that administering cyclooxygenase (COX) inhibitors to block PGE(2) production increased the total length of Purkinje cell dendrites, the number of dendritic spines, and the level of spinophilin protein, which is enriched in dendritic spines. Correspondingly, PGE(2) administration into the cerebellum decreased spinophilin protein content. We now report that PGE(2) stimulates estradiol synthesis in the immature rat cerebellum via enhanced activity of the aromatase enzyme. Treatment with cyclooxygenase inhibitors reduced cerebellar aromatase activity and estradiol content whereas PGE(2) administration increased both. Treatment with either PGE(2) or estradiol stunted Purkinje neuron dendritic length and complexity and produced a corresponding reduction in spinophilin content. Treatment with formestane to inhibit aromatase activity led to excessive sprouting of the dendritic tree, whereas elevated estradiol had the opposite effect. Electrophysiological measurements from Purkinje neurons revealed novel sex differences in input resistance and membrane capacitance that were abolished by estradiol exposure, whereas a sex difference in the amplitude of the afterhyperpolarization after an action potential was not. Correlated changes in action potential threshold suggest that prolonged alterations in neuronal firing activity could be a consequence of increased estradiol content during the second week of life. These findings reveal a previously unappreciated role for PG-stimulated steroidogenesis in the developing brain and a new potential route for inflammation-mediated disruption of neuronal maturation.

Dendritic hold and read: a gated mechanism for short term information storage and retrieval.

Two contrasting theories have been proposed to explain the mechanistic basis of short term memory. One theory posits that short term memory is represented by persistent neural activity supported by reverberating feedback networks. An alternate, more recent theory posits that short term memory can be supported by feedforward networks. While feedback driven memory can be implemented by well described mechanisms of synaptic plasticity, little is known of possible molecular and cellular mechanisms that can implement feedforward driven memory. Here we report such a mechanism in which the memory trace exists in the form of glutamate-bound but Mg(2+)-blocked NMDA receptors on the thin terminal dendrites of CA1 pyramidal neurons. Because glutamate dissociates from subsets of NMDA receptors very slowly, excitatory synaptic transmission can leave a silent residual trace that outlasts the electrical activity by hundreds of milliseconds. Read-out of the memory trace is possible if a critical level of these bound-but-blocked receptors accumulates on a dendritic branch that will allow these quasi-stable receptors to sustain a regenerative depolarization when triggered by an independent gating signal. This process is referred to here as dendritic hold and read (DHR). Because the read-out of the input is not dependent on repetition of the input and information flows in a single-pass manner, DHR can potentially support a feedforward memory architecture.

An improved test for detecting multiplicative homeostatic synaptic scaling.

Homeostatic scaling of synaptic strengths is essential for maintenance of network "gain", but also poses a risk of losing the distinctions among relative synaptic weights, which are possibly cellular correlates of memory storage. Multiplicative scaling of all synapses has been proposed as a mechanism that would preserve the relative weights among them, because they would all be proportionately adjusted. It is crucial for this hypothesis that all synapses be affected identically, but whether or not this actually occurs is difficult to determine directly. Mathematical tests for multiplicative synaptic scaling are presently carried out on distributions of miniature synaptic current amplitudes, but the accuracy of the test procedure has not been fully validated. We now show that the existence of an amplitude threshold for empirical detection of miniature synaptic currents limits the use of the most common method for detecting multiplicative changes. Our new method circumvents the problem by discarding the potentially distorting subthreshold values after computational scaling. This new method should be useful in assessing the underlying neurophysiological nature of a homeostatic synaptic scaling transformation, and therefore in evaluating its functional significance.

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.

Acute restraint stress enhances hippocampal endocannabinoid function via glucocorticoid receptor activation.

Exposure to behavioural stress normally triggers a complex, multilevel response of the hypothalamic-pituitary-adrenal (HPA) axis that helps maintain homeostatic balance. Although the endocannabinoid (eCB) system (ECS) is sensitive to chronic stress, few studies have directly addressed its response to acute stress. Here we show that acute restraint stress enhances eCB-dependent modulation of GABA release measured by whole-cell voltage clamp of inhibitory postsynaptic currents (IPSCs) in rat hippocampal CA1 pyramidal cells in vitro. Both Ca(2+)-dependent, eCB-mediated depolarization-induced suppression of inhibition (DSI), and muscarinic cholinergic receptor (mAChR)-mediated eCB mobilization are enhanced following acute stress exposure. DSI enhancement is dependent on the activation of glucocorticoid receptors (GRs) and is mimicked by both in vivo and in vitro corticosterone treatment. This effect does not appear to involve cyclooxygenase-2 (COX-2), an enzyme that can degrade eCBs; however, treatment of hippocampal slices with the L-type calcium (Ca(2+)) channel inhibitor, nifedipine, reverses while an agonist of these channels mimics the effect of in vivo stress. Finally, we find that acute stress produces a delayed (by 30 min) increase in the hippocampal content of 2-arachidonoylglycerol, the eCB responsible for DSI. These results support the hypothesis that the ECS is a biochemical effector of glucocorticoids in the brain, linking stress with changes in synaptic strength.

Optogenetic release of ACh induces rhythmic bursts of perisomatic IPSCs in hippocampus.

Acetylcholine (ACh) influences a vast array of phenomena in cortical systems. It alters many ionic conductances and neuronal firing behavior, often by regulating membrane potential oscillations in populations of cells. Synaptic inhibition has crucial roles in many forms of oscillation, and cholinergic mechanisms regulate both oscillations and synaptic inhibition. In vitro investigations using bath-application of cholinergic receptor agonists, or bulk tissue electrical stimulation to release endogenous ACh, have led to insights into cholinergic function, but questions remain because of the relative lack of selectivity of these forms of stimulation. To investigate the effects of selective release of ACh on interneurons and oscillations, we used an optogenetic approach in which the light-sensitive non-selective cation channel, Channelrhodopsin2 (ChR2), was virally delivered to cholinergic projection neurons in the medial septum/diagonal band of Broca (MS/DBB) of adult mice expressing Cre-recombinase under the control of the choline-acetyltransferase (ChAT) promoter. Acute hippocampal slices obtained from these animals weeks later revealed ChR2 expression in cholinergic axons. Brief trains of blue light pulses delivered to untreated slices initiated bursts of ACh-evoked, inhibitory post-synaptic currents (L-IPSCs) in CA1 pyramidal cells that lasted for 10's of seconds after the light stimulation ceased. L-IPSC occurred more reliably in slices treated with eserine and a very low concentration of 4-AP, which were therefore used in most experiments. The rhythmic, L-IPSCs were driven primarily by muscarinic ACh receptors (mAChRs), and could be suppressed by endocannabinoid release from pyramidal cells. Finally, low-frequency oscillations (LFOs) of local field potentials (LFPs) were significantly cross-correlated with the L-IPSCs, and reversal of the LFPs near s. pyramidale confirmed that the LFPs were driven by perisomatic inhibition. This optogenetic approach may be a useful complementary technique in future investigations of endogenous ACh effects.

The depolarizing action of GABA in cultured hippocampal neurons is not due to the absence of ketone bodies.

Two recent reports propose that the depolarizing action of GABA in the immature brain is an artifact of in vitro preparations in which glucose is the only energy source. The authors argue that this does not mimic the physiological environment because the suckling rats use ketone bodies and pyruvate as major sources of metabolic energy. Here, we show that availability of physiologically relevant levels of ketone bodies has no impact on the excitatory action of GABA in immature cultured hippocampal neurons. Addition of ß-hydroxybutyrate (BHB), the primary ketone body in the neonate rat, affected neither intracellular calcium elevation nor membrane depolarizations induced by the GABA-A receptor agonist muscimol, when assessed with calcium imaging or perforated patch-clamp recording, respectively. These results confirm that the addition of ketone bodies to the extracellular environment to mimic conditions in the neonatal brain does not reverse the chloride gradient and therefore render GABA hyperpolarizing. Our data are consistent with the existence of a genuine "developmental switch" mechanism in which GABA goes from having a predominantly excitatory role in immature cells to a predominantly inhibitory one in adults.