Chronic Morphine Induces IL-18 in Ileum Myenteric Plexus Neurons Through Mu-opioid Receptor Activation in Cholinergic and VIPergic Neurons.

The gastrointestinal epithelium is critical for maintaining a symbiotic relationship with commensal microbiota. Chronic morphine exposure can compromise the gut epithelial barrier in mice and lead to dysbiosis. Recently, studies have implicated morphine-induced dysbiosis in the mechanism of antinociceptive tolerance and reward, suggesting the presence of a gut-brain axis in the pharmacological effects of morphine. However, the mechanism(s) underlying morphine-induced changes in the gut microbiome remains unclear. The pro-inflammatory cytokine, Interleukin-18 (IL-18), released by enteric neurons can modulate gut barrier function. Therefore, in the present study we investigated the effect of morphine on IL-18 expression in the mouse ileum. We observed that chronic morphine exposure in vivo induces IL-18 expression in the ileum myenteric plexus that is attenuated by naloxone. Given that mu-opioid receptors (MORs) are mainly expressed in enteric neurons, we also characterized morphine effects on the excitability of cholinergic (excitatory) and vasoactive intestinal peptide (VIP)-expressing (inhibitory) myenteric neurons. We found fundamental differences in the electrical properties of cholinergic and VIP neurons such that VIP neurons are more excitable than cholinergic neurons. Furthermore, MORs were primarily expressed in cholinergic neurons, although a subset of VIP neurons also expressed MORs and responded to morphine in electrophysiology experiments. In conclusion, these data show that morphine increases IL-18 in ileum myenteric plexus neurons via activation of MORs in a subset of cholinergic and VIP neurons. Thus, understanding the neurochemistry and electrophysiology of MOR-expressing enteric neurons can help to delineate mechanisms by which morphine perturbs the gut barrier.

Effects of subanesthetic ketamine and (2R,6R) hydroxynorketamine on working memory and synaptic transmission in the nucleus reuniens in mice.

RATIONALE: Acute cognitive impairment and abuse potential of ketamine incentivizes the search for alternatives to ketamine for clinical management of treatment-resistant depression. Recently, (2R,6R) hydroxynorketamine ((2R,6R)-HNK), a metabolite of ketamine, has shown promise due to its reported lack of ketamine-like reinforcing properties. Nonetheless, the effect of (2R,6R)-HNK on cognition has not been reported. METHOD: Adult male mice were placed in a Y-maze to measure spatial working memory (SWM) 24 h after treatment with either a single or repeated subanesthetic dose of (2R,6R)-HNK or ketamine. To determine the effect of the drug regimens on synaptic mechanisms in neural circuits deemed critical for SWM, we conducted patch-clamp electrophysiological recordings from neurons in the midline thalamic nucleus reuniens (RE) in response to optogenetic stimulation of medial prefrontal cortex (mPFC) inputs in acutely prepared brain slices. RESULTS: Single or repeated treatment with a 10 mg/kg dose of either drug did not impact performance in a Y-maze. However, single administration of a ½-log higher dose (32 mg/kg) of ketamine significantly reduced SWM. The same dose of (2R,6R)-HNK did not produce SWM deficits. Interestingly, repeated administration of either drugs at the 32 mg/kg had no effect on SWM performances. Concomitant to these effects on SWM, only single injection of 32 mg/kg of ketamine was found to increase the mPFC-driven action potential firing activity in the RE neurons. Conversely, both single and repeated administration of the 32 mg/kg dose of (2R,6R)-HNK but not ketamine, increased the input resistance of the RE neurons. CONCLUSION: Our results indicate that acute treatment of ketamine at 32 mg/kg increases mPFC-driven firing activity of RE neurons, and this contributes to the ketamine-mediated cognitive deficit. Secondly, sub-chronic treatment with the same dose of ketamine likely induces tolerance. Although single or repeated administration of the 32 mg/kg dose of (2R,6R)-HNK can alter intrinsic properties of RE neurons, this dose does not produce cognitive deficit or changes in synaptic mechanism in the RE. This article is part of the special Issue on 'Stress, Addiction and Plasticity'.

Chloride channels with ClC-1-like properties differentially regulate the excitability of dopamine receptor D1- and D2-expressing striatal medium spiny neurons.

Dynamic chloride (Cl-) regulation is critical for synaptic inhibition. In mature neurons, Cl- influx and extrusion are primarily controlled by ligand-gated anion channels (GABAA and glycine receptors) and the potassium chloride cotransporter K+-Cl- cotransporter 2 (KCC2), respectively. Here, we report for the first time, to our knowledge, a presence of a new source of Cl- influx in striatal neurons with properties similar to chloride voltage-gated channel 1 (ClC-1). Using whole cell patch-clamp recordings, we detected an outwardly rectifying voltage-dependent current that was impermeable to the large anion methanesulfonate (MsO-). The anionic current was sensitive to the ClC-1 inhibitor 9-anthracenecarboxylic acid (9-AC) and the nonspecific blocker phloretin. The mean fractions of anionic current inhibition by MsO-, 9-AC, and phloretin were not significantly different, indicating that anionic current was caused by active ClC-1-like channels. In addition, we found that Cl- current was not sensitive to the transmembrane protein 16A (TMEM16A; Ano1) inhibitor Ani9 and that the outward Cl- rectification was preserved even at a very high intracellular Ca2+ concentration (2 mM), indicating that TMEM16B (Ano2) did not contribute to the total current. Western blotting and immunohistochemical analyses confirmed the presence of ClC-1 channels in the striatum mainly localized to the somata of striatal neurons. Finally, we found that 9-AC decreased action potential firing frequencies and increased excitability in medium spiny neurons (MSNs) expressing dopamine type 1 (D1) and type 2 (D2) receptors in the brain slices, respectively. We conclude that ClC-1-like channels are preferentially located at the somata of MSNs, are functional, and can modulate neuronal excitability.

Nucleus Reuniens Afferents in Hippocampus Modulate CA1 Network Function via Monosynaptic Excitation and Polysynaptic Inhibition.

The thalamic midline nucleus reuniens modulates hippocampal CA1 and subiculum function via dense projections to the stratum lacunosum-moleculare (SLM). Previously, anatomical data has shown that reuniens inputs in the SLM form synapses with dendrites of both CA1 principal cells and inhibitory interneurons. However, the ability of thalamic inputs to excite the CA1 principal cells remains controversial. In addition, nothing is known about the impact of reuniens inputs on diverse subpopulations of interneurons in CA1. Therefore, using whole cell patch-clamp electrophysiology in ex vivo hippocampal slices of wild-type and transgenic mice, we measured synaptic responses in different CA1 neuronal subtypes to optogenetic stimulation of reuniens afferents. Our data shows that reuniens inputs mediate both excitation and inhibition of the CA1 principal cells. However, the optogenetic excitation of the reuniens inputs failed to drive action potential firing in the majority of the principal cells. While the excitatory postsynaptic currents were mediated via direct monosynaptic activation of the CA1 principal cells, the inhibitory postsynaptic currents were generated polysynaptically via activation of local GABAergic interneurons. Moreover, we demonstrate that optogenetic stimulation of reuniens inputs differentially recruit at least two distinct and non-overlapping subpopulations of local GABAergic interneurons in CA1. We show that neurogliaform cells located in SLM, and calretinin-containing interneuron-selective interneurons at the SLM/stratum radiatum border can be excited by stimulation of reuniens inputs. Together, our data demonstrate that optogenetic stimulation of reuniens afferents can mediate excitation, feedforward inhibition, and disinhibition of the postsynaptic CA1 principal cells via multiple direct and indirect mechanisms.

HIV-1 Tat and Morphine Differentially Disrupt Pyramidal Cell Structure and Function and Spatial Learning in Hippocampal Area CA1: Continuous versus Interrupted Morphine Exposure.

About half the people infected with human immunodeficiency virus (HIV) have neurocognitive deficits that often include memory impairment and hippocampal deficits, which can be exacerbated by opioid abuse. To explore the effects of opioids and HIV on hippocampal CA1 pyramidal neuron structure and function, we induced HIV-1 transactivator of transcription (Tat) expression in transgenic mice for 14 d and co-administered time-release morphine or vehicle subcutaneous implants during the final 5 d (days 9-14) to establish steady-state morphine levels. Morphine was withheld from some ex vivo slices during recordings to begin to assess the initial pharmacokinetic consequences of opioid withdrawal. Tat expression reduced hippocampal CA1 pyramidal neuronal excitability at lower stimulating currents. Pyramidal cell firing rates were unaffected by continuous morphine exposure. Behaviorally, exposure to Tat or high dosages of morphine impaired spatial memory Exposure to Tat and steady-state levels of morphine appeared to have largely independent effects on pyramidal neuron structure and function, a response that is distinct from other vulnerable brain regions such as the striatum. By contrast, acutely withholding morphine (from morphine-tolerant ex vivo slices) revealed unique and selective neuroadaptive shifts in CA1 pyramidal neuronal excitability and dendritic plasticity, including some interactions with Tat. Collectively, the results show that opioid-HIV interactions in hippocampal area CA1 are more nuanced than previously assumed, and appear to vary depending on the outcome assessed and on the pharmacokinetics of morphine exposure.

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.

HIV and opiates dysregulate K+- Cl- cotransporter 2 (KCC2) to cause GABAergic dysfunction in primary human neurons and Tat-transgenic mice.

Approximately half of people infected with HIV (PWH) exhibit HIV-associated neuropathology (neuroHIV), even when receiving combined antiretroviral therapy. Opiate use is widespread in PWH and exacerbates neuroHIV. While neurons themselves are not infected, they incur sublethal damage and GABAergic disruption is selectively vulnerable to viral and inflammatory factors released by infected/affected glia. Here, we demonstrate diminished K+-Cl- cotransporter 2 (KCC2) levels in primary human neurons after exposure to HIV-1 or HIV-1 proteins ± morphine. Resulting disruption of GABAAR-mediated hyperpolarization/inhibition was shown using genetically-encoded voltage (Archon1) and calcium (GCaMP6f) indicators. The HIV proteins Tat (acting through NMDA receptors) and R5-gp120 (acting via CCR5) but not X4-tropic gp120 (acting via CXCR4), and morphine (acting through µ-opioid receptors) all induced KCC2 loss. We demonstrate that modifying KCC2 levels or function, or antagonizing NMDAR, CCR5 or MOR rescues KCC2 and GABAAR-mediated hyperpolarization/inhibition in HIV, Tat, or gp120 ± morphine-exposed neurons. Using an inducible, Tat-transgenic mouse neuroHIV model, we found that chronic exposure to Tat also reduces KCC2. Our results identify KCC2 as a novel therapeutic target for ameliorating the pathobiology of neuroHIV, including PWH exposed to opiates.

Acetylcholine Release Inhibits Distinct Excitatory Inputs Onto Hippocampal CA1 Pyramidal Neurons via Different Cellular and Network Mechanisms.

In hippocampal CA1, muscarinic acetylcholine (ACh) receptor (mAChR) activation via exogenous application of cholinergic agonists has been shown to presynaptically inhibit Schaffer collateral (SC) glutamatergic inputs in stratum radiatum (SR), and temporoammonic (TA) and thalamic nucleus reuniens (RE) glutamatergic inputs in stratum lacunosum-moleculare (SLM). However, steady-state uniform mAChR activation may not mimic the effect of ACh release in an intact hippocampal network. To more accurately examine the effect of ACh release on glutamatergic synaptic efficacy, we measured electrically evoked synaptic responses in CA1 pyramidal cells (PCs) following the optogenetic release of ACh in genetically modified mouse brain slices. The ratio of synaptic amplitudes in response to paired-pulse SR stimulation (stimulus 2/stimulus 1) was significantly reduced by the optogenetic release of ACh, consistent with a postsynaptic decrease in synaptic efficacy. The effect of ACh release was blocked by the M3 receptor antagonist 4-DAMP, the GABAB receptor antagonist CGP 52432, inclusion of GDP-ß-S, cesium, QX314 in the intracellular patch clamp solution, or extracellular barium. These observations suggest that ACh release decreased SC synaptic transmission through an M3 muscarinic receptor-mediated increase in inhibitory interneuron excitability, which activate GABAB receptors and inwardly rectifying potassium channels on CA1 pyramidal cells. In contrast, the ratio of synaptic amplitudes in response to paired-pulse stimulation in the SLM was increased by ACh release, consistent with presynaptic inhibition. ACh-mediated effects in SLM were blocked by the M2 receptor antagonist AF-DX 116, presumably located on presynaptic terminals. Therefore, our data indicate that ACh release differentially modulates excitatory inputs in SR and SLM of CA1 through different cellular and network mechanisms.

Selective Vulnerability of Striatal D2 versus D1 Dopamine Receptor-Expressing Medium Spiny Neurons in HIV-1 Tat Transgenic Male Mice.

Despite marked regional differences in HIV susceptibility within the CNS, there has been surprisingly little exploration into the differential vulnerability among neuron types and the circuits they underlie. The dorsal striatum is especially susceptible, harboring high viral loads and displaying marked neuropathology, with motor impairment a frequent manifestation of chronic infection. However, little is known about the response of individual striatal neuron types to HIV or how this disrupts function. Therefore, we investigated the morphological and electrophysiological effects of HIV-1 trans-activator of transcription (Tat) in dopamine subtype 1 (D1) and dopamine subtype 2 (D2) receptor-expressing striatal medium spiny neurons (MSNs) by breeding transgenic Tat-expressing mice to Drd1a-tdTomato- or Drd2-eGFP-reporter mice. An additional goal was to examine neuronal vulnerability early during the degenerative process to gain insight into key events underlying the neuropathogenesis. In D2 MSNs, exposure to HIV-1 Tat reduced dendritic spine density significantly, increased dendritic damage (characterized by swellings/varicosities), and dysregulated neuronal excitability (decreased firing at 200-300 pA and increased firing rates at 450 pA), whereas insignificant morphologic and electrophysiological consequences were observed in Tat-exposed D1 MSNs. These changes were concomitant with an increased anxiety-like behavioral profile (lower latencies to enter a dark chamber in a light-dark transition task, a greater frequency of light-dark transitions, and reduced rearing time in an open field), whereas locomotor behavior was unaffected by 2 weeks of Tat induction. Our findings suggest that D2 MSNs and a specific subset of neural circuits within the dorsal striatum are preferentially vulnerable to HIV-1.SIGNIFICANCE STATEMENT Despite combination antiretroviral therapy (cART), neurocognitive disorders afflict 30-50% of HIV-infected individuals and synaptodendritic injury remains evident in specific brain regions such as the dorsal striatum. A possible explanation for the sustained neuronal injury is that the neurotoxic HIV-1 regulatory protein trans-activator of transcription (Tat) continues to be expressed in virally suppressed patients on cART. Using inducible Tat-expressing transgenic mice, we found that dopamine subtype 2 (D2) receptor-expressing medium spiny neurons (MSNs) are selectively vulnerable to Tat exposure compared with D1 receptor-expressing MSNs. This includes Tat-induced reductions in D2 MSN dendritic spine density, increased dendritic damage, and disruptions in neuronal excitability, which coincide with elevated anxiety-like behavior. These data suggest that D2 MSNs and specific circuits within the basal ganglia are preferentially vulnerable to HIV-1.

HIV-1 Tat causes cognitive deficits and selective loss of parvalbumin, somatostatin, and neuronal nitric oxide synthase expressing hippocampal CA1 interneuron subpopulations.

Memory deficits are characteristic of HIV-associated neurocognitive disorders (HAND) and co-occur with hippocampal pathology. The HIV-1 transactivator of transcription (Tat), a regulatory protein, plays a significant role in these events, but the cellular mechanisms involved are poorly understood. Within the hippocampus, diverse populations of interneurons form complex networks; even subtle disruptions can drastically alter synaptic output, resulting in behavioral dysfunction. We hypothesized that HIV-1 Tat would impair cognitive behavior and injure specific hippocampal interneuron subtypes. Male transgenic mice that inducibly expressed HIV-1 Tat (or non-expressing controls) were assessed for cognitive behavior or had hippocampal CA1 subregions evaluated via interneuron subpopulation markers. Tat exposure decreased spatial memory in a Barnes maze and mnemonic performance in a novel object recognition test. Tat reduced the percentage of neurons expressing neuronal nitric oxide synthase (nNOS) without neuropeptide Y immunoreactivity in the stratum pyramidale and the stratum radiatum, parvalbumin in the stratum pyramidale, and somatostatin in the stratum oriens, which are consistent with reductions in interneuron-specific interneuron type 3 (IS3), bistratified, and oriens-lacunosum-moleculare interneurons, respectively. The findings reveal that an interconnected ensemble of CA1 nNOS-expressing interneurons, the IS3 cells, as well as subpopulations of parvalbumin- and somatostatin-expressing interneurons are preferentially vulnerable to HIV-1 Tat. Importantly, the susceptible interneurons form a microcircuit thought to be involved in feedback inhibition of CA1 pyramidal cells and gating of CA1 pyramidal cell inputs. The identification of vulnerable CA1 hippocampal interneurons may provide novel insight into the basic mechanisms underlying key functional and neurobehavioral deficits associated with HAND.

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.

Acetylcholine release and inhibitory interneuron activity in hippocampal CA1.

Acetylcholine release in the central nervous system (CNS) has an important role in attention, recall, and memory formation. One region influenced by acetylcholine is the hippocampus, which receives inputs from the medial septum and diagonal band of Broca complex (MS/DBB). Release of acetylcholine from the MS/DBB can directly affect several elements of the hippocampus including glutamatergic and GABAergic neurons, presynaptic terminals, postsynaptic receptors, and astrocytes. A significant portion of acetylcholine's effect likely results from the modulation of GABAergic inhibitory interneurons, which have crucial roles in controlling excitatory inputs, synaptic integration, rhythmic coordination of principal neurons, and outputs in the hippocampus. Acetylcholine affects interneuron function in large part by altering their membrane potential via muscarinic and nicotinic receptor activation. This minireview describes recent data from mouse hippocampus that investigated changes in CA1 interneuron membrane potentials following acetylcholine release. The interneuron subtypes affected, the receptor subtypes activated, and the potential outcome on hippocampal CA1 network function is discussed.

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.

Defective retinal depolarizing bipolar cells in regulators of G protein signaling (RGS) 7 and 11 double null mice.

Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gß5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gß5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gß5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gß5S is undetectable, whereas levels of the photoreceptor-specific Gß5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gß5S expression in retina, the DBC-related defects in Gß5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gß5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.

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.

Sustained survival and maturation of adult neural stem/progenitor cells after transplantation into the injured brain.

Multipotent neural stem/progenitor cells (NS/NPCs) that are capable of generating neurons and glia offer enormous potential for treating neurological diseases. Adult NS/NPCs that reside in the mature mammalian brain can be isolated and expanded in vitro, and could be a potential source for autologous transplantation to replace cells lost to brain injury or disease. When these cells are transplanted into the normal brain, they can survive and become region-specific cells. However, it has not been reported whether these cells can survive for an extended period and become functional cells in an injured heterotypic environment. In this study, we tested survival, maturation fate, and electrophysiological properties of adult NS/NPCs after transplantation into the injured rat brain. NS/NPCs were isolated from the subventricular zone of adult Fisher 344 rats and cultured as a monolayer. Recipient adult Fisher 344 rats were first subjected to a moderate fluid percussive injury. Two days later, cultured NS/NPCs were injected into the injured brain in an area between the white matter tracts and peri-cortical region directly underneath the injury impact. The animals were sacrificed 2 or 4 weeks after transplantation for immunohistochemical staining or patch-clamp recording. We found that transplanted cells survived well at 2 and 4 weeks. Many cells migrated out of the injection site into surrounding areas expressing astrocyte or oligodendrocyte markers. Whole cell patch-clamp recording at 4 weeks showed that transplanted cells possessed typical mature glial cell properties. These data demonstrate that adult NS/NPCs can survive in an injured heterotypic environment for an extended period and become functional cells.

Mu opioid receptor activation normalizes temporo-ammonic pathway driven inhibition in hippocampal CA1.

The hippocampus of the mammalian brain is important for the formation of long-term memories. Hippocampal-dependent learning can be affected by a number of neurotransmitters including the activation of µ-opioid receptors (MOR). It has been shown that MOR activation can alter synaptic plasticity and network oscillations in the hippocampus, both of which are thought to be important for the encoding of information and formation of memories. One hippocampal oscillation that has been correlated with learning and memory formation is the 4-10 Hz theta rhythm. During theta rhythms, inputs to hippocampal CA1 from CA3 (Schaffer collaterals, SC) and the entorhinal cortex (perforant path) can integrate at different times within an individual theta cycle. Consequently, when excitatory inputs in the stratum lacunosum-moleculare (the temporo-ammonic pathway (TA), which includes the perforant path) are stimulated approximately one theta period before SC inputs, the TA can indirectly inhibit SC inputs. This inhibition is due to the activation of postsynaptic GABA(B) receptors on CA1 pyramidal neurons. Importantly, MOR activation has been shown to suppress GABA(B) inhibitory postsynaptic potentials in CA1 pyramidal neurons. Therefore, we examined how MOR activation affects the integration between TA inputs and SC inputs in hippocampal CA1. To do this we used voltage-sensitive dye imaging and whole cell patch clamping from acute hippocampal slices taken from young adult rats. Here we show that MOR activation has no effect on the integration between TA and SC inputs when activation of the TA precedes SC by less than one half of a theta cycle (<75 ms). However, MOR activation completely blocked the inhibitory action of TA on SC inputs when TA stimulation occurred approximately one theta cycle before SC activation (>150 ms). This MOR suppression of TA driven inhibition occurred in both the SC input layer of hippocampal CA1 (stratum radiatum) and the output layer of CA1 pyramidal neurons (stratum pyramidale). Thus MOR activation can have profound effects on the temporal integration between two primary excitatory pathways to hippocampal CA1 and subsequently the resultant output from CA1 pyramidal neurons. These data provide important information for understanding how acute or chronic MOR activation may affect the integration of activity within hippocampal CA1 during theta rhythm.

Cholinergic modulation of excitatory synaptic input integration in hippocampal CA1.

During theta rhythm, the timing of inputs to hippocampal CA1 from the perforant path (PP) of the entorhinal cortex and the Schaffer collaterals (SCs) from individual CA3 pyramidal neurons can vary within an individual theta period. Importantly, during theta rhythms these interactions occur during elevated acetylcholine concentrations. Thus, I examined the effect that PP inputs have on SC inputs in hippocampal CA1 during cholinergic receptor activation. To do this I measured the impact that a single electrical stimulus of the stratum lacunosum-moleculare (SLM, which contains the PP) had on excitation evoked by stimulation of the stratum radiatum (SR, which contains the SC) using voltage-sensitive dye imaging, field excitatory postsynaptic potentials and whole cell patch clamping in rat hippocampal brain slices. My data showed that SLM stimulation one half a theta cycle or less (25-75 ms) before SR stimulation resulted in the summation of excitatory events in SR and SP of hippocampal CA1. The summation was unaffected by cholinergic receptor activation by carbachol. SLM stimulation one theta cycle (150-225 ms) preceding SR stimulation significantly suppressed excitatory events measured in SR and SP. This SLM stimulus inhibition of SR-driven excitatory events was augmented by carbachol application. The carbachol effect was blocked by atropine and SLM-driven suppression of excitatory events was blocked by the GABA(B) receptor antagonist CGP 54626. SR field EPSP slopes were unaffected by SLM prepulses. Carbachol increased the probability of SR input to drive action potential firing in CA1 pyramidal neurons, which was inhibited by SLM prepulses (150-225 ms). Together these data provide important information regarding the integration of inputs in hippocampal CA1 during theta rhythms. More specifically, SR inputs can be differentially gated by SLM feedforward inhibition at varying temporal intervals within a theta cycle.