A novel role for the lateral habenula in fear learning.

Fear is an extreme form of aversion that underlies pathological conditions such as panic or phobias. Fear conditioning (FC) is the best-understood model of fear learning. In FC the context and a cue are independently associated with a threatening unconditioned stimulus (US). The lateral habenula (LHb) is a general encoder of aversion. However, its role in fear learning remains poorly understood. Here we studied in rats the role of the LHb in FC using optogenetics and pharmacological tools. We found that inhibition or activation of the LHb during entire FC training impaired both cued and contextual FC. In contrast, optogenetic inhibition of the LHb restricted to cue and US presentation impaired cued but not contextual FC. In either case, simultaneous activation of contextual and cued components of FC, by the presentation of the cue in the training context, recovered the conditioned fear response. Our results support the notion that the LHb is required for the formation of independent contextual and cued fear memories, a previously uncharacterized function for this structure, that could be critical in fear generalization.

Early NMDA Receptor Ablation in Interneurons Causes an Activity-Dependent E/I Imbalance in vivo in Prefrontal Cortex Pyramidal Neurons of a Mouse Model Useful for the Study of Schizophrenia.

Altered Excitatory/Inhibitory (E/I) balance of cortical synaptic inputs has been proposed as a central pathophysiological factor for psychiatric neurodevelopmental disorders, including schizophrenia (SZ). However, direct measurement of E/I synaptic balance have not been assessed in vivo for any validated SZ animal model. Using a mouse model useful for the study of SZ we show that a selective ablation of NMDA receptors (NMDAr) in cortical and hippocampal interneurons during early postnatal development results in an E/I imbalance in vivo, with synaptic inputs to pyramidal neurons shifted towards excitation in the adult mutant medial prefrontal cortex (mPFC). Remarkably, this imbalance depends on the cortical state, only emerging when theta and gamma oscillations are predominant in the network. Additional brain slice recordings and subsequent 3D morphological reconstruction showed that E/I imbalance emerges after adolescence concomitantly with significant dendritic retraction and dendritic spine re-localization in pyramidal neurons. Therefore, early postnatal ablation of NMDAr in cortical and hippocampal interneurons developmentally impacts on E/I imbalance in vivo in an activity-dependent manner.

Interneuron NMDA Receptor Ablation Induces Hippocampus-Prefrontal Cortex Functional Hypoconnectivity after Adolescence in a Mouse Model of Schizophrenia.

Although the etiology of schizophrenia is still unknown, it is accepted to be a neurodevelopmental disorder that results from the interaction of genetic vulnerabilities and environmental insults. Although schizophrenia's pathophysiology is still unclear, postmortem studies point toward a dysfunction of cortical interneurons as a central element. It has been suggested that alterations in parvalbumin-positive interneurons in schizophrenia are the consequence of a deficient signaling through NMDARs. Animal studies demonstrated that early postnatal ablation of the NMDAR in corticolimbic interneurons induces neurobiochemical, physiological, behavioral, and epidemiological phenotypes related to schizophrenia. Notably, the behavioral abnormalities emerge only after animals complete their maturation during adolescence and are absent if the NMDAR is deleted during adulthood. This suggests that interneuron dysfunction must interact with development to impact on behavior. Here, we assess in vivo how an early NMDAR ablation in corticolimbic interneurons impacts on mPFC and ventral hippocampus functional connectivity before and after adolescence. In juvenile male mice, NMDAR ablation results in several pathophysiological traits, including increased cortical activity and decreased entrainment to local gamma and distal hippocampal theta rhythms. In addition, adult male KO mice showed reduced ventral hippocampus-mPFC-evoked potentials and an augmented low-frequency stimulation LTD of the pathway, suggesting that there is a functional disconnection between both structures in adult KO mice. Our results demonstrate that early genetic abnormalities in interneurons can interact with postnatal development during adolescence, triggering pathophysiological mechanisms related to schizophrenia that exceed those caused by NMDAR interneuron hypofunction alone.SIGNIFICANCE STATEMENT NMDAR hypofunction in cortical interneurons has been linked to schizophrenia pathophysiology. How a dysfunction of GABAergic cortical interneurons interacts with maturation during adolescence has not been clarified yet. Here, we demonstrate in vivo that early postnatal ablation of the NMDAR in corticolimbic interneurons results in an overactive but desynchronized PFC before adolescence. Final postnatal maturation during this stage outspreads the impact of the genetic manipulation toward a functional disconnection of the ventral hippocampal-prefrontal pathway, probably as a consequence of an exacerbated propensity toward hippocampal-evoked depotentiation plasticity. Our results demonstrate a complex interaction between genetic and developmental factors affecting cortical interneurons and PFC function.

Distinct Properties of Layer 3 Pyramidal Neurons from Prefrontal and Parietal Areas of the Monkey Neocortex.

In primates, working memory function depends on activity in a distributed network of cortical areas that display different patterns of delay task-related activity. These differences are correlated with, and might depend on, distinctive properties of the neurons located in each area. For example, layer 3 pyramidal neurons (L3PNs) differ significantly between primary visual and dorsolateral prefrontal (DLPFC) cortices. However, to what extent L3PNs differ between DLPFC and other association cortical areas is less clear. Hence, we compared the properties of L3PNs in monkey DLPFC versus posterior parietal cortex (PPC), a key node in the cortical working memory network. Using patch-clamp recordings and biocytin cell filling in acute brain slices, we assessed the physiology and morphology of L3PNs from monkey DLPFC and PPC. The L3PN transcriptome was studied using laser microdissection combined with DNA microarray or quantitative PCR. We found that in both DLPFC and PPC, L3PNs were divided into regular spiking (RS-L3PNs) and bursting (B-L3PNs) physiological subtypes. Whereas regional differences in single-cell excitability were modest, B-L3PNs were rare in PPC (RS-L3PN:B-L3PN, 94:6), but were abundant in DLPFC (50:50), showing greater physiological diversity. Moreover, DLPFC L3PNs display larger and more complex basal dendrites with higher dendritic spine density. Additionally, we found differential expression of hundreds of genes, suggesting a transcriptional basis for the differences in L3PN phenotype between DLPFC and PPC. These data show that the previously observed differences between DLPFC and PPC neuron activity during working memory tasks are associated with diversity in the cellular/molecular properties of L3PNs.SIGNIFICANCE STATEMENT In the human and nonhuman primate neocortex, layer 3 pyramidal neurons (L3PNs) differ significantly between dorsolateral prefrontal (DLPFC) and sensory areas. Hence, L3PN properties reflect, and may contribute to, a greater complexity of computations performed in DLPFC. However, across association cortical areas, L3PN properties are largely unexplored. We studied the physiology, dendrite morphology and transcriptome of L3PNs from macaque monkey DLPFC and posterior parietal cortex (PPC), two key nodes in the cortical working memory network. L3PNs from DLPFC had greater diversity of physiological properties and larger basal dendrites with higher spine density. Moreover, transcriptome analysis suggested a molecular basis for the differences in the physiological and morphological phenotypes of L3PNs from DLPFC and PPC.

Presynaptic Effects of N-Methyl-D-Aspartate Receptors Enhance Parvalbumin Cell-Mediated Inhibition of Pyramidal Cells in Mouse Prefrontal Cortex.

BACKGROUND: Testing hypotheses regarding the role of N-methyl-D-aspartate receptor (NMDAR) hypofunction in schizophrenia requires understanding the mechanisms of NMDAR regulation of prefrontal cortex (PFC) circuit function. NMDAR antagonists are thought to produce pyramidal cell (PC) disinhibition. However, inhibitory parvalbumin-positive basket cells (PVBCs) have modest NMDAR-mediated excitatory drive and thus are unlikely to participate in NMDAR antagonist-mediated disinhibition. Interestingly, recent studies demonstrated that presynaptic NMDARs enhance transmitter release at central synapses. Thus, if presynaptic NMDARs enhance gamma-aminobutyric acid release at PVBC-to-PC synapses, they could participate in NMDAR-dependent PC disinhibition. Here, we examined whether presynaptic NMDAR effects could modulate gamma-aminobutyric acid release at PVBC-to-PC synapses in mouse PFC. METHODS: Using whole-cell recordings from synaptically connected pairs in mouse PFC, we determined whether NMDA or NMDAR antagonist application affects PVBC-to-PC inhibition in a manner consistent with a presynaptic mechanism. RESULTS: NMDAR activation enhanced by ∼40% the synaptic current at PVBC-to-PC pairs. This effect was consistent with a presynaptic mechanism given that it was 1) observed with postsynaptic NMDARs blocked by intracellular MK801, 2) associated with a lower rate of transmission failures and a higher transmitter release probability, and 3) blocked by intracellular MK801 in the PVBC. NMDAR antagonist application did not affect the synaptic currents in PVBC-to-PC pairs, but it reduced the inhibitory currents elicited in PCs with simultaneous glutamate release by extracellular stimulation. CONCLUSIONS: We demonstrate that NMDAR activation enhances PVBC-to-PC inhibition in a manner consistent with presynaptic mechanisms, and we suggest that the functional impact of this presynaptic effect depends on the activity state of the PFC network.

Binocular deprivation induces both age-dependent and age-independent forms of plasticity in parvalbumin inhibitory neuron visual response properties.

Activity of cortical inhibitory interneurons is rapidly reduced in response to monocular deprivation during the critical period for ocular dominance plasticity and in response to salient events encountered during learning. In the case of primary sensory cortex, a decrease in mean evoked firing rate of parvalbumin-positive (PV) inhibitory neurons is causally linked to a reorganization of excitatory networks following sensory perturbation. Converging evidence indicates that it is deprivation, and not an imbalance between open- and closed-eye inputs, that triggers rapid plasticity in PV neurons. However, this has not been directly tested in vivo. Using two-photon guided cell-attached recording, we examined the impact of closing both eyes for 24 h on PV neuron response properties in mouse primary visual cortex. We found that binocular deprivation induces a 30% reduction in stimulus-evoked mean firing rate and that this reduction is specific to critical period-aged mice. The number of PV neurons showing detectable tuning to orientation increased after 24 h of deprivation, and this effect was also specific to critical period-aged mice. In contrast to evoked mean firing rate and orientation tuning, measurements of trial-to-trial variability revealed that stimulus-driven decreases in variability are significantly dampened by deprivation during both the critical period and the postcritical period. These data establish that open-eye inputs are not required to drive deprivation-induced weakening of PV neuron evoked activity and that other aspects of in vivo PV neuron activity are malleable throughout life. NEW & NOTEWORTHY Parvalbumin-positive (PV) neurons in sensory cortex are generally considered to be mediators of experience-dependent plasticity, and their plasticity is restricted to the critical period. However, in regions outside of sensory cortex, accumulating evidence demonstrates that PV neurons are plastic in adults, raising the possibility that aspects of PV response properties may be plastic throughout life. Here we identify a feature of in vivo PV neuron activity that remains plastic past the critical period.

Top-Down-Mediated Facilitation in the Visual Cortex Is Gated by Subcortical Neuromodulation.

Response properties in primary sensory cortices are highly dependent on behavioral state. For example, the nucleus basalis of the forebrain plays a critical role in enhancing response properties of excitatory neurons in primary visual cortex (V1) during active exploration and learning. Given the strong reciprocal connections between hierarchically arranged cortical regions, how are increases in sensory response gain constrained to prevent runaway excitation? To explore this, we used in vivo two-photon guided cell-attached recording in conjunction with spatially restricted optogenetic photo-inhibition of higher-order visual cortex in mice. We found that the principle feedback projection to V1 originating from the lateral medial area (LM) facilitated visual responses in layer 2/3 excitatory neurons by ∼20%. This facilitation was reduced by half during basal forebrain activation due to differential response properties between LM and V1. Our results demonstrate that basal-forebrain-mediated increases in response gain are localized to V1 and are not propagated to LM and establish that subcortical modulation of visual cortex is regionally distinct.

Functional Maturation of GABA Synapses During Postnatal Development of the Monkey Dorsolateral Prefrontal Cortex.

Development of inhibition onto pyramidal cells may be crucial for the emergence of cortical network activity, including gamma oscillations. In primate dorsolateral prefrontal cortex (DLPFC), inhibitory synaptogenesis starts in utero and inhibitory synapse density reaches adult levels before birth. However, in DLPFC, the expression levels of γ-aminobutyric acid (GABA) synapse-related gene products changes markedly during development until young adult age, suggesting a highly protracted maturation of GABA synapse function. Therefore, we examined the development of GABA synapses by recording GABAAR-mediated inhibitory postsynaptic currents (GABAAR-IPSCs) from pyramidal cells in the DLPFC of neonatal, prepubertal, peripubertal, and adult macaque monkeys. We found that the decay of GABAAR-IPSCs, possibly including those from parvalbumin-positive GABA neurons, shortened by prepubertal age, while their amplitude increased until the peripubertal period. Interestingly, both GABAAR-mediated quantal response size, estimated by miniature GABAAR-IPSCs, and the density of GABAAR synaptic appositions, measured with immunofluorescence microscopy, were stable with age. Simulations in a computational model network with constant GABA synapse density showed that the developmental changes in GABAAR-IPSC properties had a significant impact on oscillatory activity and predicted that, whereas DLPFC circuits can generate gamma frequency oscillations by prepubertal age, mature levels of gamma band power are attained at late stages of development.

Cholinergic modulation of neuronal excitability and recurrent excitation-inhibition in prefrontal cortex circuits: implications for gamma oscillations.

Cholinergic neuromodulation in neocortical networks is required for gamma oscillatory activity associated with working memory and other cognitive processes. Importantly, the cholinergic agonist carbachol (CCh) induces gamma oscillations in vitro, via mechanisms that may be shared with in vivo gamma oscillations and that are consistent with the pyramidal interneuron network gamma (PING) model. In PING oscillations, pyramidal cells (PCs), driven by asynchronous excitatory input, recruit parvalbumin-positive fast-spiking interneurons (FSNs), which then synchronize the PCs via feedback inhibition. Whereas the PING model is favoured by current data, how cholinergic neuromodulation contributes to gamma oscillation production is poorly understood. We thus studied the effects of cholinergic modulation on circuit components of the PING model in mouse medial prefrontal cortex (mPFC) brain slices. CCh depolarized and evoked action potential firing in a fraction of PCs and increased excitatory synaptic input onto FSNs. In synaptically connected pairs, CCh reduced the short-term depression at FSN-PC and PC-FSN synapses, equalizing synaptic strength during repetitive presynaptic firing while simultaneously increasing the failure probability. Interestingly, when PCs or FSNs fired in response to gamma frequency oscillatory inputs, CCh increased the firing probability per cycle. Combined with the equalization of synaptic strength, an increase by CCh in the fraction of neurons recruited per oscillation cycle may support oscillatory synchrony of similar strength during relatively long oscillation episodes such as those observed during working memory tasks, suggesting a significant functional impact of cholinergic modulation of mPFC circuit components crucial for the PING model.

Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: a theoretical study.

A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008]. The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback). The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release.

A volume regulatory response can be triggered by nucleosides in human erythrocytes, a perfect osmometer no longer.

Human erythrocytes have been regarded as perfect osmometers, which swell or shrink as dictated by their osmotic environment. In contrast, in most other cells, swelling elicits a regulatory volume decrease (RVD) modulated by the activation of purinic and pyrimidinic receptors (P receptors). For human erythrocytes this modulation has not been tested, and we thus investigated whether P receptor activation can induce RVD in these cells. Further, because ectonucleotidases may scavenge ATP or ADP or act as a source for extracellular adenosine and therefore modulate P receptor activation and RVD, we also determined their activity in intact erythrocytes. We found relatively low ectoATPase but significant ectoADPase and ectoAMPase activities. When erythrocytes were exposed to hypotonic medium alone, they swelled as expected for an osmometric response and showed no RVD. Activation of P2 receptors by exogenous ATP or ADP did not trigger RVD, whereas P1 agonists adenosine and adenosine-5'-N-ethylcarboxamide induced significant RVD. The effect of adenosine-5'-N-ethylcarboxamide was dose-dependent (maximal RVD of 27%; apparent K((1/2)) of 1.6 +/- 1.7 microM). The RVD induced by adenosine was blocked 80% with the non-selective P1 antagonist 8-(p-sulfophenyl theophylline) or the P1-A(2B) inhibitor MRS1754, but not by inhibitors of P1 subtypes A(1), A(2A), and A(3). In addition, forskolin (an inducer of intracellular cAMP formation) could mimic the effect of adenosine, supporting the idea of P1-A(2B) receptor activation. In conclusion, we report a novel P1-A(2B) receptor-mediated RVD activation in mature human erythrocytes and thus indicate that these long held perfect osmometers are not so perfect after all.

Extracellular ADP regulates lesion-induced in vivo cell proliferation and death in the zebrafish retina.

Regeneration and growth that occur in the adult teleost retina by neurogenesis have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. In this report, we demonstrate that endogenous purinergic signals regulate cell proliferation induced by a cytotoxic injury of the adult zebrafish retina which mainly damages inner retinal layers. Particularly, we found that ADP but not ATP or adenosine significantly enhanced cell division as assessed by 5-bromo-2'-deoxyuridine incorporation following injury, during the degenerative and proliferative phase of the regeneration process. This effect of ADP occurs via P2Y1 metabotropic receptors as shown by intra-ocular injection of selective antagonists. Additionally, we describe a role for purinergic signals in regulating cell death induced by injury. Scavenging of extracellular nucleotides significantly increased cell death principally seen in the inner retinal layers. This effect is partially reproduced by blocking P2Y1 receptors suggesting a neuroprotective function for ADP, which is derived from extracellular ATP probably released by dying cells as a consequence of the ouabain treatment. This study demonstrates a crucial role for ADP as a paracrine signal in the repair of retinal tissue following injury.

Kinetics of extracellular ATP from goldfish hepatocytes: a lesson from mathematical modeling.

In goldfish hepatocytes, hypotonic exposure leads to cell swelling, followed by a compensatory shrinkage termed RVD. It has been previously shown that ATP is accumulated in the extracellular medium of swollen cells in a non-linear fashion, and that extracellular ATP (ATPe) is an essential intermediate to trigger RVD. Thus, to understand how RVD proceeds in goldfish hepatocytes, we developed two mathematical models accounting for the experimental ATPe kinetics reported recently by Pafundo et al. in Am. J. Physiol. 294, R220-R233, 2008. Four different equations for ATPe fluxes were built to account for the release of ATP by lytic (J(L)) and nonlytic mechanisms (J(NL)), ATPe diffusion (J(D)), and ATPe consumption by ectonucleotidases (J(V)). Particular focus was given to J(NL), defined as the product of a time function (J(R)) and a positive feedback mechanism whereby ATPe amplifies J(NL). Several J (R) functions (Constant, Step, Impulse, Gaussian, and Lognormal) were studied. Models were tested without (model 1) or with (model 2) diffusion of ATPe. Mathematical analysis allowed us to get a general expression for each of the models. Subsequently, by using model dependent fit (simulations) as well as model analysis at infinite time, we observed that: - use of J(D) does not lead to improvements of the models. - Constant and Step time functions are only applicable when J(R)=0 (and thus, J(NL)=0), so that the only source of ATPe would be J(L), a result incompatible with experimental data. - use of impulse, Gaussian, and lognormal J(R)s in the models led to reasonable good fits to experimental data, with the lognormal function in model 1 providing the best option. Finally, the predictive nature of model 1 loaded with a lognormal J(R) was tested by simulating different putative in vivo scenarios where J(V) and J(NL) were varied over ample ranges.

Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes.

In most animal cells, hypotonic swelling is followed by a regulatory volume decrease (RVD) thought to prevent cell death. In contrast, goldfish hepatocytes challenged with hypotonic medium (180 mosM, HYPO) increase their volume 1.7 times but remain swollen and viable for at least 5 h. Incubation with ATPgammaS (an ATP analog) in HYPO triggers a 42% volume decrease. This effect is concentration dependent (K(1/2) = 760 nM) and partially abolished by P2 receptor antagonists (64% inhibition). A similar induction of RVD is observed with ATP, UTP, and UDP, whereas adenosine inhibits RVD. Goldfish hepatocytes release more than 500 nM ATP during the first minutes of HYPO with no induction of RVD. The fact that similar concentrations of ATPgammaS did trigger RVD could be explained by showing that ATPgammaS induced ATP release. Finally, we observed that in a very small extracellular volume, hepatocytes do show a 56% RVD. This response was diminished by P2 receptor antagonists (73%) and increased (73%) when the extracellular ATP hydrolysis was inhibited 72%. Using a mathematical model, we predict that during the first 2 min of HYPO exposure the extracellular [ATP] is mainly governed by ATP diffusion and by both nonlytic and lytic ATP release, with almost no contribution from ecto-ATPase activity. We show that goldfish hepatocytes under standard HYPO (large volume) do not display RVD unless this is triggered by the addition of micromolar concentrations of nucleotides. However, under very low assay volumes, sufficient endogenous extracellular [ATP] can build up to induce RVD.

Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes.

The role of cytoskeletal elements in volume regulation was studied in trout hepatocytes by investigating changes in F-actin distribution during anisotonic exposure and assessing the impact of cytoskeleton disruption on volume regulatory responses. Hypotonic challenge caused a significant decrease in the ratio of cortical to cytoplasmic F-actin, whereas this ratio was unaffected in hypertonic saline. Disruption of microfilaments with cytochalasin B (CB) or cytochalasin D significantly slowed volume recovery following hypo- and hypertonic exposure in both attached and suspended cells. The decrease of net proton release and the intracellular acidification elicited by hypotonicity were unaltered by CB, whereas the increase of proton release in hypertonic saline was dramatically reduced. Because amiloride almost completely blocked the hypertonic increase of proton release and cytoskeleton disruption diminished the associated increase of intracellular pH (pH(i)), we suggest that F-actin disruption affected Na(+)/H(+) exchanger activity. In line with this, pH(i) recovery after an ammonium prepulse was significantly inhibited in CB-treated cells. The increase of cytosolic Na(+) under hypertonic conditions was not diminished but, rather, enhanced by F-actin disruption, presumably due to inhibited Na(+)-K(+)-ATPase activity and stimulated Na(+) channel activity. The elevation of cytosolic Ca(2+) in hypertonic medium was significantly reduced by CB. Altogether, our results indicate that the F-actin network is of crucial importance in the cellular responses to anisotonic conditions, possibly via interaction with the activity of ion transporters and with signalling cascades responsible for their activation. Disruption of microtubules with colchicine had no effect on any of the parameters investigated.