Adult-onset depletion of sulfatide leads to axonal degeneration with relative myelin sparing.

3-O-sulfogalactosylceramide (sulfatide) constitutes a class of sphingolipids that comprise about 4% of myelin lipids in the central nervous system. Previously, our group characterized a mouse with sulfatide's synthesizing enzyme, cerebroside sulfotransferase (CST), constitutively disrupted. Using these mice, we demonstrated that sulfatide is required for establishment and maintenance of myelin, axoglial junctions, and axonal domains and that sulfatide depletion results in structural pathologies commonly observed in Multiple Sclerosis (MS). Interestingly, sulfatide is reduced in regions of normal appearing white matter (NAWM) of MS patients. Sulfatide reduction in NAWM suggests depletion occurs early in disease development and consistent with functioning as a driving force of disease progression. To closely model MS, an adult-onset disease, our lab generated a "floxed" CST mouse and mated it against the PLP-creERT mouse, resulting in a double transgenic mouse that provides temporal and cell-type specific ablation of the Cst gene (Gal3st1). Using this mouse, we demonstrate adult-onset sulfatide depletion has limited effects on myelin structure but results in the loss of axonal integrity including deterioration of domain organization accompanied by axonal degeneration. Moreover, structurally preserved myelinated axons progressively lose the ability to function as myelinated axons, indicated by the loss of the N1 peak. Together, our findings indicate that sulfatide depletion, which occurs in the early stages of MS progression, is sufficient to drive the loss of axonal function independent of demyelination and that axonal pathology, which is responsible for the irreversible loss of neuronal function that is prevalent in MS, may occur earlier than previously recognized.

Chronic adolescent stress causes sustained impairment of cognitive flexibility and hippocampal synaptic strength in female rats.

Females that experience chronic stress during development, particularly adolescence, are the most vulnerable group to stress-induced disease. While considerable attention has been devoted to stress-induced manifestation of anxiety, depression, and PTSD, evidence indicates that a history of chronic stress is also a risk factor for cognitive decline and dementia - with females again in a higher risk group. This interplay between sex and stress history indicates specific mechanisms drive neural dysfunction across the lifespan. The presence of sex and stress steroid receptors in the hippocampus provides a point of influence for these variables to drive changes in cognitive function. Here, we used a rodent model of chronic adolescent stress (CAS) to determine the extent to which CAS modifies glutamatergic signaling resulting in cognitive dysfunction. Male and female Wistar rats born in-house remained non-stressed (NS), unmanipulated aside from standard cage cleaning, or were exposed to either physical restraint (60 min) or social defeat (CAS) each day (6 trials each), along with social isolation, throughout the adolescent period (PND 35-47). Cognition was assessed in adult (PND 80-130) male and female rats (n = 10-12) using the Barnes Maze task and the Attention Set-Shift task. Whole hippocampi were extracted from a second cohort of male and female rats (NS and CAS; n = 9-10) and processed for RNA sequencing. Brain tissue from the first cohort (n = 6) was processed for density of glutamatergic synaptic markers (GluA1, NMDA1a, and synaptophysin) or whole-cell patch clamping (n = 4) to determine glutamatergic activity in the hippocampus. Females with a history of chronic stress had shorter latencies to locate the goal box than NS controls during acquisition learning but showed an increased latency to locate the new goal box during reversal learning. This reversal deficit persisted across domains as females with a history of stress required more trials to reach criterion during the reversal phases of the Attention Set-Shift task compared to controls. Ovariectomy resulted in greater performance variability overall during reversal learning with CAS females showing worse performance. Males showed no effects of CAS history on learning or memory performance. Bioinformatic prediction using gene ontology categorization indicated that in females, postsynaptic membrane gene clusters, specifically genes related to glutamatergic synapse remodeling, were enriched with a history of stress. Structural analysis indicated that CAS did not alter glutamate receptor density in females. However, functionally, CAS females had a decreased AMPA/NMDA-dependent current ratio compared to controls indicating a weakening in synaptic strength in the hippocampus. Males showed only a slight change in density of NMDA1a labeling in the CA3 region with a history of stress. The data observed here suggest that females are at risk for impaired cognitive flexibility following a history of adolescent stress, possibly driven by changes in glutamatergic signaling.

Heterologous expression of the invertebrate FMRFamide-gated sodium channel as a mechanism to selectively activate mammalian neurons.

Considerable effort has been directed toward the development of methods to selectively activate specific subtypes of neurons. Focus has been placed on the heterologous expression of proteins that are capable of exciting neurons in which they are expressed. Here we describe the heterologous expression of the invertebrate FMRFamide (H-phenylalanine-methionine-arginine-phenylalanine-NH2) -gated sodium channel from Helix aspersa (HaFaNaC) in hippocampal slice cultures. HaFaNaC was co-expressed with a fluorescent protein (green fluorescent protein (GFP), red fluorescent protein from Discosoma sp (dsRed) or mutated form of red fluorescent protein from Discosoma sp (tdTomato)) in CA3 pyramidal neurons of rat hippocampal slice cultures using single cell electroporation. Pressure application of the agonist FMRFamide to HaFaNaC-expressing neuronal somata produced large prolonged depolarizations and bursts of action potentials (APs). FMRFamide responses were inhibited by amiloride (100 microM). In contrast, pressure application of FMRFamide to the axons of neurons expressing HaFaNaC produced no response. Fusion of GFP to the N-terminus of HaFaNaC showed that GFP-HaFaNaC was absent from axons. Bath application of FMRFamide produced persistent AP firing in HaFaNaC-expressing neurons. This FMRFamide-induced increase in the frequency of APs was dose-dependent. The concentrations of FMRFamide required to activate HaFaNaC-expressing neurons were below that required to activate the homologous acid sensing ion channel normally found in mammalian neurons. Furthermore, the mammalian neuropeptides neuropeptide FF and RFamide-related peptide-1, which have amidated RF C-termini, did not affect HaFaNaC-expressing neurons. Antagonists of NPFF receptors (BIBP3226) also had no effect on HaFaNaC. Therefore, we suggest that heterologous-expression of HaFaNaC in mammalian neurons could be a useful method to selectively and persistently excite specific subtypes of neurons in intact nervous tissue.

Layer selective presynaptic modulation of excitatory inputs to hippocampal cornu Ammon 1 by mu-opioid receptor activation.

Chronic and acute activation of mu-opioid receptors (MOR) in hippocampal cornu Ammon 1 (CA1) disrupts rhythmic activity, alters activity-dependent synaptic plasticity and impairs spatial memory formation. In CA1, MORs act by hyperpolarizing inhibitory interneurons and suppressing inhibitory synaptic transmission. MOR modulation of inhibitory synaptic function translates into an increase in excitatory activity in all layers of CA1. However, the exact anatomical sites for MOR actions are not completely known. Therefore, we used voltage-sensitive dye imaging, whole cell patch clamping, photolysis of alpha-carboxy-2-nitrobenzyl ester, trifluoroacetic acid salt (CNB) -caged GABA, and micro-sectioned slices of rat hippocampus to investigate the effect of MOR activation in CA1. First, we investigated the effect of MOR activation using a MOR agonist [d-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO) on the direct activation of GABA receptors by photolysis of CNB-caged GABA in all layers of CA1. MOR activation did not affect hyperpolarizations due to direct GABA receptor activation in any layer of CA1, but MOR activation did suppress GABAergic inhibitory postsynaptic potentials suggesting that MOR activation acts by presynaptically inhibiting interneuron function. We next examined whether MOR activation was equivalently effective in all anatomical layers of CA1. To do this, cuts were made between anatomical layers of CA1 and isolated layers were stimulated electrically (five pulses at 20 Hz) to produce excitatory postsynaptic potentials (EPSPs). Under these conditions, MOR activation significantly increased EPSP areas in stratum radiatum (SR), stratum pyramidale (SP) and stratum oriens (SO) relative to stratum lacunosum-moleculare (SLM). When compared with the effect of GABA(A) and GABA(B) receptor antagonists on EPSP areas, the effect of DAMGO was proportionately larger in SR, SP and SO than in SLM. We conclude that MOR activation is more effective at directly modulating activity in SR, SP and SO, and the smaller effect in SLM is likely due to a smaller MOR inhibition of GABA release in SLM.

Electrophysiology of interneurons in the glomerular layer of the rat olfactory bulb.

In the mammalian olfactory bulb, glomeruli are surrounded by a heterogeneous population of interneurons called juxtaglomerular neurons. As they receive direct input from olfactory receptor neurons and connect with mitral cells, they are involved in the initial stages of olfactory information processing, but little is known about their detailed physiological properties. Using whole cell patch-clamp techniques, we recorded from juxtaglomerular neurons in rat olfactory bulb slices. Based on their response to depolarizing pulses, juxtaglomerular neurons could be divided into two physiological classes: bursting and standard firing. When depolarized, the standard firing neurons exhibited a range of responses: accommodating, nonaccommodating, irregular firing, and delayed to firing patterns of action potentials. Although the firing pattern was not rigorously predictive of a particular neuronal morphology, most short axon cells fired accommodating trains of action potentials, while most delayed to firing cells were external tufted cells. In contrast to the standard firing neurons, bursting neurons produced a calcium-channel-dependent low-threshold spike when depolarized either by current injection or by spontaneous or evoked postsynaptic potentials. Bursting neurons also could oscillate spontaneously. Most bursting cells were either periglomerular cells or external tufted cells. Based on their mode of firing and placement in the bulb circuit, these bursting cells are well situated to drive synchronous oscillations in the olfactory bulb.

Muscarinic receptor activity has multiple effects on the resting membrane potentials of CA1 hippocampal interneurons.

Inhibitory interneurons appear to be an important target for the muscarinic actions of cholinergic inputs to the hippocampus. We investigated the effect of muscarinic receptor activity on the membrane potential (V(m)) and currents of rat hippocampal CA1 interneurons using whole-cell recording from visually identified CA1 interneurons. The predominant response observed was a muscarinic depolarization that was detected in interneurons from all layers of CA1. This depolarization was mediated by at least two mechanisms: a reduction in a potassium current and a mechanism that depended on extracellular sodium. Other interneurons responded to muscarinic agonists with a hyperpolarization or a biphasic response (hyperpolarization followed by depolarization). Hyperpolarizations and biphasic responses were found in all layers of CA1 but more frequently in stratum radiatum and stratum lacunosum moleculare. Muscarinic hyperpolarization was caused by the activation of a barium- and cesium-sensitive inwardly rectifying potassium channel. A small number of interneurons, primarily in or bordering the stratum pyramidale, produced slow membrane potential (0.04 Hz) oscillations. Many interneurons did not respond to muscarinic activity at all; half of these were in the stratum oriens. There was no strong correlation between any changes in V(m) response to muscarine and morphology, as determined by reconstruction of the interneurons. It was not possible to predict the morphology or the layer distribution of an interneuron based on the type of muscarinic membrane potential response it had. This lack of correlation between muscarinic function and morphology implies a greater complexity of interneuron function than has been realized previously.

Muscarinic receptor activity induces an afterdepolarization in a subpopulation of hippocampal CA1 interneurons.

Cholinergic input to the hippocampus may be involved in important behavioral functions and the pathophysiology of neurodegenerative diseases. Muscarinic receptor activity in interneurons of the hippocampus may play a role in these actions. In this study, we investigated the effects of muscarinic receptor activity on the excitability of different subtypes of interneurons in rat hippocampal CA1. Most interneurons displayed an afterhyperpolarizing potential (AHP) after depolarization by injected current or synaptic stimulation. In the presence of a muscarinic agonist, the AHP of a subset of these interneurons was replaced by an afterdepolarization (ADP), often of sufficient magnitude to evoke action potentials in the absence of further stimulation. The ADP was insensitive to cadmium and low extracellular calcium. It was blocked by low extracellular sodium but not by tetrodotoxin or low concentrations of amiloride. Muscarinic ADPs were sometimes observed in isolation but were often accompanied by depolarizing, hyperpolarizing, or biphasic changes in the membrane potential. Interneurons with muscarinic ADPs were found in all strata of CA1 and did not fall into a single morphological classification. The potential functions of the prolonged action potential output of interneurons produced by the ADP could include changes in hippocampal circuit properties and facilitation of the release of peptide cotransmitters in these interneurons.

Nicotinic receptor activation excites distinct subtypes of interneurons in the rat hippocampus.

We examined the function of nicotinic acetylcholine receptors (nAChRs) in interneurons of area CA1 of the rat hippocampus. CA1 interneurons could be classified into three categories based on nicotinic responses. The first class was depolarized by alpha7 nAChRs, found in all layers of CA1 and as a group, had axonal projections to all neuropil layers of CA1. The second class had both fast alpha7 and slow non-alpha7 nAChR depolarizing responses, was localized primarily to the stratum oriens, and had axonal projections to the stratum lacunosum-moleculare. The third group had no nicotinic response. This group was found in or near the stratum pyramidale and had axonal projections almost exclusively within and around this layer. Low concentrations (500 nM) of nicotine desensitized fast and slow nAChR responses. These findings demonstrate that there are distinct subsets of interneurons with regard to nicotinic receptor expression and with predictable morphological properties that suggest potential cellular actions for nicotinic receptor activation in normal CNS function and during nicotine abuse.

Neuropeptide Y2 receptors inhibit the frequency of spontaneous but not miniature EPSCs in CA3 pyramidal cells of rat hippocampus.

1. Neuropeptide Y (NPY) inhibits synaptic excitation in hippocampal area CA3. We studied its site of action with the use of whole cell patch-clamp recordings from CA3 pyramidal cells of rat hippocampal slices in vitro. 2. Spontaneous excitatory postsynaptic currents (sEPSCs) were isolated with picrotoxin, to block gamma-aminobutyric acid-A receptors, whereas miniature excitatory postsynaptic currents (mEPSCs) were isolated by additionally treating the slice with tetrodotoxin (TTX) and/or Cd2+, sEPSCs and mEPSCs were eliminated by the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and DL-2-amino-5-phosphonovaleric acid (50 microM), and were thus solely attributable to glutamate release. 3. The interval and amplitude distributions of sEPSCS and (TTX-isolated) mEPSCS were analyzed. Either NPY or the rapidly reversible, Y2-receptor-selective agonist [6-aminohexanoic5-24] NPY, ([ahx5-24]NPY) sharply increased the inter-sEPSC intervals in 16 of 16 neurons tested. In 11 of these cells, these agonists also simultaneously shifted the sEPSC amplitude distribution to somewhat smaller amplitudes, whereas in the remaining 5 cells, no concurrent effect on amplitudes was observed. By contrast, in 15 separate neurons treated with 1 microM TTX, neither NPY nor [ahx5-24]NPY altered either mEPSC amplitude or interval distributions of the mEPSCs. 4. To directly compare the effects of Y2 receptor activation on sEPSC and mEPSC properties, we applied [ahx5-24]NPY to the same cell in the absence and presence of TTX (n = 7). sEPSC intervals were characteristically increased by the Y2 agonist in all cells; in six of seven cells the sEPSC distribution was also shifted to smaller amplitudes. TTX application reduced the mean amplitude of the synaptic events more than did [ahx5-24]NPY, while increasing their intervals. [ahx5-24]NPY had no effect in TTX. 5. NPY, acting on a Y2 receptor, inhibits impulse-dependent synaptic excitation of CA3 pyramidal cells of the rat hippocampus by an entirely presynaptic action.

Neuropeptide Y1 receptors inhibit N-type calcium currents and reduce transient calcium increases in rat dentate granule cells.

Neuropeptide Y (NPY) is far more abundant in the dentate gyrus than elsewhere in the hippocampal formation, but it does not alter the synaptic excitation of dentate granule cells (DGCs) as it does for pyramidal cells in areas CA1 and CA3. NPY inhibited depolarization-induced increases in intracellular Ca2+ concentrations ([Ca2+]i) in DGCs in hippocampal slices, without altering the resting [Ca2+]i. NPY inhibited Ca2+ currents (ICa) via a Y1 receptor in 84% of acutely isolated DGCs and via a Y2 receptor in 31% of the NPY-responsive cells tested. ICa inhibition was completely occluded by omega-conotoxin-GVIA but not by nimodipine. The inhibition of ICa was accompanied by a change in the time course of ICa activation in only 27% of NPY-responsive cells. Only 23% of DGCs responded to NPY when Ba2+ was substituted for extracellular Ca2+ and when [Ca2+]i was strongly buffered. Therefore, NPY inhibits an N-type ICa in DGCs, mainly via Y1 receptors. Furthermore, it seems that more than one mechanism, one of which may be sensitive to [Ca2+]i, may couple NPY receptors to the Ca2+ channels in DGCs. Because the release of dynorphin from DGCs depends in part on N-type currents, NPY receptors are poised to regulate the release of opioid peptides from DGC somata and dendrites.

Neuropeptide Y does not alter NMDA conductances in CA3 pyramidal neurons: a slice-patch study.

Neuropeptide Y (NPY) has been reported to inhibit excitatory neurotransmission in hippocampus presynaptically. Recently, it has been suggested that NPY also potentiates N-methyl-D-aspartate (NMDA)-mediated excitatory responses in hippocampus, by action at a sigma or phencyclidine (PCP) binding site. We tested this hypothesis by examining the action of NPY on CA3 pyramidal cells using slice-patch voltage clamp recordings. NPY did not affect inward currents elicited by iontophoresis of NMDA onto the proximal dendrites of these cells under two different conditions, but did reduce the excitatory postsynaptic currents elicited by mossy fiber stimulation. NPY therefore does not appear to directly alter the postsynaptic NMDA response in CA3 cells.