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.

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.

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.

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.

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.

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.

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.

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.

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.

Optogenetic acidification of synaptic vesicles and lysosomes.

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

Enhanced dopamine-dependent hippocampal plasticity after single MK-801 application.

Dopaminergic hyperfunction and N-methyl-D-aspartate receptor (NMDAR) hypofunction have both been implicated in psychosis. Dopamine-releasing drugs and NMDAR antagonists replicate symptoms associated with psychosis in healthy humans and exacerbate symptoms in patients with schizophrenia. Though hippocampal dysfunction contributes to psychosis, the impact of NMDAR hypofunction on hippocampal plasticity remains poorly understood. Here, we used an NMDAR antagonist rodent model of psychosis to investigate hippocampal long-term potentiation (LTP). We found that single systemic NMDAR antagonism results in a region-specific, presynaptic LTP at hippocampal CA1-subiculum synapses that is induced by activation of D1/D5 dopamine receptors and modulated by L-type voltage-gated Ca(2+) channels. Thereby, our findings may provide a cellular mechanism how NMDAR antagonism can lead to an enhanced hippocampal output causing activation of the hippocampus-ventral tegmental area-loop and overdrive of the dopamine system.

Role of RIM1α in short- and long-term synaptic plasticity at cerebellar parallel fibres.

The presynaptic terminals of synaptic connections are composed of a complex network of interacting proteins that collectively ensure proper synaptic transmission and plasticity characteristics. The key components of this network are the members of the RIM protein family. Here we show that RIM1α can influence short-term plasticity at cerebellar parallel-fibre synapses. We demonstrate that the loss of a single RIM isoform, RIM1α, leads to reduced calcium influx in cerebellar granule cell terminals, decreased release probability and consequently an enhanced short-term facilitation. In contrast, we find that presynaptic long-term plasticity is fully intact in the absence of RIM1α, arguing against its necessary role in the expression of this important process. Our data argue for a universal role of RIM1α in setting release probability via interaction with voltage-dependent calcium channels at different connections instead of synapse-specific functions.

Activity-mediated AMPA receptor remodeling, driven by alternative splicing in the ligand-binding domain.

The AMPA-type glutamate receptor (AMPAR) subunit composition shapes synaptic transmission and varies throughout development and in response to different input patterns. Here, we show that chronic activity deprivation gives rise to synaptic AMPAR responses with enhanced fidelity. Extrasynaptic AMPARs exhibited changes in kinetics and pharmacology associated with splicing of the alternative flip/flop exons. AMPAR mRNA indeed exhibited reprogramming of the flip/flop exons for GluA1 and GluA2 subunits in response to activity, selectively in the CA1 subfield. However, the functional changes did not directly correlate with the mRNA expression profiles but result from altered assembly of GluA1/GluA2 subunit splice variants, uncovering an additional regulatory role for flip/flop splicing in excitatory signaling. Our results suggest that activity-dependent AMPAR remodeling underlies changes in short-term synaptic plasticity and provides a mechanism for neuronal homeostasis.

Group II metabotropic glutamate receptors depress synaptic transmission onto subicular burst firing neurons.

The subiculum (SUB) is a pivotal structure positioned between the hippocampus proper and various cortical and subcortical areas. Despite the growing body of anatomical and intrinsic electrophysiological data of subicular neurons, modulation of synaptic transmission in the SUB is not well understood. In the present study we investigated the role of group II metabotropic glutamate receptors (mGluRs), which have been shown to be involved in the regulation of synaptic transmission by suppressing presynaptic cAMP activity. Using field potential and patch-clamp whole cell recordings we demonstrate that glutamatergic transmission at CA1-SUB synapses is depressed by group II mGluRs in a cell-type specific manner. Application of the group II mGluR agonist (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV) led to a significantly higher reduction of excitatory postsynaptic currents in subicular bursting cells than in regular firing cells. We further used low-frequency stimulation protocols and brief high-frequency bursts to test whether synaptically released glutamate is capable of activating presynaptic mGluRs. However, neither frequency facilitation is enhanced in the presence of the group II mGluR antagonist LY341495, nor is a test stimulus given after a high-frequency burst. In summary, we present pharmacological evidence for presynaptic group II mGluRs targeting subicular bursting cells, but both low- and high-frequency stimulation protocols failed to activate presynaptically located mGluRs.

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.

Cyclic AMP signaling control of action potential firing rate and molecular circadian pacemaking in the suprachiasmatic nucleus.

Circadian pacemaking in suprachiasmatic nucleus (SCN) neurons revolves around transcriptional/posttranslational feedback loops, driven by protein products of "clock" genes. These loops are synchronized and sustained by intercellular signaling, involving vasoactive intestinal peptide (VIP) via its VPAC2 receptor, which positively regulates cAMP synthesis. In turn, SCN cells communicate circadian time to the brain via a daily rhythm in electrophysiological activity. To investigate the mechanisms whereby VIP/VPAC2/cAMP signaling controls SCN molecular and electrical pacemaking, we combined bioluminescent imaging of circadian gene expression and whole-cell electrophysiology in organotypic SCN slices. As a potential direct target of cAMP, we focused on hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. Mutations of VIP-ergic signaling compromised the SCN molecular pacemaker, diminishing the amplitude and intercellular synchrony of circadian gene expression. These deficits were transiently reversed by elevation of cAMP. Similarly, cellular synchrony in electrical firing rates was lost in SCN slices lacking the VPAC2 receptor for VIP. Whole-cell current-clamp recordings in wild-type (WT) slices revealed voltage responses shaped by the conductance I(h), which is mediated by HCN channel activity. The influence of I(h) on voltage responses showed a modest peak in early circadian day, identifying HCN channels as a putative mediator of cAMP-dependent circadian effects on firing rate. I(h), however, was unaffected by loss of VIP-ergic signaling in VPAC2-null slices, and inhibition of cAMP synthesis had no discernible effect on I(h) but did suppress gene expression and SCN firing rates. Moreover, only sustained but not acute, pharmacological blockade of HCN channels reduced action potential (AP) firing. Thus, our evidence suggests that in the SCN, cAMP-mediated signaling is not a principal regulator of HCN channel function and that HCN is not a determinant of AP firing rate. VIP/cAMP-dependent signaling sustains the SCN molecular oscillator and action potential firing via mechanisms yet to be identified.

Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings.

Patch-clamp recording techniques have revolutionized understanding of the function and sub-cellular location of ion channels in excitable cells. The cell-attached patch-clamp configuration represents the method of choice to describe the endogenous properties of voltage-activated ion channels in the axonal, somatic and dendritic membrane of neurons, without disturbance of the intracellular milieu. Here, we directly examine the errors associated with cell-attached patch-clamp measurement of ensemble ion channel activity. We find for a number of classes of voltage-activated channels, recorded from the soma and dendrites of neurons in acute brain-slices and isolated cells, that the amplitude and kinetics of ensemble ion channel activity recorded in cell-attached patches is significantly distorted by transmembrane voltage changes generated by the flow of current through the activated ion channels. We outline simple error-correction procedures that allow a more accurate description of the density and properties of voltage-activated channels to be incorporated into computational models of neurons.