Changes in the Proportion of Inhibitory Interneuron Types from Sensory to Executive Areas of the Primate Neocortex: Implications for the Origins of Working Memory Representations.

Neuronal spiking activity encoding working memory (WM) is robust in primate association cortices but weak or absent in early sensory cortices. This may be linked to changes in the proportion of neuronal types across areas that influence circuits' ability to generate recurrent excitation. We recorded neuronal activity from areas middle temporal (MT), medial superior temporal (MST), and the lateral prefrontal cortex (LPFC) of monkeys performing a WM task and classified neurons as narrow (NS) and broad spiking (BS). The ratio NS/BS decreased from MT > MST > LPFC. We analyzed the Allen Institute database of ex vivo mice/human intracellular recordings to interpret our data. Our analysis suggests that NS neurons correspond to parvalbumin (PV) or somatostatin (SST) interneurons while BS neurons are pyramidal (P) cells or vasoactive intestinal peptide (VIP) interneurons. We labeled neurons in monkey tissue sections of MT/MST and LPFC and found that the proportion of PV in cortical layers 2/3 decreased, while the proportion of CR cells increased from MT/MST to LPFC. Assuming that primate CR/CB/PV cells perform similar computations as mice VIP/SST/PV cells, our results suggest that changes in the proportion of CR and PV neurons in layers 2/3 cells may favor the emergence of activity encoding WM in association areas.

CRF Mediates Stress-Induced Pathophysiological High-Frequency Oscillations in Traumatic Brain Injury.

It is not known why there is increased risk to have seizures with increased anxiety and stress after traumatic brain injury (TBI). Stressors cause the release of corticotropin-releasing factor (CRF) both from the hypothalamic pituitary adrenal (HPA) axis and from CNS neurons located in the central amygdala and GABAergic interneurons. We have previously shown that CRF signaling is plastic, becoming excitatory instead of inhibitory after the kindling model of epilepsy. Here, using Sprague Dawley rats we have found that CRF signaling increased excitability after TBI. Following TBI, CRF type 1 receptor (CRFR1)-mediated activity caused abnormally large electrical responses in the amygdala, including fast ripples, which are considered to be epileptogenic. After TBI, we also found the ripple (120-250 Hz) and fast ripple activity (>250 Hz) was cross-frequency coupled with θ (3-8 Hz) oscillations. CRFR1 antagonists reduced the incidence of phase coupling between ripples and fast ripples. Our observations indicate that pathophysiological signaling of the CRFR1 increases the incidence of epileptiform activity after TBI. The use for CRFR1 antagonist may be useful to reduce the severity and frequency of TBI associated epileptic seizures.

Identification of a novel synaptic protein, TMTC3, involved in periventricular nodular heterotopia with intellectual disability and epilepsy.

Defects in neuronal migration cause brain malformations, which are associated with intellectual disability (ID) and epilepsy. Using exome sequencing, we identified compound heterozygous variants (p.Arg71His and p. Leu729ThrfsTer6) in TMTC3, encoding transmembrane and tetratricopeptide repeat containing 3, in four siblings with nocturnal seizures and ID. Three of the four siblings have periventricular nodular heterotopia (PVNH), a common brain malformation caused by failure of neurons to migrate from the ventricular zone to the cortex. Expression analysis using patient-derived cells confirmed reduced TMTC3 transcript levels and loss of the TMTC3 protein compared to parental and control cells. As TMTC3 function is currently unexplored in the brain, we gathered support for a neurobiological role for TMTC3 by generating flies with post-mitotic neuron-specific knockdown of the highly conserved Drosophila melanogaster TMTC3 ortholog, CG4050/tmtc3. Neuron-specific knockdown of tmtc3 in flies resulted in increased susceptibility to induced seizures. Importantly, this phenotype was rescued by neuron-specific expression of human TMTC3, suggesting a role for TMTC3 in seizure biology. In addition, we observed co-localization of TMTC3 in the rat brain with vesicular GABA transporter (VGAT), a presynaptic marker for inhibitory synapses. TMTC3 is localized at VGAT positive pre-synaptic terminals and boutons in the rat hypothalamus and piriform cortex, suggesting a role for TMTC3 in the regulation of GABAergic inhibitory synapses. TMTC3 did not co-localize with Vglut2, a presynaptic marker for excitatory neurons. Our data identified TMTC3 as a synaptic protein that is involved in PVNH with ID and epilepsy, in addition to its previously described association with cobblestone lissencephaly.

Neuroinflammatory targets and treatments for epilepsy validated in experimental models.

A large body of evidence that has accumulated over the past decade strongly supports the role of inflammation in the pathophysiology of human epilepsy. Specific inflammatory molecules and pathways have been identified that influence various pathologic outcomes in different experimental models of epilepsy. Most importantly, the same inflammatory pathways have also been found in surgically resected brain tissue from patients with treatment-resistant epilepsy. New antiseizure therapies may be derived from these novel potential targets. An essential and crucial question is whether targeting these molecules and pathways may result in anti-ictogenesis, antiepileptogenesis, and/or disease-modification effects. Therefore, preclinical testing in models mimicking relevant aspects of epileptogenesis is needed to guide integrated experimental and clinical trial designs. We discuss the most recent preclinical proof-of-concept studies validating a number of therapeutic approaches against inflammatory mechanisms in animal models that could represent novel avenues for drug development in epilepsy. Finally, we suggest future directions to accelerate preclinical to clinical translation of these recent discoveries.

BK Channels Mediate Synaptic Plasticity Underlying Habituation in Rats.

Habituation is a basic form of implicit learning and represents a sensory filter that is disrupted in autism, schizophrenia, and several other mental disorders. Despite extensive research in the past decades on habituation of startle and other escape responses, the underlying neural mechanisms are still not fully understood. There is evidence from previous studies indicating that BK channels might play a critical role in habituation. We here used a wide array of approaches to test this hypothesis. We show that BK channel activation and subsequent phosphorylation of these channels are essential for synaptic depression presumably underlying startle habituation in rats, using patch-clamp recordings and voltage-sensitive dye imaging in slices. Furthermore, positive modulation of BK channels in vivo can enhance short-term habituation. Although results using different approaches do not always perfectly align, together they provide convincing evidence for a crucial role of BK channel phosphorylation in synaptic depression underlying short-term habituation of startle. We also show that this mechanism can be targeted to enhance short-term habituation and therefore to potentially ameliorate sensory filtering deficits associated with psychiatric disorders.SIGNIFICANCE STATEMENT Short-term habituation is the most fundamental form of implicit learning. Habituation also represents a filter for inundating sensory information, which is disrupted in autism, schizophrenia, and other psychiatric disorders. Habituation has been studied in different organisms and behavioral models and is thought to be caused by synaptic depression in respective pathways. The underlying molecular mechanisms, however, are poorly understood. We here identify, for the first time, a BK channel-dependent molecular synaptic mechanism leading to synaptic depression that is crucial for habituation, and we discuss the significance of our findings for potential treatments enhancing habituation.

A switch in G protein coupling for type 1 corticotropin-releasing factor receptors promotes excitability in epileptic brains.

Anxiety and stress increase the frequency of epileptic seizures. These behavioral states induce the secretion of corticotropin-releasing factor (CRF), a 40-amino acid neuropeptide neurotransmitter that coordinates many behavioral responses to stress in the central nervous system. In the piriform cortex, which is one of the most seizurogenic regions of the brain, CRF normally dampens excitability. By contrast, CRF increased the excitability of the piriform cortex in rats subjected to kindling, a model of temporal lobe epilepsy. In nonkindled rats, CRF activates its receptor, a G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor, and signals through a Gαq/11-mediated pathway. After seizure induction, CRF signaling occurred through a pathway involving Gαs This change in signaling was associated with reduced abundance of regulator of G protein signaling protein type 2 (RGS2), which has been reported to inhibit Gαs-dependent signaling. RGS2 knockout mice responded to CRF in a similar manner as epileptic rats. These observations indicate that seizures produce changes in neuronal signaling that can increase seizure occurrence by converting a beneficial stress response into an epileptic trigger.

Hippocampal hyperexcitability in fetal alcohol spectrum disorder: Pathological sharp waves and excitatory/inhibitory synaptic imbalance.

Prenatal alcohol exposure (PAE) can lead to long-lasting neurological alterations that may predispose individuals to seizures and neurobehavioral dysfunction. To date, there exists limited information regarding the underlying pathophysiological mechanisms. The hippocampal CA3 region generates excitatory population activity, called sharp waves (SPWs), that provide an ideal model to study perturbations in neuronal excitability at the network and cellular levels. In the present study, we utilized a mouse model of PAE and used dual extracellular and whole-cell patch-clamp recordings from CA3 hippocampal pyramidal cells to evaluate the effect of 1st trimester-equivalent ethanol exposure (10% v/v) on SPW activity and excitatory/inhibitory balance. We observed that PAE significantly altered in vitro SPW waveforms, with an increased duration and amplitude, when compared to controls. In addition, PAE slices exhibited reduced pharmacological inhibition by the GABA-A receptor antagonist bicuculline (BMI) on SPW activity, and increased population spike paired-pulse ratios, all indicative of network disinhibition within the PAE hippocampus. Evaluation of PAE CA3 pyramidal cell activity associated with SPWs, revealed increased action potential cell firing, which was accompanied by an imbalance of excitatory/inhibitory synaptic drive, shifted in favor of excitation. Moreover, we observed intrinsic changes in CA3 pyramidal activity in PAE animals, including increased burst firing and instantaneous firing rate. This is the first study to provide evidence for hippocampal dysfunction in the ability to maintain network homeostasis and underlying cellular hyperexcitability in a model of PAE. These circuit and cellular level alterations may contribute to the increased propensity for seizures and neurobehavioral dysfunction observed in patients with a history of PAE.

Differential expression of astrocytic connexins in a mouse model of prenatal alcohol exposure.

Maternal alcohol consumption during gestation can cause serious injury to the fetus, and may result in a range of physiological and behavioral impairments, including increased seizure susceptibility, that are collectively termed fetal alcohol spectrum disorder (FASD). The cellular mechanisms underlying increased seizure susceptibility in FASD are not well understood, but could involve altered excitatory coupling of neuronal populations mediated by gap junction proteins. We utilized a mouse model of the prenatal alcohol exposure (PAE) to study the expression pattern of connexin (Cx) major components of gap junctions, and pannexin proteins, which form membrane channels, in the brain of 2-3weeks old PAE and control postnatal offspring. PAE during the first trimester-equivalent period of pregnancy in mice resulted in significant up-regulation of Cx30 mRNA and Cx30 total protein in the hippocampus of PAE animals compared to age-matched controls. Surface level expression of both dimeric and monomeric Cx30 were also found to be significantly up-regulated in both hippocampus and cerebral cortex of PAE animals compared to age-matched controls. On the membrane surface, the fast migrating form of Cx43 was found to be up-regulated in the hippocampus of PAE mice. However, we did not see any up-regulation of the phosphorylated forms of Cx43 on the membrane surface. These results indicate that the expression and processing of astrocytic connexins (Cx30, Cx43) are up-regulated in the brain of PAE offspring, and these changes could play a role in the cerebral hyperexcitability observed in these animals.

Suppression of piriform cortex activity in rat by corticotropin-releasing factor 1 and serotonin 2A/C receptors.

The piriform cortex (PC) is richly innervated by corticotropin-releasing factor (CRF) and serotonin (5-HT) containing axons arising from central amygdala and Raphe nucleus. CRFR1 and 5-HT2A/2CRs have been shown to interact in manner where CRFR activation subsequently potentiates the activity of 5-HT2A/2CRs. The purpose of this study was to determine how the activation of CRFR1 and/or 5-HT2Rs modulates PC activity at both the circuit and cellular level. Voltage sensitive dye imaging showed that CRF acting through CRFR1 dampened activation of the Layer II of PC and interneurons of endopiriform nucleus. Application of the selective 5-HT2A/CR agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) following CRFR1 activation potentiated this effect. Blocking the interaction between CRFR1 and 5-HT2R with a Tat-CRFR1-CT peptide abolished this potentiation. Application of forskolin did not mimic CRFR1 activity but instead blocked it, while a protein kinase A antagonist had no effect. However, activation and antagonism of protein kinase C (PKC) either mimicked or blocked CRF modulation, respectively. DOI had no effect when applied alone indicating that the prior activation of CRFR1 receptors was critical for DOI to show significant effects similar to CRF. Patch clamp recordings showed that both CRF and DOI reduced the synaptic responsiveness of Layer II pyramidal neurons. CRF had highly variable effects on interneurons within Layer III, both increasing and decreasing their excitability, but DOI had no effect on the excitability of this group of neurons. These data show that CRF and 5-HT, acting through both CRFR1 and 5-HT2A/CRs, reduce the activation of the PC. This modulation may be an important blunting mechanism of stressor behaviors mediated through the olfactory cortex.

Gender and brain regions specific differences in brain derived neurotrophic factor protein levels of depressed individuals who died through suicide.

Considerable evidence supports the view that depressive illness and suicidal behaviour stem from perturbations of neuroplasticity. Presently, we assessed whether depressed individuals who died by suicide displayed brain region-specific changes in brain derived neurotrophic factor (BDNF) and whether such effects varied by gender. Using postmortem samples from non-psychiatric controls and depressed individuals who died by suicide, BDNF protein levels were assessed within the hippocampus and frontopolar prefrontal cortex using Western blot. As expected, BDNF levels were reduced within the frontopolar prefrontal cortex among female depressed suicides; however, males showed no such effect. Contrastingly, within the hippocampus, depressed male but not female suicides displayed significant reductions of BDNF protein levels. Although the mechanisms driving the gender and brain region specific BDNF changes are unclear, our data do support the notion that complex alterations of neuroplasticity may be fundamentally involved in the illness.

Metalloproteinase inhibition prevents inhibitory synapse reorganization and seizure genesis.

The integrity and stability of interneurons in a cortical network are essential for proper network function. Loss of interneuron synaptic stability and precise organization can lead to disruptions in the excitation/inhibition balance, a characteristic of epilepsy. This study aimed to identify alterations to the GABAergic interneuron network in the piriform cortex (PC: a cortical area believed to be involved in the development of seizures) after kindling-induced seizures. Immunohistochemistry was used to mark perineuronal nets (PNNs: structures in the extracellular matrix that provide synaptic stability and restrict reorganization of inhibitory interneurons) and interneuron nerve terminals in control and kindled tissues. We found that PNNs were significantly decreased around parvalbumin-positive interneurons after the induction of experimental epilepsy. Additionally, we found layer-specific increases in GABA release sites originating from calbindin, calretinin, and parvalbumin interneurons, implying that there is a re-wiring of the interneuronal network. This increase in release sites was matched by an increase in GABAergic post-synaptic densities. We hypothesized that the breakdown of the PNN could be due to the activity of matrix metalloproteinases (MMP) and that the prevention of PNN breakdown may reduce the rewiring of interneuronal circuits and suppress seizures. To test this hypothesis we employed doxycycline, a broad spectrum MMP inhibitor, to stabilize PNNs in kindled rats. We found that doxycycline prevented PNN breakdown, re-organization of the inhibitory innervation, and seizure genesis. Our observations indicate that PNN degradation may be necessary for the development of seizures by facilitating interneuron plasticity and increased GABAergic activity.

Catechol-O-methyltransferase Val158Met polymorphism and altered COMT gene expression in the prefrontal cortex of suicide brains.

Catechol-O-methyltransferase (COMT) plays a key role in the degradation of catecholamine neurotransmitters within the brain. A functional polymorphism COMT Val158Met has been associated with psychiatric disorders including suicidal behavior. In the present study we examined whether this polymorphism was related to COMT mRNA expression in frontal cortical regions, and whether the expression of COMT differed between depressed suicide victims and psychiatric healthy controls. The Val158Met polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. The levels of COMT mRNA expression in the frontopolar cortex (FPC; 29 suicides vs. 27 controls) and orbital frontal cortex (OFC; 19 suicides vs. 15 controls) were significantly increased among depressed individuals that died by suicide relative to those of controls, being up-regulated by approximately 60% and 65% in the FPC and OFC, respectively. Furthermore, among individuals with the Met allele (Met/Met and Met/Val genotypes) who died by suicide COMT mRNA expression was elevated relative to that of the nondepressed Met allele carriers. However, significant differences were not detected between suicides (n=49) and controls (n=72) with respect to the Val158Met genotypic distribution and allelic frequencies. These results are consistent with the perspective that altered COMT mRNA expression in frontal cortical brain regions might contribute to suicide and/or depression, further supporting the role of dysregulation of catecholaminergic pathway genes in the pathophysiology of suicide behaviors.

Gain control of γ frequency activation by a novel feed forward disinhibitory loop: implications for normal and epileptic neural activity.

The inhibition of excitatory (pyramidal) neurons directly dampens their activity resulting in a suppression of neural network output. The inhibition of inhibitory cells is more complex. Inhibitory drive is known to gate neural network synchrony, but there is also a widely held view that it may augment excitability by reducing inhibitory cell activity, a process termed disinhibition. Surprisingly, however, disinhibition has never been demonstrated to be an important mechanism that augments or drives the activity of excitatory neurons in a functioning neural circuit. Using voltage sensitive dye imaging (VSDI) we show that 20-80 Hz stimulus trains, ß-γ activation, of the olfactory cortex pyramidal cells in layer II leads to a subsequent reduction in inhibitory interneuron activity that augments the efficacy of the initial stimulus. This disinhibition occurs with a lag of about 150-250 ms after the initial excitation of the layer 2 pyramidal cell layer. In addition, activation of the endopiriform nucleus also arises just before the disinhibitory phase with a lag of about 40-80 ms. Preventing the spread of action potentials from layer II stopped the excitation of the endopiriform nucleus, abolished the disinhibitory activity, and reduced the excitation of layer II cells. After the induction of experimental epilepsy the disinhibition was more intense with a concomitant increase in excitatory cell activity. Our observations provide the first evidence of feed forward disinhibition loop that augments excitatory neurotransmission, a mechanism that could play an important role in the development of epileptic seizures.

A novel anticonvulsant modulates voltage-gated sodium channel inactivation and prevents kindling-induced seizures.

Here, we explore the mechanism of action of isoxylitone (ISOX), a molecule discovered in the plant Delphinium denudatum, which has been shown to have anticonvulsant properties. Patch-clamp electrophysiology assayed the activity of ISOX on voltage-gated sodium channels (VGSCs) in both cultured neurons and brain slices isolated from controls and rats with experimental epilepsy(kindling model). Quantitative transcription polymerase chain reaction (qRT-PCR) (QPCR) assessed brain-derived neurotrophic factor (BDNF) mRNA expression in kindled rats, and kindled rats treated with ISOX. ISOX suppressed sodium current (I(Na)) showing an IC50 value of 185 nM in cultured neurons. ISOX significantly slowed the recovery from inactivation (ISOX τ = 18.7 ms; Control τ = 9.4 ms; p < 0.001). ISOX also enhanced the development of inactivation by shifting the Boltzmann curve to more hyperpolarized potentials by -11.2 mV (p < 0.05). In naive and electrically kindled cortical neurons, the IC50 for sodium current block was identical to that found in cultured neurons. ISOX prevented kindled stage 5 seizures and decreased the enhanced BDNF mRNA expression that is normally associated with kindling (p < 0.05). Overall, our data show that ISOX is a potent inhibitor of VGSCs that stabilizes steady-state inactivation while slowing recovery and enhancing inactivation development. Like many other sodium channel blocker anti-epileptic drugs, the suppression of BDNF mRNA expression that usually occurs with kindling is likely a secondary outcome that nevertheless would suppress epileptogenesis. These data show a new class of anti-seizure compound that inhibits sodium channel function and prevents the development of epileptic seizures.

Molecular pathway reconstruction and analysis of disturbed gene expression in depressed individuals who died by suicide.

Molecular mechanisms behind the etiology and pathophysiology of major depressive disorder and suicide remain largely unknown. Recent molecular studies of expression of serotonin, GABA and CRH receptors in various brain regions have demonstrated that molecular factors may contribute to the development of depressive disorder and suicide behaviour. Here, we used microarray analysis to examine the expression of genes in brain tissue (frontopolar cortex) of individuals who had been diagnosed with major depressive disorder and died by suicide, and those who had died suddenly without a history of depression. We analyzed the list of differentially expressed genes using pathway analysis, which is an assumption-free approach to analyze microarray data. Our analysis revealed that the differentially expressed genes formed functional networks that were implicated in cell to cell signaling related to synapse maturation, neuronal growth and neuronal complexity. We further validated these data by randomly choosing (100 times) similarly sized gene lists and subjecting these lists to the same analyses. Random gene lists did not provide highly connected gene networks like those generated by the differentially expressed list derived from our samples. We also found through correlational analysis that the gene expression of control participants was more highly coordinated than in the MDD/suicide group. These data suggest that among depressed individuals who died by suicide, wide ranging perturbations of gene expression exist that are critical for normal synaptic connectively, morphology and cell to cell communication.

The loss of interneuron functional diversity in the piriform cortex after induction of experimental epilepsy.

Interneuronal functional diversity is thought to be an important factor in the control of neural network oscillations in many brain regions. Specifically, interneuron action potential firing patterns are thought to modulate brain rhythms. In neurological disorders such as epilepsy where brain rhythms are significantly disturbed interneuron function is largely unexplored. Thus the purpose of this study was to examine the functional diversity of piriform cortex interneurons (PC; an area of the brain that easily supports seizures) before and after kindling-induced epilepsy. Using cluster analysis, we found five control firing behaviors. These groups were termed: non-adapting very high frequency (NAvHF), adapting high frequency (AHF), adapting low frequency (ALF), strongly adapting low frequency (sALF), and weakly adapting low frequency (wALF). A morphological analysis showed these spiking patterns were not associated with any specific interneuronal morphology although we found that most of the cells displaying NAvHF firing pattern were multipolar. After kindling about 40% of interneuronal firing pattern changed, and neither the NAvHF nor the wALF phenotypes were found. We also found that in multipolar interneurons a long-lasting potassium current was increased. A qPCR analysis indicated Kv1.6 subtype was up-regulated after kindling. An immunocytochemical analysis showed that Kv1.6 protein expression on parvalbumin (multipolar) interneurons increased by greater than 400%. We also examined whether these changes could be due to the selective death of a subset of interneurons but found that there was no change in cell number. These data show an important loss of the functional diversity of interneurons in the PC. Our data suggest that under pathophysiological condition interneurons are plastic resulting in the attenuation of high frequency network oscillations in favor of low frequency network activity. This may be an important new mechanism by which network synchrony is disturbed in epileptic seizures.

Kindling alters neurosteroid-induced modulation of phasic and tonic GABAA receptor-mediated currents: role of phosphorylation.

We have previously shown that after kindling (a model of temporal lobe epilepsy), the neuroactive steroid tetrahydrodeoxycorticosterone (THDOC) was unable to augment GABA type A receptor (GABA(A))-mediated synaptic currents occurring on pyramidal cells of the piriform cortex. Phosphorylation of GABA(A) receptors has been shown previously to alter the activity of THDOC, so we tested the hypothesis that kindling induces changes in the phosphorylation of GABA(A) receptors and this accounts for the loss in efficacy. To assay whether GABA(A) receptors are more phosphorylated after kindling, we examined the phosphorylation state of the ß3 subunit and found that it was increased. Incubation of brain slices with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (100 nM) also increased phosphorylation in the same assay. In patch clamp, recordings from non-kindled rat brain slices PMA also reduced the activity of THDOC in a manner that was identical to what is observed after kindling. We also found that the tonic current was no longer augmented by THODC after kindling and PMA treatment. The protein kinase C (PKC) antagonist bisindolylmaleimide I blocked the effects PMA on the synaptic but not the tonic currents. However, the broad spectrum PKC antagonist staurosporine blocked the effects of PMA on the tonic currents, implying that different PKC isoforms phosphorylate GABA(A) receptors responsible for phasic and tonic currents. The phosphatase activator Li(+) palmitate restored the 'normal' activity of THDOC on synaptic currents in kindled brain slices but not the tonic currents. These data demonstrate that kindling enhances the phosphorylation state of GABA(A) receptors expressed in pyramidal neurons reducing THDOC efficacy.