Histamine Release in the Prefrontal Cortex Excites Fast-Spiking Interneurons while GABA Released from the Same Axons Inhibits Pyramidal Cells.

We studied how histamine and GABA release from axons originating from the hypothalamic tuberomammillary nucleus (TMN) and projecting to the prefrontal cortex (PFC) influence circuit processing. We optostimulated histamine/GABA from genetically defined TMN axons that express the histidine decarboxylase gene (TMNHDC axons). Whole-cell recordings from PFC neurons in layer 2/3 of prelimbic, anterior cingulate, and infralimbic regions were used to monitor excitability before and after optostimulated histamine/GABA release in male and female mice. We found that histamine-GABA release influences the PFC through actions on distinct neuronal types: the histamine stimulates fast-spiking interneurons; and the released GABA enhances tonic (extrasynaptic) inhibition on pyramidal cells (PyrNs). For fast-spiking nonaccommodating interneurons, histamine released from TMNHDC axons induced additive gain changes, which were blocked by histamine H1 and H2 receptor antagonists. The excitability of other fast-spiking interneurons in the PFC was not altered. In contrast, the GABA released from TMNHDC axons predominantly produced divisive gain changes in PyrNs, increasing their resting input conductance, and decreasing the slope of the input-output relationship. This inhibitory effect on PyrNs was not blocked by histamine receptor antagonists but was blocked by GABAA receptor antagonists. Across the adult life span (from 3 to 18 months of age), the GABA released from TMNHDC axons in the PFC inhibited PyrN excitability significantly more in older mice. For individuals who maintain cognitive performance into later life, the increases in TMNHDC GABA modulation of PyrNs during aging could enhance information processing and be an adaptive mechanism to buttress cognition.SIGNIFICANCE STATEMENT The hypothalamus controls arousal state by releasing chemical neurotransmitters throughout the brain to modulate neuronal excitability. Evidence is emerging that the release of multiple types of neurotransmitters may have opposing actions on neuronal populations in key cortical regions. This study demonstrates for the first time that the neurotransmitters histamine and GABA are released in the prefrontal cortex from axons originating from the tuberomammillary nucleus of the hypothalamus. This work demonstrates how hypothalamic modulation of neuronal excitability is maintained throughout adult life, highlighting an unexpected aspect of the aging process that may help maintain cognitive abilities.

A Role for Thalamic Projection GABAergic Neurons in Circadian Responses to Light.

The thalamus is an important hub for sensory information and participates in sensory perception, regulation of attention, arousal and sleep. These functions are executed primarily by glutamatergic thalamocortical neurons that extend axons to the cortex and initiate cortico-thalamocortical connectional loops. However, the thalamus also contains projection GABAergic neurons that do not extend axons toward the cortex. Here, we have harnessed recent insight into the development of the intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (LGv) to specifically target and manipulate thalamic projection GABAergic neurons in female and male mice. Our results show that thalamic GABAergic neurons of the IGL and LGv receive retinal input from diverse classes of retinal ganglion cells (RGCs) but not from the M1 intrinsically photosensitive retinal ganglion cell (ipRGC) type. We describe the synergistic role of the photoreceptor melanopsin and the thalamic neurons of the IGL/LGv in circadian entrainment to dim light. We identify a requirement for the thalamic IGL/LGv neurons in the rapid changes in vigilance states associated with circadian light transitions.SIGNIFICANCE STATEMENT The intergeniculate leaflet (IGL) and ventral lateral geniculate nucleus (LGv) are part of the extended circadian system and mediate some nonimage-forming visual functions. Here, we show that each of these structures has a thalamic (dorsal) as well as prethalamic (ventral) developmental origin. We map the retinal input to thalamus-derived cells in the IGL/LGv complex and discover that while RGC input is dominant, this is not likely to originate from M1ipRGCs. We implicate thalamic cells in the IGL/LGv in vigilance state transitions at circadian light changes and in overt behavioral entrainment to dim light, the latter exacerbated by concomitant loss of melanopsin expression.

Bottom-Up versus Top-Down Induction of Sleep by Zolpidem Acting on Histaminergic and Neocortex Neurons.

Zolpidem, a GABAA receptor-positive modulator, is the gold-standard drug for treating insomnia. Zolpidem prolongs IPSCs to decrease sleep latency and increase sleep time, effects that depend on α2 and/or α3 subunit-containing receptors. Compared with natural NREM sleep, zolpidem also decreases the EEG power, an effect that depends on α1 subunit-containing receptors, and which may make zolpidem-induced sleep less optimal. In this paper, we investigate whether zolpidem needs to potentiate only particular GABAergic pathways to induce sleep without reducing EEG power. Mice with a knock-in F77I mutation in the GABAA receptor γ2 subunit gene are zolpidem-insensitive. Using these mice, GABAA receptors in the frontal motor neocortex and hypothalamic (tuberomammillary nucleus) histaminergic-neurons of γ2I77 mice were made selectively sensitive to zolpidem by genetically swapping the γ2I77 subunits with γ2F77 subunits. When histamine neurons were made selectively zolpidem-sensitive, systemic administration of zolpidem shortened sleep latency and increased sleep time. But in contrast to the effect of zolpidem on wild-type mice, the power in the EEG spectra of NREM sleep was not decreased, suggesting that these EEG power-reducing effects of zolpidem do not depend on reduced histamine release. Selective potentiation of GABAA receptors in the frontal cortex by systemic zolpidem administration also reduced sleep latency, but less so than for histamine neurons. These results could help with the design of new sedatives that induce a more natural sleep. SIGNIFICANCE STATEMENT: Many people who find it hard to get to sleep take sedatives. Zolpidem (Ambien) is the most widely prescribed "sleeping pill." It makes the inhibitory neurotransmitter GABA work better at its receptors throughout the brain. The sleep induced by zolpidem does not resemble natural sleep because it produces a lower power in the brain waves that occur while we are sleeping. We show using mouse genetics that zolpidem only needs to work on specific parts and cell types of the brain, including histamine neurons in the hypothalamus, to induce sleep but without reducing the power of the sleep. This knowledge could help in the design of sleeping pills that induce a more natural sleep.

Altered activity in the central medial thalamus precedes changes in the neocortex during transitions into both sleep and propofol anesthesia.

How general anesthetics cause loss of consciousness is unknown. Some evidence points toward effects on the neocortex causing "top-down" inhibition, whereas other findings suggest that these drugs act via subcortical mechanisms, possibly selectively stimulating networks promoting natural sleep. To determine whether some neuronal circuits are affected before others, we used Morlet wavelet analysis to obtain high temporal resolution in the time-varying power spectra of local field potentials recorded simultaneously in discrete brain regions at natural sleep onset and during anesthetic-induced loss of righting reflex in rats. Although we observed changes in the local field potentials that were anesthetic-specific, there were some common changes in high-frequency (20-40 Hz) oscillations (reductions in frequency and increases in power) that could be detected at, or before, sleep onset and anesthetic-induced loss of righting reflex. For propofol and natural sleep, these changes occur first in the thalamus before changes could be detected in the neocortex. With dexmedetomidine, the changes occurred simultaneously in the thalamus and neocortex. In addition, the phase relationships between the low-frequency (1-4 Hz) oscillations in thalamic nuclei and neocortical areas are essentially the same for natural sleep and following dexmedetomidine administration, but a sudden change in phase, attributable to an effect in the central medial thalamus, occurs at the point of dexmedetomidine loss of righting reflex. Our data are consistent with the central medial thalamus acting as a key hub through which general anesthesia and natural sleep are initiated.

Two-pore domain potassium channels enable action potential generation in the absence of voltage-gated potassium channels.

In this study, we explored the possibility that two-pore domain potassium (K2P) channels are sufficient to support action potential (AP) generation in the absence of conventional voltage-gated potassium (KV) channels. Hodgkin-Huxley parameters were used to mimic the presence of voltage-gated sodium (NaV) channels in HEK-293 cells. Recombinant expression of either TREK-1 or TASK-3 channels was then used to generate a hyperpolarised resting membrane potential (RMP) leading to the characteristic non-linear current-voltage relationship expected of a K2P-mediated conductance. During conductance simulation experiments, both TASK-3 and TREK-1 channels were able to repolarise the membrane once AP threshold was reached, and at physiologically relevant current densities, this K2P-mediated conductance supported sustained AP firing. Moreover, the magnitude of the conductance correlated with the speed of the AP rise in a manner predicted from our computational studies. We discuss the physiological impact of axonal K2P channels and speculate on the possible clinical relevance of K2P channel modulation when considering the actions of general and local anaesthetics.

Copper block of extrasynaptic GABAA receptors in the mature cerebellum and striatum.

Inhibition of GABAA receptors by Cu(2+) has been appreciated for some time, but differences between synaptic and extrasynaptic GABAA receptors have not been explored. We show that Cu(2+) potently blocks steady-state GABA currents mediated by extrasynaptic δ subunit-containing GABAA receptors (δ-GABAARs) with an IC50 of 65 nM. This compares with an IC50 of 85 µM for synaptic γ subunit-containing GABAARs (γ-GABAARs). To test the significance of this subunit selectivity, we examined the blocking action of Cu(2+) on neurons of the mouse cerebellum and striatum, brain regions that are known to express both types of receptor. Cu(2+) was shown to significantly reduce tonic inhibition mediated by extrasynaptic δ-GABAARs with little action on phasic inhibition mediated by conventional synaptic γ-GABAARs. We speculate on the implications of these observations for conditions, such as Wilson's disease, that can involve raised Cu(2+) levels in the brain.

The type of inhibition provided by thalamic interneurons alters the input selectivity of thalamocortical neurons.

A fundamental problem in neuroscience is how neurons select for their many inputs. A common assumption is that a neuron's selectivity is largely explained by differences in excitatory synaptic input weightings. Here we describe another solution to this important problem. We show that within the first order visual thalamus, the type of inhibition provided by thalamic interneurons has the potential to alter the input selectivity of thalamocortical neurons. To do this, we developed conductance injection protocols to compare how different types of synchronous and asynchronous GABA release influence thalamocortical excitability in response to realistic patterns of retinal ganglion cell input. We show that the asynchronous GABA release associated with tonic inhibition is particularly efficient at maintaining information content, ensuring that thalamocortical neurons can distinguish between their inputs. We propose a model where alterations in GABA release properties results in rapid changes in input selectivity without requiring structural changes in the network.

Are extrasynaptic GABAA receptors important targets for sedative/hypnotic drugs?

High-affinity extrasynaptic GABA(A) receptors are persistently activated by the low ambient GABA levels that are known to be present in extracellular space. The resulting tonic conductance generates a form of shunting inhibition that is capable of altering cellular and network behavior. It has been suggested that this tonic inhibition will be enhanced by neurosteroids, antiepileptics, and sedative/hypnotic drugs. However, we show that the ability of sedative/hypnotic drugs to enhance tonic inhibition in the mouse cerebellum will critically depend on ambient GABA levels. For example, we show that the intravenous anesthetic propofol enhances tonic inhibition only when ambient GABA levels are <100 nm. More surprisingly, the actions of the sleep-promoting drug 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) are attenuated at ambient GABA levels of just 20 nm. In contrast, our data suggest that neurosteroid enhancement of tonic inhibition will be greater at high ambient GABA concentrations. We present a model that takes into account realistic estimates of ambient GABA levels and predicted extrasynaptic GABA(A) receptor numbers when considering the ability of sedative/hypnotic drugs to enhance tonic inhibition. These issues will be important when considering drug strategies designed to target extrasynaptic GABA(A) receptors in the treatment of sleep disorders and other neurological conditions.

GABAergic inhibition of histaminergic neurons regulates active waking but not the sleep-wake switch or propofol-induced loss of consciousness.

The activity of histaminergic neurons in the tuberomammillary nucleus (TMN) of the hypothalamus correlates with an animal's behavioral state and maintains arousal. We examined how GABAergic inputs onto histaminergic neurons regulate this behavior. A prominent hypothesis, the "flip-flop" model, predicts that increased and sustained GABAergic drive onto these cells promotes sleep. Similarly, because of the histaminergic neurons' key hub-like place in the arousal circuitry, it has also been suggested that anesthetics such as propofol induce loss of consciousness by acting primarily at histaminergic neurons. We tested both these hypotheses in mice by genetically removing ionotropic GABA(A) or metabotropic GABA(B) receptors from histidine decarboxylase-expressing neurons. At the cellular level, histaminergic neurons deficient in synaptic GABA(A) receptors were significantly more excitable and were insensitive to the anesthetic propofol. At the behavioral level, EEG profiles were recorded in nontethered mice over 24 h. Surprisingly, GABAergic transmission onto histaminergic neurons had no effect in regulating the natural sleep-wake cycle and, in the case of GABA(A) receptors, for propofol-induced loss of righting reflex. The latter finding makes it unlikely that the histaminergic TMN has a central role in anesthesia. GABA(B) receptors on histaminergic neurons were dispensable for all behaviors examined. Synaptic inhibition of histaminergic cells by GABA(A) receptors, however, was essential for habituation to a novel environment.

Profound desensitization by ambient GABA limits activation of δ-containing GABAA receptors during spillover.

High-affinity extrasynaptic GABA(A) receptors (GABA(A)Rs) are a prominent feature of cerebellar granule neurons and thalamic relay neurons. In both cell types, the presence of synaptic glomeruli would be expected to promote activation of these GABA(A)Rs, contributing to phasic spillover-mediated currents and tonic inhibition. However, the precise role of different receptor subtypes in these two phenomena is unclear. To address this question, we made recordings from neurons in acute brain slices from mice, and from tsA201 cells expressing recombinant GABA(A)Rs. We found that δ subunit-containing GABA(A)Rs of both cerebellar granule neurons and thalamic relay neurons of the lateral geniculate nucleus contributed to tonic conductance caused by ambient GABA but not to spillover-mediated currents. In the presence of a low "ambient" GABA concentration, recombinant "extrasynaptic" δ subunit-containing GABA(A)Rs exhibited profound desensitization, rendering them insensitive to brief synaptic- or spillover-like GABA transients. Together, our results demonstrate that phasic spillover and tonic inhibition reflect the activation of distinct receptor populations.

An unexpected role for TASK-3 potassium channels in network oscillations with implications for sleep mechanisms and anesthetic action.

TASK channels are acid-sensitive and anesthetic-activated members of the family of two-pore-domain potassium channels. We have made the surprising discovery that the genetic ablation of TASK-3 channels eliminates a specific type of theta oscillation in the cortical electroencephalogram (EEG) resembling type II theta (4-9 Hz), which is thought to be important in processing sensory stimuli before initiating motor activity. In contrast, ablation of TASK-1 channels has no effect on theta oscillations. Despite the absence of type II theta oscillations in the TASK-3 knockout (KO) mice, the related type I theta, which has certain neuronal pathways in common and is involved in exploratory behavior, is unaffected. In addition to the absence of type II theta oscillations, the TASK-3 KO animals show marked alterations in both anesthetic sensitivity and natural sleep behavior. Their sensitivity to halothane, a potent activator of TASK channels, is greatly reduced, whereas their sensitivity to cyclopropane, which does not activate TASK-3 channels, is unchanged. The TASK-3 KO animals exhibit a slower progression from their waking to sleeping states and, during their sleeping period, their sleep episodes as well as their REM theta oscillations are more fragmented. These results imply a previously unexpected role for TASK-3 channels in the cellular mechanisms underlying these behaviors and suggest that endogenous modulators of these channels may regulate theta oscillations.

Signaling across the synapse: a role for Wnt and Dishevelled in presynaptic assembly and neurotransmitter release.

Proper dialogue between presynaptic neurons and their targets is essential for correct synaptic assembly and function. At central synapses, Wnt proteins function as retrograde signals to regulate axon remodeling and the accumulation of presynaptic proteins. Loss of Wnt7a function leads to defects in the localization of presynaptic markers and in the morphology of the presynaptic axons. We show that loss of function of Dishevelled-1 (Dvl1) mimics and enhances the Wnt7a phenotype in the cerebellum. Although active zones appear normal, electrophysiological recordings in cerebellar slices from Wnt7a/Dvl1 double mutant mice reveal a defect in neurotransmitter release at mossy fiber-granule cell synapses. Deficiency in Dvl1 decreases, whereas exposure to Wnt increases, synaptic vesicle recycling in mossy fibers. Dvl increases the number of Bassoon clusters, and like other components of the Wnt pathway, it localizes to synaptic sites. These findings demonstrate that Wnts signal across the synapse on Dvl-expressing presynaptic terminals to regulate synaptic assembly and suggest a potential novel function for Wnts in neurotransmitter release.

Dual midbrain and forebrain origins of thalamic inhibitory interneurons.

The ubiquitous presence of inhibitory interneurons in the thalamus of primates contrasts with the sparsity of interneurons reported in mice. Here, we identify a larger than expected complexity and distribution of interneurons across the mouse thalamus, where all thalamic interneurons can be traced back to two developmental programmes: one specified in the midbrain and the other in the forebrain. Interneurons migrate to functionally distinct thalamocortical nuclei depending on their origin: the abundant, midbrain-derived class populates the first and higher order sensory thalamus while the rarer, forebrain-generated class is restricted to some higher order associative regions. We also observe that markers for the midbrain-born class are abundantly expressed throughout the thalamus of the New World monkey marmoset. These data therefore reveal that, despite the broad variability in interneuron density across mammalian species, the blueprint of the ontogenetic organisation of thalamic interneurons of larger-brained mammals exists and can be studied in mice.

The involvement of hypothalamic sleep pathways in general anesthesia: testing the hypothesis using the GABAA receptor beta3N265M knock-in mouse.

The GABA(A) receptor has been identified as the single most important target for the intravenous anesthetic propofol. How effects at this receptor are then translated into a loss of consciousness, however, remains a mystery. One possibility is that anesthetics act on natural sleep pathways. Here, we test this hypothesis by exploring the anesthetic sensitivities of GABAergic synaptic currents in three specific brain nuclei that are known to be involved in sleep. Using whole-cell electrophysiology, we have recorded GABAergic IPSCs from the tuberomammillary nucleus (TMN), the perifornical area (Pef), and the locus ceruleus (LC) in brain slices from both wild-type mice and mice that carry a specific mutation in the GABA(A) receptor beta(3) subunit (N265M), which greatly reduces their sensitivity to propofol, but not to the neurosteroid alphaxalone. We find that this in vivo pattern of anesthetic sensitivity is mirrored in the hypothalamic TMN and Pef nuclei, consistent with their role as direct anesthetic targets. In contrast, anesthetic sensitivity in the LC was unaffected by the beta(3)N265M mutation, ruling out this nucleus as a major target for propofol. In support of the hypothesis that orexinergic neurons in the Pef are involved in propofol anesthesia, we further show that these neurons are selectively inhibited by GABAergic drugs in vivo during anesthesia, and that a modulation in the activity of Pef neurons alone can affect loss of righting reflex. Overall, our results support the idea that GABAergic anesthetics such as propofol exert their effects, at least in part, by modulating hypothalamic sleep pathways.

Synaptic release generates a tonic GABA(A) receptor-mediated conductance that modulates burst precision in thalamic relay neurons.

Tonic inhibition has emerged as a key regulator of neuronal excitability in the CNS. Thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) exhibit a tonic GABA(A) receptor (GABA(A)R)-mediated conductance that is correlated with delta-subunit expression. Indeed, consistent with the absence of delta-subunit expression, no tonic conductance is found in the adjacent ventral LGN. We show that, in contrast to the situation in cerebellar granule cells, thalamic delta-subunit-containing GABA(A)Rs (delta-GABA(A)Rs) do not contribute to a spillover component of IPSCs in dLGN. However, tonic activation of thalamic delta-GABA(A)Rs is sensitive to the global level of inhibition, showing an absolute requirement on the synaptic release of GABA. Thus, the tonic conductance is abolished when transmitter release probability is reduced or action potential-evoked release is blocked. We further show that continuous activation of delta-GABA(A)Rs introduces variability into the timing of low-threshold rebound bursts. Hence, activation of delta-GABA(A)Rs could act to destabilize thalamocortical oscillations and therefore have an important impact on behavioral state.

TASK-3 two-pore domain potassium channels enable sustained high-frequency firing in cerebellar granule neurons.

The ability of neurons, such as cerebellar granule neurons (CGNs), to fire action potentials (APs) at high frequencies during sustained depolarization is usually explained in relation to the functional properties of voltage-gated ion channels. Two-pore domain potassium (K(2P)) channels are considered to simply hyperpolarize the resting membrane potential (RMP) by increasing the potassium permeability of the membrane. However, we find that CGNs lacking the TASK-3 type K(2P) channel exhibit marked accommodation of action potential firing. The accommodation phenotype was not associated with any change in the functional properties of the underlying voltage-gated sodium channels, nor could it be explained by the more depolarized RMP that resulted from TASK-3 channel deletion. A functional rescue, involving the introduction of a nonlinear leak conductance with a dynamic current clamp, was able to restore wild-type firing properties to adult TASK-3 knock-out CGNs. Thus, in addition to the accepted role of TASK-3 channels in limiting neuronal excitability, by increasing the resting potassium conductance TASK-3 channels also increase excitability by supporting high-frequency firing once AP threshold is reached.

Acting locally but sensing globally: impact of GABAergic synaptic plasticity on phasic and tonic inhibition in the thalamus.

We have discovered that adult thalamocortical relay neurones exhibit a sustained enhancement of synaptic inhibition triggered by transient action potential firing of a single thalamic relay neurone. The sustained activity-dependent increase in IPSC frequency (+48.3 +/- 11.4%, n = 32) was blocked by chelating calcium inside an individual cell, by scavenging nitric oxide or by blocking NMDA receptor activation in the thalamus. Surprisingly, the tonic inhibition that is known to result from extrasynaptic GABA(A) receptor activation in these cells was unaffected by this local form of plasticity. However, tonic inhibition was increased (+131.9 +/- 56.5%, n = 13) following widespread changes in GABA release across the thalamus. These data suggest that thalamocortical sleep-state oscillations requiring membrane hyperpolarization will be influenced by global sensing of GABA release acting through extrasynaptic GABA(A) receptors. In contrast, local changes in GABA release of the type observed following this novel form of activity-dependent plasticity will influence local integration of sensory information without changing levels of tonic inhibition.

Combining mGRASP and Optogenetics Enables High-Resolution Functional Mapping of Descending Cortical Projections.

We have applied optogenetics and mGRASP, a light microscopy technique that labels synaptic contacts, to map the number and strength of defined corticocollicular (CC) connections. Using mGRASP, we show that CC projections form small, medium, and large synapses, and both the number and the distribution of synapse size vary among the IC regions. Using optogenetics, we show that low-frequency stimulation of CC axons expressing channelrhodopsin produces prolonged elevations of the CC miniature EPSC (mEPSC) rate. Functional analysis of CC mEPSCs reveals small-, medium-, and large-amplitude events that mirror the synaptic distributions observed with mGRASP. Our results reveal that descending ipsilateral projections dominate CC feedback via an increased number of large synaptic contacts, especially onto the soma of IC neurons. This study highlights the feasibility of combining microscopy (i.e., mGRASP) and optogenetics to reveal synaptic weighting of defined projections at the level of single neurons, enabling functional connectomic mapping in diverse neural circuits.

Exploring the significance of morphological diversity for cerebellar granule cell excitability.

The relatively simple and compact morphology of cerebellar granule cells (CGCs) has led to the view that heterogeneity in CGC shape has negligible impact upon the integration of mossy fibre (MF) information. Following electrophysiological recording, 3D models were constructed from high-resolution imaging data to identify morphological features that could influence the coding of MF input patterns by adult CGCs. Quantification of MF and CGC morphology provided evidence that CGCs could be connected to the multiple rosettes that arise from a single MF input. Predictions from our computational models propose that MF inputs could be more densely encoded within the CGC layer than previous models suggest. Moreover, those MF signals arriving onto the dendrite closest to the axon will generate greater CGC excitation. However, the impact of this morphological variability on MF input selectivity will be attenuated by high levels of CGC inhibition providing further flexibility to the MF → CGC pathway. These features could be particularly important when considering the integration of multimodal MF sensory input by individual CGCs.