Cholinergic modulation shifts the response of CA1 pyramidal cells to depolarizing ramps via TRPM4 channels with potential implications for place field firing.

A synergistic combination of in vitro electrophysiology and multicompartmental modeling of rat CA1 pyramidal neurons identified TRPM4 channels as major drivers of cholinergic modulation of the firing rate during a triangular current ramp, which emulates the bump in synaptic input received while traversing the place field. In control, fewer spikes at lower frequencies are elicited on the down-ramp compared to the up-ramp due to long-term inactivation of the NaV channel. The cholinergic agonist carbachol (CCh) removes or even reverses this spike rate adaptation, causing more spikes to be elicited on the down-ramp than the up-ramp. CCh application during Schaffer collateral stimulation designed to simulate a ramp produces similar shifts in the center of mass of firing to later in the ramp. The non-specific TRP antagonist flufenamic acid and the TRPM4-specific blockers CBA and 9-phenanthrol, but not the TRPC-specific antagonist SKF96365, reverse the effect of CCh; this implicates the Ca2+-activated nonspecific cation current, ICAN, carried by TRPM4 channels. The cholinergic shift of the center of mass of firing is prevented by strong intracellular Ca2+ buffering but not by antagonists for IP3 and ryanodine receptors, ruling out a role for known mechanisms of release from intracellular Ca2+ stores. Pharmacology combined with modeling suggest that [Ca2+] in a nanodomain near the TRPM4 channel is elevated through an unknown source that requires both muscarinic receptor activation and depolarization-induced Ca2+ influx during the ramp. Activation of the regenerative inward TRPM4 current in the model qualitatively replicates and provides putative underlying mechanisms for the experimental observations.

Long-Term Inactivation of Sodium Channels as a Mechanism of Adaptation in CA1 Pyramidal Neurons.

Many hippocampal CA1 pyramidal cells function as place cells, increasing their firing rate when a specific place field is traversed. The dependence of CA1 place cell firing on position within the place field is asymmetric. We investigated the source of this asymmetry by injecting triangular depolarizing current ramps to approximate the spatially tuned, temporally diffuse depolarizing synaptic input received by these neurons while traversing a place field. Ramps were applied to CA1 pyramidal neurons from male rats in vitro (slice electrophysiology) and in silico (multicompartmental NEURON model). Under control conditions, CA1 neurons fired more action potentials at higher frequencies on the up-ramp versus the down-ramp. This effect was more pronounced for dendritic compared with somatic ramps. We incorporated a four-state Markov scheme for NaV1.6 channels into our model and calibrated the spatial dependence of long-term inactivation according to the literature; this spatial dependence was sufficient to explain the difference in dendritic versus somatic ramps. Long-term inactivation reduced the firing frequency by decreasing open-state occupancy, and reduced spike amplitude during trains by decreasing occupancy in the closed state, which comprises the available pool. PKC activator phorbol-dibutyrate, known to reduce NaV long-term inactivation, removed spike amplitude attenuation in vitro more visibly in dendrites and greatly reduced adaptation, consistent with our hypothesized mechanism. Intracellular application of a peptide inducing long-term NaV inactivation elicited spike amplitude attenuation during spike trains in the soma and greatly enhanced adaptation. Our synergistic experimental/computational approach shows that long-term inactivation of NaV1.6 is a key mechanism of adaptation in CA1 pyramidal cells.SIGNIFICANCE STATEMENT The hippocampus plays an important role in certain types of memory, in part through context-specific firing of "place cells"; these cells were first identified in rodents as being particularly active when an animal is in a specific location in an environment, called the place field of that neuron. In this in vitro/in silico study, we found that long-term inactivation of sodium channels causes adaptation in the firing rate that could potentially skew the firing of CA1 hippocampal pyramidal neurons earlier within a place field. A computational model of the sodium channel revealed differential regulation of spike frequency and amplitude by long-term inactivation, which may be a general mechanism for spike frequency adaptation in the CNS.

Ih from synapses to networks: HCN channel functions and modulation in neurons.

Hyperpolarization-activated cyclic nucleotide gated (HCN) channels and the current they carry, Ih, are widely and diversely distributed in the central nervous system (CNS). The distribution of the four subunits of HCN channels is variable within the CNS, within brain regions, and often within subcellular compartments. The precise function of Ih can depend heavily on what other channels are co-expressed. In this review, we give an overview of HCN channel structure, distribution, and modulation by cyclic adenosine monophosphate (cAMP). We then discuss HCN channel and Ih functions, where we have parsed the roles into two main effects: a steady effect on maintaining the resting membrane potential at relatively depolarized values, and slow channel dynamics. Within this framework, we discuss Ih involvement in resonance, synaptic integration, transmitter release, plasticity, and point out a special case, where the effects of Ih on the membrane potential and its slow channel dynamics have dual roles in thalamic neurons.

Intrinsic Mechanisms of Frequency Selectivity in the Proximal Dendrites of CA1 Pyramidal Neurons.

Gamma oscillations are thought to play a role in learning and memory. Two distinct bands, slow (25-50 Hz) and fast (65-100 Hz) gamma, have been identified in area CA1 of the rodent hippocampus. Slow gamma is phase locked to activity in area CA3 and presumably driven by the Schaffer collaterals (SCs). We used a combination of computational modeling and in vitro electrophysiology in hippocampal slices of male rats to test whether CA1 neurons responded to SC stimulation selectively at slow gamma frequencies and to identify the mechanisms involved. Both approaches demonstrated that, in response to temporally precise input at SCs, CA1 pyramidal neurons fire preferentially in the slow gamma range regardless of whether the input is at fast or slow gamma frequencies, suggesting frequency selectivity in CA1 output with respect to CA3 input. In addition, phase locking, assessed by the vector strength, was more precise for slow gamma than fast gamma input. This frequency selectivity was greatly attenuated when the slow Ca2+-dependent K+ (SK) current was removed from the model or blocked in vitro with apamin. Perfusion of slices with BaCl2 to block A-type K+ channels tightened this frequency selectivity. Both the broad-spectrum cholinergic agonist carbachol and the muscarinic agonist oxotremorine-M greatly attenuated the selectivity. The more precise firing at slower frequencies persisted throughout all of the pharmacological manipulations conducted. We propose that these intrinsic mechanisms provide a means by which CA1 phase locks to CA3 at different gamma frequencies preferentially in vivo as physiological conditions change with behavioral demands.SIGNIFICANCE STATEMENT Gamma frequency activity, one of multiple bands of synchronous activity, has been suggested to underlie various aspects of hippocampal function. Multisite recordings within the rat hippocampal formation indicate that different behavioral tasks are associated with synchronized activity between areas CA3 and CA1 at two different gamma bands: slow and fast gamma. In this study, we examine the intrinsic mechanisms that may allow CA1 to selectively "listen" to CA3 at slow compared with fast gamma and suggest mechanisms that gate this selectivity. Identifying the intrinsic mechanisms underlying differential gamma preference may help to explain the distinct types of CA3-CA1 synchronization observed in vivo under different behavioral conditions.

Differential Contribution of Ca2+-Dependent Mechanisms to Hyperexcitability in Layer V Neurons of the Medial Entorhinal Cortex.

Temporal lobe epilepsy is characterized by recurrent seizures in one or both temporal lobes of the brain; some in vitro models show that epileptiform discharges initiate in entorhinal layer V neurons and then spread into other areas of the temporal lobe. We previously found that, in the presence of GABAA receptor antagonists, stimulation of afferent fibers, terminating both at proximal and distal dendritic locations, initiated hyperexcitable bursts in layer V medial entorhinal neurons. We investigated the differential contribution of Ca2+-dependent mechanisms to the plateaus underlying these bursts at proximal and distal synapses. We found that the NMDA glutamatergic antagonist D,L-2-amino-5-phosphonovaleric acid (APV; 50 µM) reduced both the area and duration of the bursts at both proximal and distal synapses by about half. The L-type Ca2+ channel blocker nimodipine (10 µM) and the R- and T-type Ca2+ channel blocker NiCl2 (200 µM) decreased the area of the bursts to a lesser extent; none of these effects appeared to be location-dependent. Remarkably, the perfusion of flufenamic acid (FFA; 100 µM), to block Ca2+-activated non-selective cation currents (ICAN) mediated by transient receptor potential (TRP) channels, had a location-dependent effect, by abolishing burst firing and switching the suprathreshold response to a single action potential (AP) for proximal stimulation, but only minimally affecting the bursts evoked by distal stimulation. A similar outcome was found when FFA was pressure-applied locally around the proximal dendrite of the recorded neurons and in the presence of a selective blocker of melastatin TRP (TRPM) channels, 9-phenanthrol (100 µM), whereas a selective blocker of canonical TRP (TRPC) channels, SKF 96365, did not affect the bursts. These results indicate that different mechanisms might contribute to the initiation of hyperexcitability in layer V neurons at proximal and distal synapses and could shed light on the initiation of epileptiform activity in the entorhinal cortex.

Low prevalence of Chagas parasite infection in a nonhuman primate colony in Louisiana.

Chagas disease, an important cause of heart disease in Latin America, is caused by the parasite Trypanosoma cruzi, which typically is transmitted to humans by triatomine insects. Although autochthonous transmission of the Chagas parasite to humans is rare in the United States, triatomines are common, and more than 20 species of mammals are infected with the Chagas parasite in the southern United States. Chagas disease has also been detected in colonies of nonhuman primates (NHP) in Georgia and Texas, and heart abnormalities consistent with Chagas disease have occurred at our NHP center in Louisiana. To determine the level of T. cruzi infection, we serologically tested 2157 of the approximately 4200 NHP at the center; 34 of 2157 primates (1.6%) tested positive. Presence of the T. cruzi parasite was confirmed by hemoculture in 4 NHP and PCR of the cultured parasites. These results strongly suggest local transmission of T. cruzi, because most of the infected NHP were born and raised at this site. All 3 species of NHP tested yielded infected animals, with significantly higher infection prevalence in pig-tailed macaques, suggesting possible exploration of this species as a model organism. The local T. cruzi strain isolated during this study would enhance such investigations. The NHP at this center are bred for use in scientific research, and the effects of the Chagas parasite on infected primates could confuse the interpretation of other studies.

Modest alterations in patterns of motor neuron dendrite morphology in the Fmr1 knockout mouse model for fragile X.

Fragile X, an inheritable form of mental retardation, is caused by the inactivation of a gene on the X chromosome, FMR1 which codes for an RNA binding protein, fragile X mental retardation protein. Loss of this protein is associated with reduced complexities of neuronal dendrites and alterations in spine morphology in a number of cortical brain regions, and these deficits may underlie the cognitive impairment observed in fragile X patients. Among the many symptoms of fragile X are altered motor functions, although the neuronal basis for these remains unclear. In this study we investigated whether knockout of Fmr1 in the mouse model of fragile X altered dendrite morphology in developing spinal cord motor neurons. We find that Fmr1 knockout leads to modest alterations in the distribution of dendritic arbor across the span of the motor neuron dendritic tree in 2- and 4-week-old mice, compared to wild-type controls, consistent with slower rates of extension and abnormal pruning of intermediate dendritic segments. These studies suggest that some motor deficits in fragile X patients may be due to abnormal maturation of dendritic patterning within spinal motor neurons, and suggest that strategies aimed at preventing motor impairment in fragile X patients may be targeted at motor functions during early development.

IFN-gamma-producing dendritic cells are important for priming of gut intraepithelial lymphocyte response against intracellular parasitic infection.

The importance of intraepithelial lymphocytes (IEL) in immunoprotection against orally acquired pathogens is being increasingly recognized. Recent studies have demonstrated that Ag-specific IEL can be generated and can provide an important first line of defense against pathogens acquired via oral route. However, the mechanism involved in priming of IEL remains elusive. Our current study, using a microsporidial model of infection, demonstrates that priming of IEL is dependent on IFN-gamma-producing dendritic cells (DC) from mucosal sites. DC from mice lacking the IFN-gamma gene are unable to prime IEL, resulting in failure of these cells to proliferate and lyse pathogen-infected targets. Also, treatment of wild-type DC from Peyer's patches with Ab to IFN-gamma abrogates their ability to prime an IEL response against Encephalitozoon cuniculi in vitro. Moreover, when incubated with activated DC from IFN-gamma knockout mice, splenic CD8(+) T cells are not primed efficiently and exhibit reduced ability to home to the gut compartment. These data strongly suggest that IFN-gamma-producing DC from mucosal sites play an important role in the generation of an Ag-specific IEL response in the small intestine. To our knowledge, this report is the first demonstrating a role for IFN-gamma-producing DC from Peyer's patches in the development of Ag-specific IEL population and their trafficking to the gut epithelium.

Lack of IL-15 results in the suboptimal priming of CD4+ T cell response against an intracellular parasite.

IFN-gamma-producing CD4+ T cells, although important for protection against acute Toxoplasma gondii infection, can cause gut pathology, which may prove to be detrimental for host survival. Here we show that mice lacking IL-15 gene develop a down-regulated IFN-gamma-producing CD4+ T cell response against the parasite, which leads to a reduction in gut necrosis and increased level of survival against infection. Moreover, transfer of immune CD4+ T cells from WT to IL-15-/- mice reversed inhibition of gut pathology and caused mortality equivalent to levels of parental WT mice. Down-regulated CD4+ T cell response in the absence of IL-15, manifested as reduced antigen-specific proliferation, was due to defective priming of the T cell subset by dendritic cells (DCs) of these animals. When stimulated with antigen-pulsed DCs from WT mice, CD4+ T cells from IL-15-/- mice were primed optimally, and robust proliferation of these cells was observed. A defect in the DCs of knockout mice was further confirmed by their reduced ability to produce IL-12 upon stimulation with Toxoplasma lysate antigen. Addition of exogenous IL-15 to DC cultures from knockout mice led to increased IL-12 production by these cells and restored their ability to prime an optimal parasite-specific CD4+ T cell response. To our knowledge, this is the first demonstration of the role of IL-15 in the development of CD4+ T cell immunity against an intracellular pathogen. Furthermore, based on these observations, targeting of IL-15 should have a beneficial effect on individuals suffering from CD4+ T cell-mediated autoimmune diseases.

NK cells help to induce CD8(+)-T-cell immunity against Toxoplasma gondii in the absence of CD4(+) T cells.

CD8(+) T-cell immunity plays an important role in protection against intracellular infections. Earlier studies have shown that CD4(+) T-cell help was needed for launching in vivo CD8(+) T-cell activity against these pathogens and tumors. However, recently CD4(+) T-cell-independent CD8 responses during several microbial infections including those with Toxoplasma gondii have been described, although the mechanism is not understood. We now demonstrate that, in the absence of CD4(+) T cells, T. gondii-infected mice exhibit an extended NK cell response, which is mediated by continued interleukin-12 (IL-12) secretion. This prolonged NK cell response is critical for priming parasite-specific CD8(+) T-cell immunity. Depletion of NK cells inhibited the generation of CD8(+) T-cell immunity in CD4(-/-) mice. Similarly neutralization of IL-12 reduces NK cell numbers in infected animals and leads to the down-regulation of CD8(+) T-cell immunity against T. gondii. Adoptive transfer of NK cells into the IL-12-depleted animals restored their CD8(+) T-cell immune response, and animals exhibited reduced mortality. NK cell gamma interferon was essential for cytotoxic T-lymphocyte priming. Our studies for the first time demonstrate that, in the absence of CD4(+) T cells, NK cells can play an important role in induction of primary CD8(+) T-cell immunity against an intracellular infection. These observations have therapeutic implications for immunocompromised individuals, including those with human immunodeficiency virus infection.