The Entorhinal Cortical Alvear Pathway Differentially Excites Pyramidal Cells and Interneuron Subtypes in Hippocampal CA1.

The entorhinal cortex alvear pathway is a major excitatory input to hippocampal CA1, yet nothing is known about its physiological impact. We investigated the alvear pathway projection and innervation of neurons in CA1 using optogenetics and whole cell patch clamp methods in transgenic mouse brain slices. Using this approach, we show that the medial entorhinal cortical alvear inputs onto CA1 pyramidal cells (PCs) and interneurons with cell bodies located in stratum oriens were monosynaptic, had low release probability, and were mediated by glutamate receptors. Optogenetic theta burst stimulation was unable to elicit suprathreshold activation of CA1 PCs but was capable of activating CA1 interneurons. However, different subtypes of interneurons were not equally affected. Higher burst action potential frequencies were observed in parvalbumin-expressing interneurons relative to vasoactive-intestinal peptide-expressing or a subset of oriens lacunosum-moleculare (O-LM) interneurons. Furthermore, alvear excitatory synaptic responses were observed in greater than 70% of PV and VIP interneurons and less than 20% of O-LM cells. Finally, greater than 50% of theta burst-driven inhibitory postsynaptic current amplitudes in CA1 PCs were inhibited by optogenetic suppression of PV interneurons. Therefore, our data suggest that the alvear pathway primarily affects hippocampal CA1 function through feedforward inhibition of select interneuron subtypes.

Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via α4ß2* nicotinic receptor activation.

Acetylcholine (ACh) release onto nicotinic receptors directly activates subsets of inhibitory interneurons in hippocampal CA1. However, the specific interneurons activated and their effect on the hippocampal network is not completely understood. Therefore, we investigated subsets of hippocampal CA1 interneurons that respond to ACh release through the activation of nicotinic receptors and the potential downstream effects this may have on hippocampal CA1 network function. ACh was optogenetically released in mouse hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated viral mediated transfection. The actions of optogenetically released ACh were assessed on both pyramidal neurons and different interneuron subtypes via whole cell patch clamp methods. Vasoactive intestinal peptide (VIP)-expressing interneurons that selectively innervate other interneurons (VIP/IS) were excited by ACh through the activation of nicotinic receptors containing α4 and ß2 subunits (α4ß2*). ACh release onto VIP/IS was presynaptically inhibited by M2 muscarinic autoreceptors. ACh release produced spontaneous inhibitory postsynaptic current (sIPSC) barrages blocked by dihydro-ß-erythroidine in interneurons but not pyramidal neurons. Optogenetic suppression of VIP interneurons did not inhibit these sIPSC barrages suggesting other interneuron-selective interneurons were also excited by α4ß2* nicotinic receptor activation. In contrast, interneurons that innervate pyramidal neuron perisomatic regions were not activated by ACh release onto nicotinic receptors. Therefore, we propose ACh release in CA1 facilitates disinhibition through activation of α4ß2* nicotinic receptors on interneuron-selective interneurons whereas interneurons that innervate pyramidal neurons are less affected by nicotinic receptor activation.

Activation of muscarinic receptors by ACh release in hippocampal CA1 depolarizes VIP but has varying effects on parvalbumin-expressing basket cells.

KEY POINTS: Optogenetically released acetylcholine (ACh) from medial septal afferents activates muscarinic receptors on both vasoactive intestinal peptide-expressing (VIP) and parvalbumin-expressing (PV) basket cells (BCs) in mouse hippocampal CA1. ACh release depolarized VIP BCs whereas PV BCs depolarized, hyperpolarized or produced biphasic responses. Depolarizing responses in VIP or PV BCs resulted in increased amplitudes and frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons. The instantaneous frequency of sIPSCs that result from excitation of VIP or PV BCs primarily occurred within the low gamma frequency band (25-50 Hz). We investigated the effect of acetylcholine release on mouse hippocampal CA1 perisomatically projecting interneurons. Acetylcholine was optogenetically released in hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated virally mediated transfection. The effect of optogenetically released acetylcholine was assessed on interneurons expressing Cre recombinase in vasoactive intestinal peptide (VIP) or parvalbumin (PV) interneurons using whole cell patch clamp methods. Acetylcholine released onto VIP interneurons that innervate pyramidal neuron perisomatic regions (basket cells, BCs) were depolarized by muscarinic receptors. Although PV BCs were also excited by muscarinic receptor activation, they more frequently responded with hyperpolarizing or biphasic responses. Muscarinic receptor activation resulting from ACh release increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in downstream hippocampal CA1 pyramidal neurons with peak instantaneous frequencies occurring in both the gamma and theta bandwidths. Both PV and VIP BCs contributed to the increased sIPSC frequency in pyramidal neurons and optogenetic suppression of PV or VIP BCs inhibited sIPSCs occurring in the gamma range. Therefore, we propose acetylcholine release in CA1 has a complex effect on CA1 pyramidal neuron output through varying effects on perisomatically projecting interneurons.

Synaptic muscarinic response types in hippocampal CA1 interneurons depend on different levels of presynaptic activity and different muscarinic receptor subtypes.

Depolarizing, hyperpolarizing and biphasic muscarinic responses have been described in hippocampal inhibitory interneurons, but the receptor subtypes and activity patterns required to synaptically activate muscarinic responses in interneurons have not been completely characterized. Using optogenetics combined with whole cell patch clamp recordings in acute slices, we measured muscarinic responses produced by endogenously released acetylcholine (ACh) from cholinergic medial septum/diagonal bands of Broca inputs in hippocampal CA1. We found that depolarizing responses required more cholinergic terminal stimulation than hyperpolarizing ones. Furthermore, elevating extracellular ACh with the acetylcholinesterase inhibitor physostigmine had a larger effect on depolarizing versus hyperpolarizing responses. Another subpopulation of interneurons responded biphasically, and periodic release of ACh entrained some of these interneurons to rhythmically burst. M4 receptors mediated hyperpolarizing responses by activating inwardly rectifying K(+) channels, whereas the depolarizing responses were inhibited by the nonselective muscarinic antagonist atropine but were unaffected by M1, M4 or M5 receptor modulators. In addition, activation of M4 receptors significantly altered biphasic interneuron firing patterns. Anatomically, interneuron soma location appeared predictive of muscarinic response types but response types did not correlate with interneuron morphological subclasses. Together these observations suggest that the hippocampal CA1 interneuron network will be differentially affected by cholinergic input activity levels. Low levels of cholinergic activity will preferentially suppress some interneurons via hyperpolarization and increased activity will recruit other interneurons to depolarize, possibly because of elevated extracellular ACh concentrations. These data provide important information for understanding how cholinergic therapies will affect hippocampal network function in the treatment of some neurodegenerative diseases.

Optogenetic stimulation of the corticothalamic pathway affects relay cells and GABAergic neurons differently in the mouse visual thalamus.

The dorsal lateral geniculate nucleus (dLGN) serves as the primary conduit of retinal information to visual cortex. In addition to retinal input, dLGN receives a large feedback projection from layer VI of visual cortex. Such input modulates thalamic signal transmission in different ways that range from gain control to synchronizing network activity in a stimulus-specific manner. However, the mechanisms underlying such modulation have been difficult to study, in part because of the complex circuitry and diverse cell types this pathway innervates. To address this and overcome some of the technical limitations inherent in studying the corticothalamic (CT) pathway, we adopted a slice preparation in which we were able to stimulate CT terminal arbors in the visual thalamus of the mouse with blue light by using an adeno-associated virus to express the light-gated ion channel, ChIEF, in layer VI neurons. To examine the postsynaptic responses evoked by repetitive CT stimulation, we recorded from identified relay cells in dLGN, as well as GFP expressing GABAergic neurons in the thalamic reticular nucleus (TRN) and intrinsic interneurons of dLGN. Relay neurons exhibited large glutamatergic responses that continued to increase in amplitude with each successive stimulus pulse. While excitatory responses were apparent at postnatal day 10, the strong facilitation noted in adult was not observed until postnatal day 21. GABAergic neurons in TRN exhibited large initial excitatory responses that quickly plateaued during repetitive stimulation, indicating that the degree of facilitation was much larger for relay cells than for TRN neurons. The responses of intrinsic interneurons were smaller and took the form of a slow depolarization. These differences in the pattern of excitation for different thalamic cell types should help provide a framework for understanding how CT feedback alters the activity of visual thalamic circuitry during sensory processing as well as different behavioral or pathophysiological states.

Utility of ultrasound and MRI in prenatal diagnosis of placenta accreta: a pilot study.

OBJECTIVE: The purpose of this study was to evaluate transabdominal pelvic ultrasound and MRI for the prenatal diagnosis of placenta accreta. MATERIALS AND METHODS: A historical cohort pilot study was performed at our institution to identify women at risk of placenta accreta who had undergone both prenatal ultrasound and MRI. Findings at ultrasound and MRI were compared with the final diagnosis, which was established with clinical findings at delivery and pathologic examination of specimens. Volume measurements were made of low-signal-intensity intraplacental bands on T2-weighted MR images. Risk factors for placental insufficiency were recorded. RESULTS: Thirteen patients at risk of placenta accreta underwent both sonography and MRI. Nine of these patients had abnormal placentation. With ultrasound, abnormal placentation was correctly identified in six of nine patients (67%) and the absence of accreta in two of four patients (50%). With MRI, abnormal placentation was correctly identified in seven of nine patients (78%) and the absence of accreta in three of four patients (75%). The volumes of low-signal-intensity bands were significantly different in the patients with abnormal placentation and those without placenta accreta (p = 0.047), and band volumes were significantly different among patients with accreta, increta, and percreta (p < 0.0005). CONCLUSION: The accuracy of MRI may improve if volumes of low-signal-intensity bands are calculated, MRI is performed before 30 weeks' gestation, and risk factors for placental insufficiency are recognized.

Nicotinic excitatory postsynaptic potentials in hippocampal CA1 interneurons are predominantly mediated by nicotinic receptors that contain α4 and ß2 subunits.

In the hippocampus, activation of nicotinic receptors that include α4 and ß2 subunits (α4ß2*) facilitates memory formation. α4ß2* receptors may also play a role in nicotine withdrawal, and their loss may contribute to cognitive decline in aging and Alzheimer's disease (AD). However, little is known about their cellular function in the hippocampus. Therefore, using optogenetics, whole cell patch clamping and voltage-sensitive dye (VSD) imaging, we measured nicotinic excitatory postsynaptic potentials (EPSPs) in hippocampal CA1. In a subpopulation of inhibitory interneurons, release of ACh resulted in slow depolarizations (rise time constant 33.2 ± 6.5 ms, decay time constant 138.6 ± 27.2 ms) mediated by the activation of α4ß2* nicotinic receptors. These interneurons had somata and dendrites located in the stratum oriens (SO) and stratum lacunosum-moleculare (SLM). Furthermore, α4ß2* nicotinic EPSPs were largest in the SLM. Thus, our data suggest that nicotinic EPSPs in hippocampal CA1 interneurons are predominantly mediated by α4ß2* nicotinic receptors and their activation may preferentially affect extrahippocampal inputs in SLM of hippocampal CA1.

MAPK recruitment by beta-amyloid in organotypic hippocampal slice cultures depends on physical state and exposure time.

Elevated beta-amyloid is thought to trigger the onset of Alzheimer's disease. Alzheimer's disease is marked by progressive loss of cognitive function, an early symptom of which is episodic memory deficits. Impairment of episodic memory is linked to hippocampal pathology. We investigated the signal transduction consequences of exposure to nanomolar to low micromolar concentrations of aggregate forms of beta-amyloid in the hippocampus. We found that, in addition to activation of ERK MAPK and its downstream target ribosomal S6 kinase in hippocampal slice cultures following acute exposure to oligomeric beta-amyloid(1-42), ERK activation also requires phosphoinositide-3 kinase activity. These effects were contingent on the alpha7 subtype of nicotinic acetylcholine receptor. Hippocampal slice cultures treated acutely with oligomeric beta-amyloid(1-42) did not exhibit JNK MAPK activation; however, chronic exposure to oligomers or high molecular weight aggregates of beta-amyloid(1-42) led to JNK MAPK activation coincident with ERK MAPK down-regulation. In contrast to the effects of acute application of oligomeric beta-amyloid(1-42), nicotine activated ERK MAPK via alpha7 nicotinic acetylcholine receptors utilizing protein kinase A as an intermediate. In conclusion, we found that both the physical state and duration of exposure to beta-amyloid are determinants of MAPK recruitment in hippocampus. We also found that nicotine and beta-amyloid activate ERK MAPK via alpha7 nicotinic acetylcholine receptors but use distinct intermediate kinases. These data indicate the existence of differential coupling of alpha7 to downstream targets depending on the type of ligand that leads to receptor activation.

beta -Amyloid peptide activates alpha 7 nicotinic acetylcholine receptors expressed in Xenopus oocytes.

The alpha7 nicotinic acetylcholine receptor is highly expressed in hippocampus and in cholinergic projection neurons from the basal forebrain, structures that are particularly vulnerable to the ravages of Alzheimer's disease. Previous work suggests that beta-amyloid peptide can interact with alpha7 nicotinic acetylcholine receptors, although the nature of this interaction has not been well characterized. To test whether beta-amyloid peptide can activate alpha7 nicotinic acetylcholine receptors, we expressed these receptors in Xenopus oocytes and performed two-electrode voltage clamp recordings, characterizing the response to beta-amyloid peptide 1-42 applied at concentrations ranging from 1 pm to 100 nm. In alpha7-expressing oocytes, beta-amyloid peptide 1-42 elicits inward currents at low concentrations (1-100 pm), whereas at higher concentrations (nm), less effective receptor activation is observed, indicative of receptor desensitization. Preincubation with the alpha7-selective agents, the antagonist methyllycaconatine, and the agonist 4-OH-GTS-21 blocked beta-amyloid peptide-induced receptor activation. beta-amyloid peptide 1-42 at low concentrations was able to activate the L250T mutant alpha7 receptor. The endogenous Ca(2+)-activated chloride current in Xenopus oocytes is recruited upon receptor activation since replacing Ca(2+) with Ba(2+) in the recording solution reduced current amplitude. Thus, when beta-amyloid peptide activation of alpha7 receptors occurs, these currents are comprised, at least in part, of Ca(2+).

Salpingitis, salpingoliths, and serous tumors of the ovaries: is there a connection?

We have observed luminal and mucosal calcifications frequently surrounded by a mantle of bland epithelium in the fallopian tubes ("salpingoliths") of women with serous tumors of the ovaries. These lesions resemble noninvasive peritoneal "implants" in women with advanced stage atypical proliferative serous tumors (APSTs) and micropapillary serous carcinomas (MPSCs). The presence of salpingitis and salpingoliths was prospectively evaluated in 358 women with a variety of nonneoplastic and neoplastic ovarian conditions and compared with 87 previously reported women with APSTs/MPSCs in an effort to determine whether these lesions were specifically associated with serous tumors. The frequency of chronic salpingitis among women without ovarian pathology was 27%, and the frequency of salpingoliths was 4%. Serous epithelial tumors (cystadenomas, APST/MPSC, and carcinomas) were significantly more often associated with chronic salpingitis (53%) and salpingoliths (32%) than all other cases with or without ovarian neoplasms (p<0.01). APSTs/MPSCs were associated with salpingoliths significantly more frequently than all other groups (p<0.001). For patients with APSTs/MPSCs, salpingoliths were found significantly more often in advanced stage (FIGO II and III) patients (51%) than stage I patients (24%) (p<0.01), but salpingitis, present in 60% of these patients, was not stage-dependent (p>0.05). Chronic salpingitis was identified in 66% of women with endometriosis, which was significantly more frequent than those with normal ovaries (27%) (p<0.001). In conclusion, fallopian tube abnormalities may be related to both the high frequency of infertility and the noninvasive peritoneal implants in women with APSTs/MPSCs. Whether the fallopian tubes with salpingoliths are the source of the peritoneal "implants," the recipient of implants, or are independent is unknown. In addition, the high frequency of salpingitis in women with endometriosis may be related to the mechanism of endometriosis-associated infertility.