Microglial cannabinoid receptor type 1 mediates social memory deficits in mice produced by adolescent THC exposure and 16p11.2 duplication.

Adolescent cannabis use increases the risk for cognitive impairments and psychiatric disorders. Cannabinoid receptor type 1 (Cnr1) is expressed not only in neurons and astrocytes, but also in microglia, which shape synaptic connections during adolescence. However, the role of microglia in mediating the adverse cognitive effects of delta-9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, is not fully understood. Here, we report that in mice, adolescent THC exposure produces microglial apoptosis in the medial prefrontal cortex (mPFC), which was exacerbated in a model of 16p11.2 duplication, a representative copy number variation (CNV) risk factor for psychiatric disorders. These effects are mediated by microglial Cnr1, leading to reduction in the excitability of mPFC pyramidal-tract neurons and deficits in social memory in adulthood. Our findings suggest the microglial Cnr1 may contribute to adverse effect of cannabis exposure in genetically vulnerable individuals.

Microglial cannabinoid receptor type 1 mediates social memory deficits produced by adolescent THC exposure and 16p11.2 duplication.

Adolescent cannabis use increases the risk for cognitive impairments and psychiatric disorders. Cannabinoid receptor type 1 (Cnr1) is expressed not only in neurons and astrocytes, but also in microglia, which shape synaptic connections during adolescence. Nonetheless, until now, the role of microglia in mediating the adverse cognitive effects of delta-9-tetrahydrocannabinol (THC), the principal psychoactive constituent of cannabis, has been unexplored. Here, we report that adolescent THC exposure produces microglial apoptosis in the medial prefrontal cortex (mPFC), which was exacerbated in the mouse model of 16p11.2 duplication, a representative copy number variation (CNV) risk factor for psychiatric disorders. These effects are mediated by microglial Cnr1, leading to reduction in the excitability of mPFC pyramidal-tract neurons and deficits in social memory in adulthood. Our findings highlight the importance of microglial Cnr1 to produce the adverse effect of cannabis exposure in genetically vulnerable individuals.

Neural activity in the mouse claustrum in a cross-modal sensory selection task.

The claustrum, a subcortical nucleus forming extensive connections with the neocortex, has been implicated in sensory selection. Sensory-evoked claustrum activity is thought to modulate the neocortex's context-dependent response to sensory input. Recording from claustrum neurons while mice performed a tactile-visual sensory-selection task, we found that neurons in the anterior claustrum, including putative optotagged claustrocortical neurons projecting to the primary somatosensory cortex (S1), were rarely modulated by sensory input. Rather, they exhibited different types of direction-tuned motor responses. Furthermore, we found that claustrum neurons encoded upcoming movement during intertrial intervals and that pairs of claustrum neurons exhibiting synchronous firing were enriched for pairs preferring contralateral lick directions, suggesting that the activity of specific ensembles of similarly tuned claustrum neurons may modulate cortical activity. Chemogenetic inhibition of claustrocortical neurons decreased lick responses to inappropriate sensory stimuli. Altogether, our data indicate that the claustrum is integrated into higher-order premotor circuits recently implicated in decision-making.

Mechanisms Underlying Target Selectivity for Cell Types and Subcellular Domains in Developing Neocortical Circuits.

The cerebral cortex contains numerous neuronal cell types, distinguished by their molecular identity as well as their electrophysiological and morphological properties. Cortical function is reliant on stereotyped patterns of synaptic connectivity and synaptic function among these neuron types, but how these patterns are established during development remains poorly understood. Selective targeting not only of different cell types but also of distinct postsynaptic neuronal domains occurs in many brain circuits and is directed by multiple mechanisms. These mechanisms include the regulation of axonal and dendritic guidance and fine-scale morphogenesis of pre- and postsynaptic processes, lineage relationships, activity dependent mechanisms and intercellular molecular determinants such as transmembrane and secreted molecules, many of which have also been implicated in neurodevelopmental disorders. However, many studies of synaptic targeting have focused on circuits in which neuronal processes target different lamina, such that cell-type-biased connectivity may be confounded with mechanisms of laminar specificity. In the cerebral cortex, each cortical layer contains cell bodies and processes from intermingled neuronal cell types, an arrangement that presents a challenge for the development of target-selective synapse formation. Here, we address progress and future directions in the study of cell-type-biased synaptic targeting in the cerebral cortex. We highlight challenges to identifying developmental mechanisms generating stereotyped patterns of intracortical connectivity, recent developments in uncovering the determinants of synaptic target selection during cortical synapse formation, and current gaps in the understanding of cortical synapse specificity.

Control of neurogenic competence in mammalian hypothalamic tanycytes.

Hypothalamic tanycytes, radial glial cells that share many features with neuronal progenitors, can generate small numbers of neurons in the postnatal hypothalamus, but the identity of these neurons and the molecular mechanisms that control tanycyte-derived neurogenesis are unknown. In this study, we show that tanycyte-specific disruption of the NFI family of transcription factors (Nfia/b/x) robustly stimulates tanycyte proliferation and tanycyte-derived neurogenesis. Single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analysis reveals that NFI (nuclear factor I) factors repress Sonic hedgehog (Shh) and Wnt signaling in tanycytes and modulation of these pathways blocks proliferation and tanycyte-derived neurogenesis in Nfia/b/x-deficient mice. Nfia/b/x-deficient tanycytes give rise to multiple mediobasal hypothalamic neuronal subtypes that can mature, fire action potentials, receive synaptic inputs, and selectively respond to changes in internal states. These findings identify molecular mechanisms that control tanycyte-derived neurogenesis, which can potentially be targeted to selectively remodel the hypothalamic neural circuitry that controls homeostatic physiological processes.

The development of local circuits in the neocortex: recent lessons from the mouse visual cortex.

Precise synaptic connections among neurons in the neocortex generate the circuits that underlie a broad repertoire of cortical functions including perception, learning and memory, and complex problem solving. The specific patterns and properties of these synaptic connections are fundamental to the computations cortical neurons perform. How such specificity arises in cortical circuits has remained elusive. Here, we first consider the cell-type, subcellular and synaptic specificity required for generating mature patterns of cortical connectivity and responses. Next, we focus on recent progress in understanding how the synaptic connections among excitatory cortical projection neurons are established during development using the primary visual cortex of the mouse as a model.

A Non-canonical Feedback Circuit for Rapid Interactions between Somatosensory Cortices.

Sensory perception depends on interactions among cortical areas. These interactions are mediated by canonical patterns of connectivity in which higher areas send feedback projections to lower areas via neurons in superficial and deep layers. Here, we probed the circuit basis of interactions among two areas critical for touch perception in mice, whisker primary (wS1) and secondary (wS2) somatosensory cortices. Neurons in layer 4 of wS2 (S2L4) formed a major feedback pathway to wS1. Feedback from wS2 to wS1 was organized somatotopically. Spikes evoked by whisker deflections occurred nearly as rapidly in wS2 as in wS1, including among putative S2L4 → S1 feedback neurons. Axons from S2L4 → S1 neurons sent stimulus orientation-specific activity to wS1. Optogenetic excitation of S2L4 neurons modulated activity across both wS2 and wS1, while inhibition of S2L4 reduced orientation tuning among wS1 neurons. Thus, a non-canonical feedback circuit, originating in layer 4 of S2, rapidly modulates early tactile processing.

New Breakthroughs in Understanding the Role of Functional Interactions between the Neocortex and the Claustrum.

Almost all areas of the neocortex are connected with the claustrum, a nucleus located between the neocortex and the striatum, yet the functions of corticoclaustral and claustrocortical connections remain largely obscure. As major efforts to model the neocortex are currently underway, it has become increasingly important to incorporate the corticoclaustral system into theories of cortical function. This Mini-Symposium was motivated by a series of recent studies which have sparked new hypotheses regarding the function of claustral circuits. Anatomical, ultrastructural, and functional studies indicate that the claustrum is most highly interconnected with prefrontal cortex, suggesting important roles in higher cognitive processing, and that the organization of the corticoclaustral system is distinct from the driver/modulator framework often used to describe the corticothalamic system. Recent findings supporting roles in detecting novel sensory stimuli, directing attention and setting behavioral states, were the subject of the Mini-Symposium at the 2017 Society for Neuroscience Annual Meeting.

Corrigendum: Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

This corrects the article DOI: 10.1038/nature23663.

Changes in the Excitability of Neocortical Neurons in a Mouse Model of Amyotrophic Lateral Sclerosis Are Not Specific to Corticospinal Neurons and Are Modulated by Advancing Disease.

Cell type-specific changes in neuronal excitability have been proposed to contribute to the selective degeneration of corticospinal neurons in amyotrophic lateral sclerosis (ALS) and to neocortical hyperexcitability, a prominent feature of both inherited and sporadic variants of the disease, but the mechanisms underlying selective loss of specific cell types in ALS are not known. We analyzed the physiological properties of distinct classes of cortical neurons in the motor cortex of hSOD1G93A mice of both sexes and found that they all exhibit increases in intrinsic excitability that depend on disease stage. Targeted recordings and in vivo calcium imaging further revealed that neurons adapt their functional properties to normalize cortical excitability as the disease progresses. Although different neuron classes all exhibited increases in intrinsic excitability, transcriptional profiling indicated that the molecular mechanisms underlying these changes are cell type specific. The increases in excitability in both excitatory and inhibitory cortical neurons show that selective dysfunction of neuronal cell types cannot account for the specific vulnerability of corticospinal motor neurons in ALS. Furthermore, the stage-dependent alterations in neuronal function highlight the ability of cortical circuits to adapt as disease progresses. These findings show that both disease stage and cell type must be considered when developing therapeutic strategies for treating ALS.SIGNIFICANCE STATEMENT It is not known why certain classes of neurons preferentially die in different neurodegenerative diseases. It has been proposed that the enhanced excitability of affected neurons is a major contributor to their selective loss. We show using a mouse model of amyotrophic lateral sclerosis (ALS), a disease in which corticospinal neurons exhibit selective vulnerability, that changes in excitability are not restricted to this neuronal class and that excitability does not increase monotonically with disease progression. Moreover, although all neuronal cell types tested exhibited abnormal functional properties, analysis of their gene expression demonstrated cell type-specific responses to the ALS-causing mutation. These findings suggest that therapies for ALS may need to be tailored for different cell types and stages of disease.

Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep-wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.

Cell-type specific differences in promoter activity of the ALS-linked C9orf72 mouse ortholog.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Both loss-of-function and gain-of-function mechanisms have been proposed to underlie this disease, but the pathogenic pathways are not fully understood. To better understand the involvement of different cell types in the pathogenesis of ALS, we systematically analyzed the distribution of promoter activity of the mouse ortholog of C9orf72 in the central nervous system. We demonstrate that C9orf72 promoter activity is widespread in both excitatory and inhibitory neurons as well as in oligodendrocytes and oligodendrocyte precursor cells. In contrast, few microglia and astrocytes exhibit detectable C9orf72 promoter activity. Although at a gross level, the distribution of C9orf72 promoter activity largely follows overall cellular density, we found that it is selectively enriched in subsets of neurons and glial cells that degenerate in ALS. Specifically, we show that C9orf72 promoter activity is enriched in corticospinal and spinal motor neurons as well as in oligodendrocytes in brain regions that are affected in ALS. These results suggest that cell autonomous changes in both neurons and glia may contribute to C9orf72-mediated disease, as has been shown for mutations in superoxide dismutase-1 (SOD1).

Synaptic Organization of the Neuronal Circuits of the Claustrum.

The claustrum, a poorly understood subcortical structure located between the cortex and the striatum, forms widespread connections with almost all cortical areas, but the cellular organization of claustral circuits remains largely unknown. Based primarily on anatomical data, it has been proposed that the claustrum integrates activity across sensory modalities. However, the extent to which the synaptic organization of claustral circuits supports this integration is unclear. Here, we used paired whole-cell recordings and optogenetic approaches in mouse brain slices to determine the cellular organization of the claustrum. We found that unitary synaptic connections among claustrocortical (ClaC) neurons were rare. In contrast, parvalbumin-positive (PV) inhibitory interneurons were highly interconnected with both chemical and electrical synapses. In addition, ClaC neurons and PV interneurons formed frequent synaptic connections. As suggested by anatomical data, we found that corticoclaustral afferents formed monosynaptic connections onto both ClaC neurons and PV interneurons. However, the responses to cortical input were comparatively stronger in PV interneurons. Consistent with this overall circuit organization, activation of corticoclaustral afferents generated monosynaptic excitatory responses as well as disynaptic inhibitory responses in ClaC neurons. These data indicate that recurrent excitatory circuits within the claustrum alone are unlikely to integrate across multiple sensory modalities. Rather, this cellular organization is typical of circuits sensitive to correlated inputs. Although single ClaC neurons may integrate corticoclaustral input from different cortical regions, these results are consistent with more recent proposals implicating the claustrum in detecting sensory novelty or in amplifying correlated cortical inputs to coordinate the activity of functionally related cortical regions. Significance statement: The function of the claustrum, a brain nucleus found in mammals, remains poorly understood. It has been proposed, based primarily on anatomical data, that claustral circuits play an integrative role and contribute to multimodal sensory integration. Here we show that the principal neurons of the claustrum, claustrocortical (ClaC) projection neurons, rarely form synaptic connections with one another and are unlikely to contribute to broad integration within the claustrum. We show that, although single ClaC neurons may integrate corticoclaustral inputs carrying information for different sensory modalities, the synaptic organization of ClaC neurons, local parvalbumin-positive interneurons within the claustrum, and cortical afferents is also consistent with recent proposals that the claustrum plays a role in detecting salient stimuli or amplifying correlated cortical inputs.

Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice.

A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.

Layer 6 corticothalamic neurons activate a cortical output layer, layer 5a.

Layer 6 corticothalamic neurons are thought to modulate incoming sensory information via their intracortical axons targeting the major thalamorecipient layer of the neocortex, layer 4, and via their long-range feedback projections to primary sensory thalamic nuclei. However, anatomical reconstructions of individual layer 6 corticothalamic (L6 CT) neurons include examples with axonal processes ramifying within layer 5, and the relative input of the overall population of L6 CT neurons to layers 4 and 5 is not well understood. We compared the synaptic impact of L6 CT cells on neurons in layers 4 and 5. We found that the axons of L6 CT neurons densely ramified within layer 5a in both visual and somatosensory cortices of the mouse. Optogenetic activation of corticothalamic neurons generated large EPSPs in pyramidal neurons in layer 5a. In contrast, excitatory neurons in layer 4 exhibited weak excitation or disynaptic inhibition. Fast-spiking parvalbumin-positive cells in both layer 5a and layer 4 were also strongly activated by L6 CT neurons. The overall effect of L6 CT activation was to suppress layer 4 while eliciting action potentials in layer 5a pyramidal neurons. Together, our data indicate that L6 CT neurons strongly activate an output layer of the cortex.

Prolonged disynaptic inhibition in the cortex mediated by slow, non-α7 nicotinic excitation of a specific subset of cortical interneurons.

Cholinergic activation of nicotinic receptors in the cortex plays a critical role in arousal, attention, and learning. Here we demonstrate that cholinergic axons from the basal forebrain of mice excite a specific subset of cortical interneurons via a remarkably slow, non-α7 nicotinic receptor-mediated conductance. In turn, these inhibitory cells generate a delayed and prolonged wave of disynaptic inhibition in neighboring cortical neurons, altering the spatiotemporal pattern of inhibition in cortical circuits.

Cell-type identity: a key to unlocking the function of neocortical circuits.

A central tenet of neuroscience is that the precise patterns of connectivity among neurons in a given brain area underlie its function. However, assigning any aspect of perception or behavior to the wiring of local circuits has been challenging. Here, we review recent work in sensory neocortex that demonstrates the power of identifying specific cell types when investigating the functional organization of brain circuits. These studies indicate that knowing the identity of both the presynaptic and postsynaptic cell type is key when analyzing neocortical circuits. Furthermore, identifying the circuit organization of particular cell types in the neocortex allows the recording and manipulation of each cell type's activity and the direct testing of its functional role in perception and behavior.

Intracortical circuits of pyramidal neurons reflect their long-range axonal targets.

Cortical columns generate separate streams of information that are distributed to numerous cortical and subcortical brain regions. We asked whether local intracortical circuits reflect these different processing streams by testing whether the intracortical connectivity among pyramidal neurons reflects their long-range axonal targets. We recorded simultaneously from up to four retrogradely labelled pyramidal neurons that projected to the superior colliculus, the contralateral striatum or the contralateral cortex to assess their synaptic connectivity. Here we show that the probability of synaptic connection depends on the functional identities of both the presynaptic and postsynaptic neurons. We first found that the frequency of monosynaptic connections among corticostriatal pyramidal neurons is significantly higher than among corticocortical or corticotectal pyramidal neurons. We then show that the probability of feed-forward connections from corticocortical neurons to corticotectal neurons is approximately three- to fourfold higher than the probability of monosynaptic connections among corticocortical or corticotectal cells. Moreover, we found that the average axodendritic overlap of the presynaptic and postsynaptic pyramidal neurons could not fully explain the differences in connection probability that we observed. The selective synaptic interactions we describe demonstrate that the organization of local networks of pyramidal cells reflects the long-range targets of both the presynaptic and postsynaptic neurons.

Endocannabinoids inhibit transmission at granule cell to Purkinje cell synapses by modulating three types of presynaptic calcium channels.

At many central synapses, endocannabinoids released by postsynaptic cells inhibit neurotransmitter release by activating presynaptic cannabinoid receptors. The mechanisms underlying this important means of synaptic regulation are not fully understood. It has been shown at several synapses that endocannabinoids inhibit neurotransmitter release by reducing calcium influx into presynaptic terminals. One hypothesis maintains that endocannabinoids indirectly reduce calcium influx by modulating potassium channels and narrowing the presynaptic action potential. An alternative hypothesis is that endocannabinoids directly and selectively inhibit N-type calcium channels in presynaptic terminals. Here we test these hypotheses at the granule cell to Purkinje cell synapse in cerebellar brain slices. By monitoring optically the presynaptic calcium influx (Ca(influx)) and measuring the EPSC amplitudes, we found that cannabinoid-mediated inhibition arises solely from reduced presynaptic Ca(influx). Next we found that cannabinoid receptor activation does not affect the time course of presynaptic calcium entry, indicating that the reduced Ca(influx) reflects inhibition of presynaptic calcium channels. Finally, we assessed the classes of presynaptic calcium channels inhibited by cannabinoid receptor activation via peptide calcium channel antagonists. Previous studies established that N-type, P/Q-type, and R-type calcium channels are all present in granule cell presynaptic boutons. We found that cannabinoid activation reduced Ca(influx) through N-type, P/Q-type, and R-type calcium channels to 29, 60, and 55% of control, respectively. Thus, rather than narrowing the presynaptic action potential or exclusively modulating N-type calcium channels, CB1 receptor activation inhibits synaptic transmission by modulating all classes of calcium channels present in the presynaptic terminal of the granule cell to Purkinje cell synapse.

Brief presynaptic bursts evoke synapse-specific retrograde inhibition mediated by endogenous cannabinoids.

Many types of neurons can release endocannabinoids that act as retrograde signals to inhibit neurotransmitter release from presynaptic terminals. Little is known, however, about the properties or role of such inhibition under physiological conditions. Here we report that brief bursts of presynaptic activity evoked endocannabinoid release, which strongly inhibited parallel fiber-to-Purkinje cell synapses in rat cerebellar slices. This retrograde inhibition was triggered by activation of either postsynaptic metabotropic or ionotropic glutamate receptors and was restricted to synapses activated with high-frequency bursts. Thus, endocannabinoids allow neurons to inhibit specific synaptic inputs in response to a burst, thereby dynamically fine-tuning the properties of synaptic integration.