NDNF is selectively expressed by neocortical, but not habenular neurogliaform cells.

The lateral habenula (LHb) is a brain structure which is known to be pathologically hyperactive in depression, whereby it shuts down the brains' reward systems. Interestingly, inhibition of the LHb has been shown to have an antidepressant effect, hence making the LHb a fascinating subject of study for developing novel antidepressant therapies. Despite this however, the exact mechanisms by which inhibitory signalling is processed within the LHb remain incompletely understood. Some studies have proposed the existence of locally targeting inhibitory interneuron populations within the LHb. One such population is believed to be akin to neocortical neurogliaform cells, yet specific molecular markers for studying these neurons are sparse and hence their function remains elusive. Recently, neuron-derived neurotrophic factor (NDNF) has been proposed as one such marker for neocortical neurogliaform cells. Using a combination of histological, physiological and optogenetic tools, we hence sought to first validate if NDNF was selectively expressed by such inhibitory neurons within the neocortex, and then if it was confined to a similar population within the LHb. While we report this to be true for the neocortex, we find no such evidence within the LHb; rather that NDNF is expressed without restriction to a particular neuronal subpopulation. These results hence indicate that molecular markers can represent broadly diverse populations of neurons on a region-to-region basis and that therefore each population as defined by molecular marker expression should be validated in each brain structure.

Effects of early life stress and subsequent re-exposure to stress on neuronal activity in the lateral habenula.

Early life stress can result in depression in humans and depressive-like behaviour in rodents. In various animal models of depression, the lateral habenula (LHb) has been shown to become hyperactive immediately after early life stress. However, whether these pathological changes persist into adulthood is less well understood. Hence, we utilised the maternal separation (MS) model of depression to study how early life stress alters LHb physiology and depressive behaviour in adult mice. We find that only a weak depressive phenotype persists into adulthood which surprisingly is underpinned by LHb hypoactivity in acute slices, accompanied by alterations in both excitatory and inhibitory signalling. However, while we find the LHb to be less active at rest, we report that the neurons reside in a sensitised state where they are more responsive to re-exposure to stress in adulthood in the form of acute restraint, thus priming them to respond to aversive events with an increase in neuronal activity mediated by changes in glutamatergic transmission. These findings thus suggest that in addition to LHb hyperactivity, hypoactivity likely also promotes an adverse phenotype. Re-exposure to stress results in the reappearance of LHb hyperactivity offering a possible mechanism to explain how depression relapses occur following previous depressive episodes.

Cannabinoids disrupt hippocampal sharp wave-ripples via inhibition of glutamate release.

Cannabis consumption results in impaired learning. The proper synchronization of neuronal activity in the mammalian hippocampus gives rise to network rhythms that are implicated in memory formation. Here, we have studied the impact of cannabinoids on hippocampal sharp waves and associated ripple oscillations using field- and whole-cell voltage-clamp recordings. We demonstrate that the activation of cannabinoid receptor 1 suppresses sharp wave-ripples (SWRs) in mice in vivo and in vitro. This suppression was paralleled by a selective reduction of SWR-associated inward but not outward charge transfer, demonstrating an impairment of excitation due to cannabinoid exposure. Adenosine, a presynaptic modulator of glutamate release, mimicked and occluded the observed consequences of cannabinoids on SWRs. We conclude that inhibition of glutamatergic feed-forward excitation can explain cannabinoid-mediated disruption of SWRs and may account for cannabinoid-induced impairment of hippocampus-dependent memory.

Specificity of synaptic connectivity between layer 1 inhibitory interneurons and layer 2/3 pyramidal neurons in the rat neocortex.

Understanding the structure and function of the neocortical microcircuit requires a description of the synaptic connectivity between identified neuronal populations. Here, we investigate the electrophysiological properties of layer 1 (L1) neurons of the rat somatosensory neocortex (postnatal day 24-36) and their synaptic connectivity with supragranular pyramidal neurons. The active and passive properties of visually identified L1 neurons (n = 266) suggested division into 4 groups according to the Petilla classification scheme with characteristics of neurogliaform cells (NGFCs) (n = 72), classical-accommodating (n = 137), fast-spiking (n = 23), and burst-spiking neurons (n = 34). Anatomical reconstructions of L1 neurons supported the existence of 4 major neuronal groups. Multiparameter unsupervised cluster analysis confirmed the existence of 4 groups, revealing a high degree of similarity with the Petilla scheme. Simultaneous recordings between synaptically connected L1 neurons and L2/3 pyramidal neurons (n = 384) demonstrated neuronal class specificity in both excitatory and inhibitory connectivity and the properties of synaptic potentials. Notably, all groups of L1 neurons received monosynaptic excitatory input from L2/3 pyramidal neurons (n = 33), with the exception of NGFCs (n = 68 pairs tested). In contrast, NGFCs strongly inhibited L2/3 pyramidal neurons (n = 12 out 27 pairs tested). These data reveal a high specificity of excitatory and inhibitory connections in the superficial layers of the neocortex.

Transcriptomically-Guided Pharmacological Experiments in Neocortical and Hippocampal NPY-Positive GABAergic Interneurons.

Cortical GABAergic interneurons have been shown to fulfil important roles by inhibiting excitatory principal neurons. Recent transcriptomic studies have confirmed seminal discoveries that used anatomic and electrophysiological methods highlighting the existence of multiple different classes of GABAergic interneurons. Although some of these studies have emphasized that inter-regional differences may exist for a given class, the extent of such differences remains unknown. To address this problem, we used single-cell Patch-RNAseq to characterize neuropeptide Y (NPY)-positive GABAergic interneurons in superficial layers of the primary auditory cortex (AC) and in distal layers of area CA3 in mice. We found that more than 300 genes are differentially expressed in NPY-positive neurons between these two brain regions. For example, the AMPA receptor (AMPAR) auxiliary subunit Shisa9/CKAMP44 and the 5HT2a receptor (5HT2aR) are significantly higher expressed in auditory NPY-positive neurons. These findings guided us to perform pharmacological experiments that revealed a role for 5HT2aRs in auditory NPY-positive neurons. Specifically, although the application of 5HT led to a depolarization of both auditory and CA3 NPY-positive neurons, the 5HT2aR antagonist ketanserin only reversed membrane potential changes in auditory NPY-positive neurons. Our study demonstrates the potential of single-cell transcriptomic studies in guiding directed pharmacological experiments.

Protease-activated receptor 2 activation induces behavioural changes associated with depression-like behaviour through microglial-independent modulation of inflammatory cytokines.

RATIONALE: Major depressive disorder (MDD) is a leading cause of disability worldwide but currently prescribed treatments do not adequately ameliorate the disorder in a significant portion of patients. Hence, a better appreciation of its aetiology may lead to the development of novel therapies. OBJECTIVES: In the present study, we have built on our previous findings indicating a role for protease-activated receptor-2 (PAR2) in sickness behaviour to determine whether the PAR2 activator, AC264613, induces behavioural changes similar to those observed in depression-like behaviour. METHODS: AC264613-induced behavioural changes were examined using the open field test (OFT), sucrose preference test (SPT), elevated plus maze (EPM), and novel object recognition test (NOR). Whole-cell patch clamping was used to investigate the effects of PAR2 activation in the lateral habenula with peripheral and central cytokine levels determined using ELISA and quantitative PCR. RESULTS: Using a blood-brain barrier (BBB) permeable PAR2 activator, we reveal that AC-264613 (AC) injection leads to reduced locomotor activity and sucrose preference in mice but is without effect in anxiety and memory-related tasks. In addition, we show that AC injection leads to elevated blood sera IL-6 levels and altered cytokine mRNA expression within the brain. However, neither microglia nor peripheral lymphocytes are the source of these altered cytokine profiles. CONCLUSIONS: These data reveal that PAR2 activation results in behavioural changes often associated with depression-like behaviour and an inflammatory profile that resembles that seen in patients with MDD and therefore PAR2 may be a target for novel antidepressant therapies.

The back and forth of dendritic plasticity.

Synapses are located throughout the often-elaborate dendritic tree of central neurons. Hebbian models of plasticity require temporal association between synaptic input and neuronal output to produce long-term potentiation of excitatory transmission. Recent studies have highlighted how active dendritic spiking mechanisms control this association. Here, we review new work showing that associative synaptic plasticity can be generated without neuronal output and that the interplay between neuronal architecture and the active electrical properties of the dendritic tree regulates synaptic plasticity.

Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration.

Cerebral selenium (Se) deficiency is associated with neurological phenotypes including seizures and ataxia. We wanted to define whether neurons require selenoprotein expression and which selenoproteins are most important, and explore the possible pathomechanism. Therefore, we abrogated the expression of all selenoproteins in neurons by genetic inactivation of the tRNA[Ser](Sec) gene. Cerebral expression of selenoproteins was significantly diminished in the mutants, and histological analysis revealed progressive neurodegeneration. Developing interneurons failed to specifically express parvalbumin (PV) in the mutants. Electrophysiological recordings, before overt cell death, showed normal excitatory transmission, but revealed spontaneous epileptiform activity consistent with seizures in the mutants. In developing cortical neuron cultures, the number of PV(+) neurons was reduced on combined Se and vitamin E deprivation, while other markers, such as calretinin (CR) and GAD67, remained unaffected. Because of the synergism between Se and vitamin E, we analyzed mice lacking neuronal expression of the Se-dependent enzyme glutathione peroxidase 4 (GPx4). Although the number of CR(+) interneurons remained normal in Gpx4-mutant mice, the number of PV(+) interneurons was reduced. Since these mice similarly exhibit seizures and ataxia, we conclude that GPx4 is a selenoenzyme modulating interneuron function and PV expression. Cerebral SE deficiency may thus act via reduced GPx4 expression.-Wirth, E. K., Conrad, M., Winterer, J., Wozny, C., Carlson, B. A., Roth, S., Schmitz, D., Bornkamm, G. W., Coppola, V., Tessarollo, L., Schomburg, L., Köhrle, J., Hatfield, D. L., Schweizer, U. Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration.

Inhibition Within the Lateral Habenula-Implications for Affective Disorders.

The lateral habenula (LHb) is a key brain region implicated in the pathology of major depressive disorder (MDD). Specifically, excitatory LHb neurons are known to be hyperactive in MDD, thus resulting in a greater excitatory output mainly to downstream inhibitory neurons in the rostromedial tegmental nucleus. This likely results in suppression of downstream dopaminergic ventral tegmental area neurons, therefore, resulting in an overall reduction in reward signalling. In line with this, increasing evidence implicates aberrant inhibitory signalling onto LHb neurons as a co-causative factor in MDD, likely as a result of disinhibition of excitatory neurons. Consistently, growing evidence now suggests that normalising inhibitory signalling within the LHb may be a potential therapeutic strategy for MDD. Despite these recent advances, however, the exact pharmacological and neural circuit mechanisms which control inhibitory signalling within the LHb are still incompletely understood. Thus, in this review article, we aim to provide an up-to-date summary of the current state of knowledge of the mechanisms by which inhibitory signalling is processed within the LHb, with a view of exploring how this may be targeted as a future therapy for MDD.

Probing subicular inputs to the medial prefrontal cortex.

The hippocampal formation is anatomically and functionally divided into a dorsal and a ventral part, being involved in processing cognitive tasks and emotional stimuli, respectively. The ventral subiculum as part of the hippocampal formation projects to the medial prefrontal cortex (mPFC), but only very little is known about connections arising from the dorsal SUB (dSUB). Here, we investigate the dSUB to mPFC connectivity in acute brain slices using electrophysiology and optogenetics. We show that the anterior cingulate cortex (ACC) is the main target of dorsal subicular projections to the mPFC, with no preference between excitatory or inhibitory neurons. In addition to superficial neurons in the ACC, the prelimbic and infralimbic PFC are also targeted by subicular fibers. Thus, these novel region- and layer-specific connections between the dSUB and the prefrontal cortices challenge existing anatomical data and refine the hippocampocortical wiring diagram.

Role of amyloid-beta glycine 33 in oligomerization, toxicity, and neuronal plasticity.

The aggregation of the amyloid-beta (Abeta) peptide plays a pivotal role in the pathogenesis of Alzheimer's disease, as soluble oligomers are intimately linked to neuronal toxicity and inhibition of hippocampal long-term potentiation (LTP). In the C-terminal region of Abeta there are three consecutive GxxxG dimerization motifs, which we could previously demonstrate to play a critical role in the generation of Abeta. Here, we show that glycine 33 (G33) of the central GxxxG interaction motif within the hydrophobic Abeta sequence is important for the aggregation dynamics of the peptide. Abeta peptides with alanine or isoleucine substitutions of G33 displayed an increased propensity to form higher oligomers, which we could attribute to conformational changes. Importantly, the oligomers of G33 variants were much less toxic than Abeta(42) wild type (WT), in vitro and in vivo. Also, whereas Abeta(42) WT is known to inhibit LTP, Abeta(42) G33 variants had lost the potential to inhibit LTP. Our findings reveal that conformational changes induced by G33 substitutions unlink toxicity and oligomerization of Abeta on the molecular level and suggest that G33 is the key amino acid in the toxic activity of Abeta. Thus, a specific toxic conformation of Abeta exists, which represents a promising target for therapeutic interventions.

Behavior: Local Lateral Habenula Interneurons Mediate Aggression.

A recent study has shown that local inhibitory GAD2-positive neurons regulate the activity of lateral habenula neurons, thereby governing aggressive behavior in male mice.

Disentangling neuronal inhibition and inhibitory pathways in the lateral habenula.

The lateral habenula (LHb) is hyperactive in depression, and thus potentiating inhibition of this structure makes an interesting target for future antidepressant therapies. However, the circuit mechanisms mediating inhibitory signalling within the LHb are not well-known. We addressed this issue by studying LHb neurons expressing either parvalbumin (PV) or somatostatin (SOM), two markers of particular sub-classes of neocortical inhibitory neurons. Here, we find that both PV and SOM are expressed by physiologically distinct sub-classes. Furthermore, we describe multiple sources of inhibitory input to the LHb arising from both local PV-positive neurons, from PV-positive neurons in the medial dorsal thalamic nucleus, and from SOM-positive neurons in the ventral pallidum. These findings hence provide new insight into inhibitory control within the LHb, and highlight that this structure is more neuronally diverse than previously thought.

Loss of Long-Term Potentiation at Hippocampal Output Synapses in Experimental Temporal Lobe Epilepsy.

Patients suffering from temporal lobe epilepsy (TLE) show severe problems in hippocampus dependent memory consolidation. Memory consolidation strongly depends on an intact dialog between the hippocampus and neocortical structures. Deficits in hippocampal signal transmission are known to provoke disturbances in memory formation. In the present study, we investigate changes of synaptic plasticity at hippocampal output structures in an experimental animal model of TLE. In pilocarpine-treated rats, we found suppressed long-term potentiation (LTP) in hippocampal and parahippocampal regions such as the subiculum and the entorhinal cortex (EC). Subsequently we focused on the subiculum, serving as the major relay station between the hippocampus proper and downstream structures. In control animals, subicular pyramidal cells express different forms of LTP depending on their intrinsic firing pattern. In line with our extracellular recordings, we could show that LTP could only be induced in a minority of subicular pyramidal neurons. We demonstrate that a well-characterized cAMP-dependent signaling pathway involved in presynaptic forms of LTP is perturbed in pilocarpine-treated animals. Our findings suggest that in TLE, disturbances of synaptic plasticity may influence the information flow between the hippocampus and the neocortex.

Differential cAMP signaling at hippocampal output synapses.

cAMP is a critical second messenger involved in synaptic transmission and synaptic plasticity. Here, we show that activation of the adenylyl cyclase by forskolin and application of the cAMP-analog Sp-5,6-DCl-cBIMPS both mimicked and occluded tetanus-induced long-term potentiation (LTP) in subicular bursting neurons, but not in subicular regular firing cells. Furthermore, LTP in bursting cells was inhibited by protein kinase A (PKA) inhibitors Rp-8-CPT-cAMP and H-89. Variations in the degree of EPSC blockade by the low-affinity competitive AMPA receptor-antagonist gamma-d-glutamyl-glycine (gamma-DGG), analysis of the coefficient of variance as well as changes in short-term potentiation suggest an increase of glutamate concentration in the synaptic cleft after expression of LTP. We conclude that presynaptic LTP in bursting cells requires activation of PKA by a calcium-dependent adenylyl cyclase while LTP in regular firing cells is independent of elevated cAMP levels. Our results provide evidence for a differential role of cAMP in LTP at hippocampal output synapses.

Two different forms of long-term potentiation at CA1-subiculum synapses.

Distinct functional roles in learning and memory are attributed to certain areas of the hippocampus and the parahippocampal region. The subiculum as a part of the hippocampal formation is the principal target of CA1 pyramidal cell axons and serves as an interface in the information processing between the hippocampus and the neocortex. Subicular pyramidal cells have been classified as bursting and regular firing cells. Here we report fundamental differences in long-term potentiation (LTP) between both cell types. Prolonged high-frequency stimulation induced NMDA receptor-dependent LTP in both cell types. While LTP relied on postsynaptic calcium in regular firing neurons, no increase in postsynaptic calcium was required in bursting cells. Furthermore, paired-pulse facilitation revealed that the site of LTP expression was postsynaptic in regular firing neurons, while presynaptic in burst firing neurons. Our findings on synaptic plasticity in the subiculum indicate that regular firing and bursting cells represent two functional units with distinct physiological roles in processing hippocampal output.

VGLUT2 Functions as a Differential Marker for Hippocampal Output Neurons.

The subiculum is the gatekeeper between the hippocampus and cortical areas. Yet, the lack of a pyramidal cell-specific marker gene has made the analysis of the subicular area very difficult. Here we report that the vesicular-glutamate transporter 2 (VGLUT2) functions as a specific marker gene for subicular burst-firing neurons, and demonstrate that VGLUT2-Cre mice allow for Channelrhodopsin-2 (ChR2)-assisted connectivity analysis.

Investigation of hippocampal synaptic transmission and plasticity in mice deficient in the actin-binding protein Drebrin.

The dynamic regulation of the actin cytoskeleton plays a key role in controlling the structure and function of synapses. It is vital for activity-dependent modulation of synaptic transmission and long-term changes in synaptic morphology associated with memory consolidation. Several regulators of actin dynamics at the synapse have been identified, of which a salient one is the postsynaptic actin stabilising protein Drebrin (DBN). It has been suggested that DBN modulates neurotransmission and changes in dendritic spine morphology associated with synaptic plasticity. Given that a decrease in DBN levels is correlated with cognitive deficits associated with ageing and dementia, it was hypothesised that DBN protein abundance instructs the integrity and function of synapses. We created a novel DBN deficient mouse line. Analysis of gross brain and neuronal morphology revealed no phenotype in the absence of DBN. Electrophysiological recordings in acute hippocampal slices and primary hippocampal neuronal cultures showed that basal synaptic transmission, and both long-term and homeostatic synaptic plasticity were unchanged, suggesting that loss of DBN is not sufficient in inducing synapse dysfunction. We propose that the overall lack of changes in synaptic function and plasticity in DBN deficient mice may indicate robust compensatory mechanisms that safeguard cytoskeleton dynamics at the synapse.

Excitatory Microcircuits within Superficial Layers of the Medial Entorhinal Cortex.

The distinctive firing pattern of grid cells in the medial entorhinal cortex (MEC) supports its role in the representation of space. It is widely believed that the hexagonal firing field of grid cells emerges from neural dynamics that depend on the local microcircuitry. However, local networks within the MEC are still not sufficiently characterized. Here, applying up to eight simultaneous whole-cell recordings in acute brain slices, we demonstrate the existence of unitary excitatory connections between principal neurons in the superficial layers of the MEC. In particular, we find prevalent feed-forward excitation from pyramidal neurons in layer III and layer II onto stellate cells in layer II, which might contribute to the generation or the inheritance of grid cell patterns.

Cellular and network properties of the subiculum in the pilocarpine model of temporal lobe epilepsy.

The subiculum was recently shown to be crucially involved in the generation of interictal activity in human temporal lobe epilepsy. Using the pilocarpine model of epilepsy, this study examines the anatomical substrates for network hyperexcitability recorded in the subiculum. Regular- and burst-spiking subicular pyramidal cells were stained with fluorescence dyes and reconstructed to analyze seizure-induced alterations of the dendritic and axonal system. In control animals burst-spiking cells outnumbered regular-spiking cells by about two to one. Regular- and burst-spiking cells were characterized by extensive axonal branching and autapse-like contacts, suggesting a high intrinsic connectivity. In addition, subicular axons projecting to CA1 indicate a CA1-subiculum-CA1 circuit. In the subiculum of pilocarpine-treated rats we found an enhanced network excitability characterized by spontaneous rhythmic activity, polysynaptic responses, and all-or-none evoked bursts of action potentials. In pilocarpine-treated rats the subiculum showed cell loss of about 30%. The ratio of regular- and burst-spiking cells was practically inverse as compared to control preparations. A reduced arborization and spine density in the proximal part of the apical dendrites suggests a partial deafferentiation from CA1. In pilocarpine-treated rats no increased axonal outgrowth of pyramidal cells was observed. Hence, axonal sprouting of subicular pyramidal cells is not mandatory for the development of the pathological events. We suggest that pilocarpine-induced seizures cause an unmasking or strengthening of synaptic contacts within the recurrent subicular network.