Increased Action Potential Firing Rate Research Articles

The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells.

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating β adrenergic receptors (βARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight increase in AP firing rate. We found that while βAR stimulation significantly altered nearly all AP waveform parameters, the increase in firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the βAR-sensitive component of If showed that it accounts for ∼41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate.

In vivo knockdown of astroglial glutamate transporters GLT-1 and GLAST increases excitatory neurotransmission in mouse infralimbic cortex: Relevance for depressive-like phenotypes

Alterations of energy metabolism and of astrocyte number/function in ventral anterior cingulate cortex (vACC) have been reported in major depressive disorder (MDD) patients and may contribute to MDD pathophysiology. We recently developed a mouse model of MDD mimicking these alterations. We knocked down the astroglial glutamate transporters GLAST and GLT-1 in infralimbic cortex (IL, rodent equivalent of vACC) using small interfering RNA (siRNA). GLAST and GLT-1 siRNA microinfusion in IL evoked a depressive-like phenotype, associated with a reduced serotonergic function and reduced forebrain BDNF expression. Neither effect occurred after siRNA application in the adjacent prelimbic cortex (PrL), thus emphasizing the critical role of vACC/IL in MDD pathogenesis. Here we examined the cellular/network basis of the changes induced in IL using intracellular recordings of layer V pyramidal neurons from mice microinjected with siRNA 24 h before. We analyzed (i) the electrophysiological characteristics of neurons; (ii) the synaptic transmission properties, by monitoring miniature, spontaneous and evoked EPSCs, and (iii) the gliotransmission, by monitoring slow inward currents (SICs), mediated by astrocytic glutamate release and activation of extra-synaptic NMDA receptors. GLT-1 and GLAST knockdown led to a more depolarized membrane potential and increased action potential firing rate of layer V pyramidal neurons, and enhanced excitatory synaptic transmission, as shown by the enhanced amplitude/frequency of spontaneous EPSCs. Gliotransmission was also increased, as indicated by the enhanced SIC amplitude/frequency. Hence, the depressive-like phenotype is associated with IL hyperactivity, likely leading to an excessive top-down inhibitory control of serotonergic activity through IL-midbrain descending pathways.

USE OF SINGLE CELL GENOMIC TECHNOLOGY TO UNDERSTAND PSYCHIATRIC DISEASE

Alterations of energy metabolism and of astrocyte number/function in ventral anterior cingulate cortex (vACC) have been reported in major depressive disorder (MDD) patients and may contribute to MDD pathophysiology. We recently developed a mouse model of MDD mimicking these alterations. We knocked down the astroglial glutamate transporters GLAST and GLT-1 in infralimbic cortex (IL, rodent equivalent of vACC) using small interfering RNA (siRNA). GLAST and GLT-1 siRNA microinfusion in IL evoked a depressive-like phenotype, associated with a reduced serotonergic function and reduced forebrain BDNF expression. Neither effect occurred after siRNA application in the adjacent prelimbic cortex (PrL), thus emphasizing the critical role of vACC/IL in MDD pathogenesis. Here we examined the cellular/network basis of the changes induced in IL using intracellular recordings of layer V pyramidal neurons from mice microinjected with siRNA 24 h before. We analyzed (i) the electrophysiological characteristics of neurons; (ii) the synaptic transmission properties, by monitoring miniature, spontaneous and evoked EPSCs, and (iii) the gliotransmission, by monitoring slow inward currents (SICs), mediated by astrocytic glutamate release and activation of extra-synaptic NMDA receptors. GLT-1 and GLAST knockdown led to a more depolarized membrane potential and increased action potential firing rate of layer V pyramidal neurons, and enhanced excitatory synaptic transmission, as shown by the enhanced amplitude/frequency of spontaneous EPSCs. Gliotransmission was also increased, as indicated by the enhanced SIC amplitude/frequency. Hence, the depressive-like phenotype is associated with IL hyperactivity, likely leading to an excessive top-down inhibitory control of serotonergic activity through IL-midbrain descending pathways.

Synaptic Regulation by OPRM1 Variants in Reward Neurocircuitry.

Mu-opioid receptors (MORs) are the primary site of action of opioid drugs, both licit and illicit. Susceptibility to opioid addiction is associated with variants in the gene encoding the MOR, OPRM1 Varying with ethnicity, ∼25% of humans carry a single nucleotide polymorphism (SNP) in OPRM1 (A118G). This SNP produces a nonsynonymous amino acid substitution, replacing asparagine (N40) with aspartate (D40), and has been linked with an increased risk for drug addiction. While a murine model of human OPRM1 A118G (A112G in mouse) recapitulates most of the phenotypes reported in humans, the neuronal mechanisms underlying these phenotypes remain elusive. Here, we investigated the impact of A118G on opioid regulation of synaptic transmission in mesolimbic VTA dopaminergic neurons. Using electrophysiology, we showed that both inhibitory and excitatory inputs to VTA dopaminergic neurons projecting to the NAc medial shell were suppressed by the MOR agonists DAMGO and morphine, which caused a shift in the excitatory/inhibitory balance and an increased action potential firing rate. Mice carrying the 112G/G allele exhibited lower sensitivity to DAMGO and morphine compared with major allele carriers (112A/A). Paradoxically, DAMGO produced facilitatory effects on mEPSCs, which were mediated by presynaptic GABAB receptors. However, this was only prominent in homozygous major allele carriers, which could explain a stronger shift in action potential firing in 112A/A mice. This study provides a better understanding on the neurobiological mechanisms that may underlie risk of addiction development in carriers of the A118G SNP in OPRM1SIGNIFICANCE STATEMENT The pandemic of opioid drug abuse is associated with many socioeconomic burdens. The primary brain target of opioid drugs is the μ-opioid receptor (MOR), encoded by the OPRM1 gene, which is highly polymorphic in humans. Using a mouse model of the human OPRM1 A118G single nucleotide polymorphism (SNP) (A112G in mice), we demonstrated that MOR and GABAB signaling coordinate in regulating mesolimbic dopamine neuronal firing via presynaptic regulation. The A118G SNP affects MOR-mediated suppression of GABA and glutamate release, showing weaker efficacy of synaptic regulation by MORs. These results may shed light on whether MOR SNPs need to be considered for devising effective therapeutic interventions.

The proinflammatory cytokine tumor necrosis factor-α excites subfornical organ neurons.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine implicated in cardiovascular and autonomic regulation via actions in the central nervous system. TNF-α-/- mice do not develop angiotensin II (ANG II)-induced hypertension, and administration of TNF-α into the bloodstream of rats increases blood pressure and sympathetic tone. Recent studies have shown that lesion of the subfornical organ (SFO) attenuates the hypertensive and autonomic effects of TNF-α, while direct administration of TNF-α into the SFO increases blood pressure, suggesting the SFO to be a key site for the actions of TNF-α. Therefore, we used patch-clamp techniques to examine both acute and long-term effects of TNF-α on the excitability of Sprague-Dawley rat SFO neurons. It was observed that acute bath application of TNF-α depolarized SFO neurons and subsequently increased action potential firing rate. Furthermore, the magnitude of depolarization and the proportion of depolarized SFO neurons were concentration dependent. Interestingly, following 24-h incubation with TNF-α, the basal firing rate of the SFO neurons was increased and the rheobase was decreased, suggesting that TNF-α elevates SFO neuron excitability. This effect was likely mediated by the transient sodium current, as TNF-α increased the magnitude of the current and lowered its threshold of activation. In contrast, TNF-α did not appear to modulate either the delayed rectifier potassium current or the transient potassium current. These data suggest that acute and long-term TNF-α exposure elevates SFO neuron activity, providing a basis for TNF-α hypertensive and sympathetic effects.NEW & NOTEWORTHY Considerable recent evidence has suggested important links between inflammation and the pathological mechanisms underlying hypertension. The present study describes cellular mechanisms through which acute and long-term exposure of tumor necrosis factor-α (TNF-α) influences the activity of subfornical organ neurons by modulating the voltage-gated transient Na+ current. This provides critical new information regarding the specific pathological mechanisms through which inflammation and TNF-α in particular may result in the development of hypertension.

Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells

cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of β-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to β-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic–computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (β-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca2+-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate.

NMDA receptors and L-type voltage-gated Ca2+ channels mediate the expression of bidirectional homeostatic intrinsic plasticity in cultured hippocampal neurons

Homeostatic plasticity is engaged when neurons need to stabilize their synaptic strength and excitability in response to acute or prolonged destabilizing changes in global activity. Compared to the extensive studies investigating the molecular mechanisms for homeostatic synaptic plasticity, the mechanism underlying homeostatic intrinsic plasticity is largely unknown. Through whole-cell patch-clamp recording in low-density cultures of dissociated hippocampal neurons, we demonstrate here that prolonged activity blockade induced by the sodium channel blocker tetrodotoxin (TTX) leads to increased action potential firing rates. Conversely, prolonged activity enhancement induced by the A-type gamma-aminobutyric acid receptor antagonist bicuculline (BC) results in decreased firing rates. Prolonged activity enhancement also enhanced potassium (K+) current through Kv1 channels, suggesting that changes in K+ current, in part, mediate stabilization of hippocampal neuronal excitability upon prolonged activity elevation. In contrast to the previous reports showing that L-type voltage-gated calcium (Ca2+) channels solely mediate homeostatic regulation of excitatory synaptic strength (Ibata et al., 2008; Goold and Nicoll, 2010), inhibition of N-Methyl-d-aspartate (NMDA) receptors alone mimics the elevation in firing frequency driven by prolonged TTX application, while the decrease in firing rates induced by prolonged BC treatment involves the activity of NMDA receptors and L-type voltage-gated Ca2+ channels. These results collectively provide strong evidence that alterations in Ca2+ influx through NMDA receptors and L-type voltage-gated Ca2+ channels mediate homeostatic intrinsic plasticity in hippocampal neurons in response to prolonged activity changes.

Reduced dendritic arborization and hyperexcitability of pyramidal neurons in a Scn1b-based model of Dravet syndrome.

Epileptic encephalopathies, including Dravet syndrome, are severe treatment-resistant epilepsies with developmental regression. We examined a mouse model based on a human β1 sodium channel subunit (Scn1b) mutation. Homozygous mutant mice shared phenotypic features and pharmaco-sensitivity with Dravet syndrome. Patch-clamp analysis showed that mutant subicular and layer 2/3 pyramidal neurons had increased action potential firing rates, presumably as a consequence of their increased input resistance. These changes were not seen in L5 or CA1 pyramidal neurons. This raised the concept of a regional seizure mechanism that was supported by data showing increased spontaneous synaptic activity in the subiculum but not CA1. Importantly, no changes in firing or synaptic properties of gamma-aminobutyric acidergic interneurons from mutant mice were observed, which is in contrast with Scn1a-based models of Dravet syndrome. Morphological analysis of subicular pyramidal neurons revealed reduced dendritic arborization. The antiepileptic drug retigabine, a K+ channel opener that reduces input resistance, dampened action potential firing and protected mutant mice from thermal seizures. These results suggest a novel mechanism of disease genesis in genetic epilepsy and demonstrate an effective mechanism-based treatment of the disease.

Insulin-Like Growth Factor-1 Receptor-Mediated Inhibition of A-type K+ Current Induces Sensory Neuronal Hyperexcitability Through the Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/2 Pathways, Independently of Akt

Although IGF-1 has been implicated in mediating hypersensitivity to pain, the underlying mechanisms remain unclear. We identified a novel functional of the IGF-1 receptor (IGF-1R) in regulating A-type K(+) currents (IA) as well as membrane excitability in small trigeminal ganglion neurons. Our results showed that IGF-1 reversibly decreased IA, whereas the sustained delayed rectifier K(+) current was unaffected. This IGF-1-induced IA decrease was associated with a hyperpolarizing shift in the voltage dependence of inactivation and was blocked by the IGF-1R antagonist PQ-401; an insulin receptor tyrosine kinase inhibitor had no such effect. An small interfering RNA targeting the IGF-1R, or pretreatment of neurons with specific phosphatidylinositol 3-kinase (PI3K) inhibitors abolished the IGF-1-induced IA decrease. Surprisingly, IGF-1-induced effects on IA were not regulated by Akt, a common downstream target of PI3K. The MAPK/ERK kinase inhibitor U0126, but not its inactive analog U0124, as well as the c-Raf-specific inhibitor GW5074, blocked the IGF-1-induced IA response. Analysis of phospho-ERK (p-ERK) showed that IGF-1 significantly activated ERK1/2 whereas p-JNK and p-p38 were unaffected. Moreover, the IGF-1-induced p-ERK1/2 increase was attenuated by PI3K and c-Raf inhibition, but not by Akt blockade. Functionally, we observed a significantly increased action potential firing rate induced by IGF-1; pretreatment with 4-aminopyridine abolished this effect. Taken together, our results indicate that IGF-1 attenuates IA through sequential activation of the PI3K- and c-Raf-dependent ERK1/2 signaling cascade. This occurred via the activation of IGF-1R and might contribute to neuronal hyperexcitability in small trigeminal ganglion neurons.

Mechanisms that match ATP supply to demand in cardiac pacemaker cells during high ATP demand

The spontaneous action potential (AP) firing rate of sinoatrial node cells (SANCs) involves high-throughput signaling via Ca(2+)-calmodulin activated adenylyl cyclases (AC), cAMP-mediated protein kinase A (PKA), and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of SR Ca(2+) cycling and surface membrane ion channel proteins. When the throughput of this signaling increases, e.g., in response to β-adrenergic receptor activation, the resultant increase in spontaneous AP firing rate increases the demand for ATP. We hypothesized that an increase of ATP production to match the increased ATP demand is achieved via a direct effect of increased mitochondrial Ca(2+) (Ca(2+)m) and an indirect effect via enhanced Ca(2+)-cAMP/PKA-CaMKII signaling to mitochondria. To increase ATP demand, single isolated rabbit SANCs were superfused by physiological saline at 35 ± 0.5°C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca(2+) and flavoprotein fluorescence in single SANC, and we measured cAMP, ATP, and O₂ consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O₂ consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca(2+)m and cAMP increased concurrently with the increase in AP firing rate. When Ca(2+)m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca(2+)m and an increase in Ca(2+) activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.

Network‐specific mechanisms may explain the paradoxical effects of carbamazepine and phenytoin

A common notion of the mechanism by which the antiepileptic drugs (AEDs) carbamazepine and phenytoin act is that they block sodium channels by binding preferentially to the inactivated state, thereby allowing normal neuronal firing while blocking ictal activity. However, these drugs have unpredictable efficacy and, in some cases, may exacerbate seizures. Previous studies have suggested that reducing sodium channel availability in the dentate gyrus (DG) paradoxically increases excitability. We used a biophysically detailed computer model of the DG to test the hypothesis that AEDs increase excitability by disproportionately reducing negative feedback mechanisms. We built a Markov model of sodium channel gating that reproduces responses to voltage clamp experiments in the presence of carbamazepine and phenytoin. We incorporated this validated Markov model into a biophysically realistic computer model of DG neurons and networks. Simulated drug concentrations were similar to those measured in cerebral spinal fluid in medicated patients. Single neuron models were stimulated with current injections, and networks were stimulated with perforant path synaptic input. In the network model, environmental effects were studied by introducing mossy fiber sprouting. As expected, drugs reduced sodium channel availability, which in turn reduced action potential amplitude. This had only a small effect on action potential (AP) firing rate during brief (100 msec) current injections. Paradoxically, long current injections (2,500 msec) increased AP firing rates. This was caused by reduced calcium entry and consequently reduced activation of calcium activated potassium channels. It is important to note that the main determinant of drug effect was resting membrane potential (RMP) and not action potential firing rate. Binding of phenytoin and carbamazepine is slow and, thus drug effects are largely determined by the long term state of the RMP. This paradoxical AP firing increase was dependent on the unusually large calcium-activated potassium conductances expressed by DG granule cells. This predicts that drug efficacy in a given network will depend on the precise makeup of conductances in the network. RMP is expected to vary with the level of activity in the network. We simulated the effects of drugs on single shot stimulus responses in networks with mossy fiber sprouting and varied the RMP in all neurons as a model for network activity. For an RMP of -50 mV, representing an active network, drugs had no effect, or in some cases, increased excitability. Drugs had an increasingly larger inhibitory effect on network responses as RMP decreased. An important prediction is that drugs will be unable to block ictal activity invading an active network. Our key findings are that drug effects depend on both intrinsic properties of the network and its behavioral state. This may explain the paradoxical and unpredictable effects of some AEDs on seizure control in some patients.

Electrophysiological changes precede morphological changes to frontal cortical pyramidal neurons in the rTg4510 mouse model of progressive tauopathy

Whole-cell patch-clamp recordings and high-resolution morphometry were used to assess functional and structural properties of layer 3 pyramidal neurons in early (<4months) and advanced (>8months) stages of tauopathy in frontal cortical slices prepared from rTg4510 tau mutant (P301L) mice. In early tauopathy, dendritic architecture is preserved. In advanced tauopathy, neurons can be categorized as either "atrophic" (58%)-exhibiting marked atrophy of the apical tuft, or "intact" (42%)-with normal apical tufts and, in some instances, proliferative sprouting of oblique branches of the apical trunk. Approximately equal numbers of atrophic and intact neurons contain neurofibrillary tangles (NFTs) or are tangle-free, lending further support to the idea that NFTs per se are not toxic. Spine density is decreased due to a specific reduction in mushroom spines, but filopodia are increased in both atrophic and intact neurons. By contrast to these morphological changes, which are robust only in the advanced stage, significant electrophysiological changes are present in the early stage and persist in the advanced stage in both atrophic and intact neurons. The most marked of these changes are: a depolarized resting membrane potential, an increased depolarizing sag potential and increased action potential firing rates-all indicative of hyperexcitability. Spontaneous excitatory postsynaptic currents are not reduced in frequency or amplitude in either stage. The difference in the time course of functionally important electrophysiological changes versus regressive morphological changes implies differences in pathogenic mechanisms underlying functional and structural changes to neurons during progressive tauopathy.

Modeling predicts that parameters shaping action potentials and synaptic responses differ in pyramidal neurons of the visual and prefrontal cortices

Pyramidal neurons in the prefrontal cortex integrate inputs that are greater in number and diversity than pyramidal neurons of the primary visual cortex [1]. We have recently characterized the morphology and physiology of layer 3 neurons from area 46 of the dorsolateral prefrontal cortex (dlPFC) and from visual area V1 in the rhesus monkey. Ultra-high resolution confocal imaging and 3D reconstruction revealed that the dendritic arbors of V1 neurons are much smaller and less complex than those of the dlPFC, and possess far fewer dendritic spines. Physiologically, whole cell patch clamp recordings demonstrated that V1 neurons have twice as high mean input resistance and significantly increased action potential firing rates compared to dlPFC neurons. Further, AMPAR-mediated spontaneous excitatory postsynaptic currents (sEPSCs) exhibit significantly faster kinetics and smaller mean amplitudes in V1 compared to dlPFC neurons. We have used compartment modeling with the NEURON simulation environment [2] to investigate the extent to which morphology accounts for the observed physiological differences. We first applied a relatively simple model, including only Hodgkin-Huxley sodium and potassium currents [3], to representative 3D reconstructions of neurons from dlPFC and V1. This model predicts that morphology alone leads to major differences in the attenuation of electrical signals coming into, and leaving, the soma in neurons from the two brain areas. It also predicts that morphology largely accounts for the increased excitability of V1 cells, but not the differences in kinetics and amplitudes of sEPSCs. The model predicts that parameters controlling passive voltage spread, the density of Hodgkin-Huxley channels, and synaptic inputs likely differ between dlPFC and V1 neurons. Yet, the simple model on which these predictions are based reproduces neither the H-current dependent ‘sag’ response during hyperpolarization, nor spike frequency adaptation, both of which are observed in dlPFC and V1 neurons. We have extended the model to include the H current [4], plus a calcium-dependent potassium channel with an associated one-pool source of intracellular calcium [5]. We have tuned the model with a combination of manual and automatic optimization techniques to match the resting potential, input resistance, and overall firing rates of the recorded dlPFC and V1 neurons. As in the simpler model, we find that morphology alone cannot account for observed physiological differences. Therefore, we predict that parameters shaping action potentials and synaptic input differ between dlPFC and V1.

Alphaherpesvirus Infection Disrupts Mitochondrial Transport in Neurons

Mitochondria are dynamic organelles that are essential for cellular metabolism but can be functionally disrupted during pathogen infection. In neurons, mitochondria are transported on microtubules via the molecular motors kinesin-1 and dynein and recruited to energy-requiring regions such as synapses. Previous studies showed that proteins from pseudorabies virus (PRV), an alphaherpesvirus, localize to mitochondria and affect mitochondrial function. We show that PRV and herpes simplex virus type 1 (HSV-1) infection of rodent superior cervical ganglion (SCG) neurons disrupts mitochondrial motility and morphology. During PRV infection, glycoprotein B (gB)-dependent fusion events result in electrical coupling of neurons and increased action potential firing rates. Consequently, intracellular [Ca(2+)] increases and alters mitochondrial dynamics through a mechanism involving the Ca(2+)-sensitive cellular protein Miro and reduced recruitment of kinesin-1 to mitochondria. This disruption in mitochondrial dynamics is required for efficient growth and spread of PRV, indicating that altered mitochondrial transport enhances alphaherpesvirus pathogenesis and infection.

Nicotinic excitation of serotonergic projections from dorsal raphe to the nucleus accumbens

Tobacco use is a major public health problem, and although many smokers report that they want to quit, only a small percentage succeed. Side effects associated with nicotine withdrawal, including depression, anxiety, and restlessness, certainly contribute to the low success rate. The dorsal raphe nucleus (DRN) is a serotonergic center with many functions, including control of mood and emotional state. We investigated the effect of nicotine on DRN neurons that project to the nucleus accumbens (NAc), an area involved in reward-related behaviors. Using a retrograde labeling method, we found that 75% of DRN-NAc projection neurons are serotonergic. In coronal slices that include the DRN, whole cell recordings were conducted on neurons identified by fluorescent backlabeling from NAc or randomly selected within the nucleus. Nicotine increased action potential firing rates in a subset of DRN neurons. Voltage-clamp recording revealed nicotinic acetylcholine receptor (nAChR)-mediated inward currents that contribute to the nicotine-induced excitation. Nicotinic receptors also indirectly affect excitability by modulating synaptic inputs to these neurons. Nicotine enhanced excitatory glutamatergic inputs to a subset of DRN-NAc projection neurons, while inhibitory γ-aminobutyric acid (GABA)ergic inputs were modulated either positively or negatively in a subset of these neurons. The net effect of nAChR activation is enhancement of serotonergic output from DRN to the NAc, which may contribute to the effects of nicotine on mood and affect.

Age-related increase of sIAHP in prefrontal pyramidal cells of monkeys: relationship to cognition

Reduced excitability, due to an increase in the slow afterhyperpolarization (and its underlying current sIAHP), occurs in CA1 pyramidal cells in aged cognitively-impaired, but not cognitively-unimpaired, rodents. We sought to determine whether similar age-related changes in the sIAHP occur in pyramidal cells in the rhesus monkey dorsolateral prefrontal cortex (dlPFC). Whole-cell patch-clamp recordings were obtained from layer 3 and layer 5 pyramidal cells in dlPFC slices prepared from young (9.6 ± 0.7 years old) and aged (22.3 ± 0.7 years old) behaviorally characterized subjects. The amplitude of the sIAHP was significantly greater in layer 3 (but not layer 5) cells from aged-impaired compared with both aged-unimpaired and young monkeys, which did not differ. Aged layer 3, but not layer 5, cells exhibited significantly increased action potential firing rates, but there was no relationship between sIAHP and firing rate. Thus, in monkey dlPFC layer 3 cells, an increase in sIAHP is associated with age-related cognitive decline; however, this increase is not associated with a reduction in excitability.

The RNA Binding Complex Translin‐Trax Mediates Pro‐Excitatory Activity in Neurons

In animal models of neurogenic hypertension the Rostral Ventrolateral Medulla (RVLM), of the brain, displays increased excitatory activity that is linked to elevated sympathetic tone and blood pressure. Analysis of gene expression in the RVLM of normotensive Wistar Kyoto (WKY) and Spontaneous Hypertensive Rat (SHR) revealed reduction of the protein Translin (TSN) in SHR. TSN and its partner Trax (TRX) form a complex that is thought to mediate dendritic trafficking of mRNA. To examine the functional effect of this complex, we dialyzed purified TSN‐TRX into cultured brain neurons from either WKY or SHR and measured its effect on the rate of action potential firing (APF). Basal APF rate was 1.4 Hz in WKY neurons and after 20 min control treated neurons showed a similar rate of 1.3 Hz. In contrast, TSN‐TRX treatment mediated a significant increase in APF rate to 1.9 Hz. Tsn alone had no effect in WKY neurons and SHR neurons showed no response to TSN‐TRX. Pretreatment with the RNA translation inhibitor, cycloheximide, abolished the excitatory effect of Tsn‐TRX in WKY neurons. Our results suggest that the excitatory effect of TSN‐TRX in WKY neurons is translation‐dependent. We hypothesize that homeostatic feedback mechanisms in SHR neurons lead to downregulation of TSN levels and the resistance of these cells to the excitatory effects of the TSN‐TRX complex. This work was supported by funding from NHLBI, NIDA of the NIH and NARSAD.

Pseudorabies Virus Infection Alters Neuronal Activity and Connectivity In Vitro

Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV), infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP) firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural function seen in PRV-infected animals.

A rodent model of schizophrenia derived from postmortem studies

There is compelling postmortem evidence that GABA cell dysfunction plays a role in the pathophysiology of schizophrenia (SZ). Based on a unique distribution of postmortem abnormalities in layer II of the anterior cingulate cortex and sectors CA3/2 of the hippocampus, we postulated that afferent fibers from the basolateral amygdala to these sites may contribute to diminished GABAergic modulation in these disorders. To test this hypothesis, picrotoxin (PICRO), a non-competitive antagonist of the GABA-A receptor, is stereotaxically infused the basolateral complex of the amygdala (BLA) to increase the flow of excitatory activity into stratum oriens (SO) of sectors CA3/2 of the hippocampus. This pharmacological manipulation results in a selective reduction of GABAergic interneurons containing parvalbumin, calbindin and calretinin in CA3/2. Using single cell recordings in a hippocampal slide preparation, these changes in PICRO-treated rats seem to be associated with a reduction in evoked and spontaneous inhibitory post-synaptic potentials (sIPSCs) recorded from pyramidal neurons in sector CA3/2, but not CA1. A lower resting membrane potential and an increased action potential firing rate have been recorded in interneurons in the SO of CA2/3, but not CA1. Additionally, currents associated with hyperpolarization-activated cationic channels (Ih), which help to control neuronal firing rates of GABA cells in the hippocampus, were also increased. Overall, these studies support the view that postmortem studies contribute information for the development of empiric models of SZ, ones that can be used as translational tools for elucidating the functional changes that may be present in GABA cell subtypes their molecular regulatory mechanisms in this disorder.

Amygdala-Dependent Regulation of Electrical Properties of Hippocampal Interneurons in a Model of Schizophrenia

Schizophrenia (SZ) involves dysfunction of gamma-aminobutyric acid (GABA)ergic transmission in the hippocampus (HIPP), particularly in sector CA2/3. Previous work using a rodent model of postmortem abnormalities in SZ demonstrated that activation of the basolateral amygdala (BLA) results in decreases of GABA currents in pyramidal neurons of CA2/3 but not CA1. In addition, a decrease of GABA cells has been reported in postmortem studies of the HIPP in SZ. In the present work we tested the hypothesis that BLA activation in this rodent model of SZ leads to changes in the electrical properties of interneurons located in sector CA2/3. Patch clamp recordings in HIPP slices were performed in rat HIPP slices after 15 days of infusion of picrotoxin into the BLA. The intrinsic and firing properties and hyperpolarization-activated currents (Ih) of interneurons were measured in stratum oriens (SO) of CA2/3 and CA1. The BLA activation was associated with a lower resting membrane potential and an increased action potential firing rate in interneurons of CA2/3 but not CA1. Recordings from interneurons further demonstrated an increase of currents associated with hyperpolarization-activated cationic channels (Ih), which help to control neuronal firing rates and oscillatory rhythms. Taken together, these results suggest that the enhanced BLA activity is capable of increasing the excitability of interneurons in SO of CA2/3 and might contribute to GABAergic dysfunction in SZ.