Septal cholinergic neurons gate hippocampal output to entorhinal cortex via oriens lacunosum moleculare interneurons.

Neuromodulation of neural networks, whereby a selected circuit is regulated by a particular modulator, plays a critical role in learning and memory. Among neuromodulators, acetylcholine (ACh) plays a critical role in hippocampus-dependent memory and has been shown to modulate neuronal circuits in the hippocampus. However, it has remained unknown how ACh modulates hippocampal output. Here, using in vitro and in vivo approaches, we show that ACh, by activating oriens lacunosum moleculare (OLM) interneurons and therefore augmenting the negative-feedback regulation to the CA1 pyramidal neurons, suppresses the circuit from the hippocampal area CA1 to the deep-layer entorhinal cortex (EC). We also demonstrate, using mouse behavior studies, that the ablation of OLM interneurons specifically impairs hippocampus-dependent but not hippocampus-independent learning. These data suggest that ACh plays an important role in regulating hippocampal output to the EC by activating OLM interneurons, which is critical for the formation of hippocampus-dependent memory.

Cholinergic modulation of the hippocampal region and memory function.

Acetylcholine (ACh) plays an important role in memory function and has been implicated in aging-related dementia, in which the impairment of hippocampus-dependent learning strongly manifests. Cholinergic neurons densely innervate the hippocampus, mediating the formation of episodic as well as semantic memory. Here, we will review recent findings on acetylcholine's modulation of memory function, with a particular focus on hippocampus-dependent learning, and the circuits involved. In addition, we will discuss the complexity of ACh actions in memory function to better understand the physiological role of ACh in memory. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Nutritional state-dependent ghrelin activation of vasopressin neurons via retrograde trans-neuronal-glial stimulation of excitatory GABA circuits.

Behavioral and physiological coupling between energy balance and fluid homeostasis is critical for survival. The orexigenic hormone ghrelin has been shown to stimulate the secretion of the osmoregulatory hormone vasopressin (VP), linking nutritional status to the control of blood osmolality, although the mechanism of this systemic crosstalk is unknown. Here, we show using electrophysiological recordings and calcium imaging in rat brain slices that ghrelin stimulates VP neurons in the hypothalamic paraventricular nucleus (PVN) in a nutritional state-dependent manner by activating an excitatory GABAergic synaptic input via a retrograde neuronal-glial circuit. In slices from fasted rats, ghrelin activation of a postsynaptic ghrelin receptor, the growth hormone secretagogue receptor type 1a (GHS-R1a), in VP neurons caused the dendritic release of VP, which stimulated astrocytes to release the gliotransmitter adenosine triphosphate (ATP). ATP activation of P2X receptors excited presynaptic GABA neurons to increase GABA release, which was excitatory to the VP neurons. This trans-neuronal-glial retrograde circuit activated by ghrelin provides an alternative means of stimulation of VP release and represents a novel mechanism of neuronal control by local neuronal-glial circuits. It also provides a potential cellular mechanism for the physiological integration of energy and fluid homeostasis.

Short-term potentiation of GABAergic synaptic inputs to vasopressin and oxytocin neurones.

The magnocellular vasopressin (VP) and oxytocin (OT) neurones undergo long-term synaptic plasticity to accommodate prolonged hormone demand. By contrast, rapidly induced,transient synaptic plasticity in response to brief stimuli could enable the activation of magnocellular neurones in response to acute challenges. Here, we report a robust short-term potentiation of asynchronous GABAergic synaptic inputs (STP(GABA)) to VP and OT neurones of the hypothalamic supraoptic nucleus elicited by repetitive extracellular electrical stimulation.The STP(GABA) required extracellular Ca2+, but did not require activation of glutamate, VP or OT receptors or nitric oxide synthesis. Presynaptic action potential generation was necessary for the induction, but not the maintenance, of STP(GABA). The STP(GABA) led to a minutes-long GABA(A)receptor-dependent increase in spike frequency in VP neurones, but not in OT neurones,consistent with an excitatory function of GABA in only VP neurones and with the generation of prolonged bursts of action potentials in VP neurones. Therefore, this short-term plasticity of GABAergic synaptic inputs is likely to play very different roles in the regulation of OT and VP neurones and their distinct patterns of physiological activation.

GABA is excitatory in adult vasopressinergic neuroendocrine cells.

Neuronal excitability in the adult brain is controlled by a balance between synaptic excitation and inhibition mediated by glutamate and GABA, respectively. While generally inhibitory in the adult brain, GABA(A) receptor activation is excitatory under certain conditions in which the GABA reversal potential is shifted positive due to intracellular Cl(-) accumulation, such as during early postnatal development and brain injury. However, the conditions under which GABA is excitatory are generally either transitory or pathological. Here, we reveal GABAergic synaptic inputs to be uniformly excitatory in vasopressin (VP)-secreting magnocellular neurons in the adult hypothalamus under normal conditions. The GABA reversal potential (E(GABA)) was positive to resting potential and spike threshold in VP neurons, but not in oxytocin (OT)-secreting neurons. The VP neurons lacked expression of the K(+)-Cl(-) cotransporter 2 (KCC2), the predominant Cl(-) exporter in the adult brain. The E(GABA) was unaffected by inhibition of KCC2 in VP neurons, but was shifted positive in OT neurons, which express KCC2. Alternatively, inhibition of the Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1), a Cl(-) importer expressed in most cell types mainly during postnatal development, caused a negative shift in E(GABA) in VP neurons, but had no effect on GABA currents in OT neurons. GABA(A) receptor blockade caused a decrease in the firing rate of VP neurons, but an increase in firing in OT neurons. Our findings demonstrate that GABA is excitatory in adult VP neurons, suggesting that the classical excitation/inhibition paradigm of synaptic glutamate and GABA control of neuronal excitability does not apply to VP neurons.

Entorhinal cortical delta oscillations drive memory consolidation.

Long-term memories are formed by creating stable memory representations via memory consolidation, which mainly occurs during sleep following the encoding of labile memories in the hippocampus during waking. The entorhinal cortex (EC) has intricate connections with the hippocampus, but its role in memory consolidation is largely unknown. Using cell-type- and input-specific in vivo neural activity recordings, here we show that the temporoammonic pathway neurons in the EC, which directly innervate the output area of the hippocampus, exhibit potent oscillatory activities during anesthesia and sleep. Using in vivo individual and populational neuronal activity recordings, we demonstrate that a subpopulation of the temporoammonic pathway neurons, which we termed sleep cells, generate delta oscillations via hyperpolarization-activated cyclic-nucleotide-gated channels during sleep. The blockade of these oscillations significantly impaired the consolidation of hippocampus-dependent memory. Together, our findings uncover a key driver of delta oscillations and memory consolidation that are found in the EC.

Recombinant Viral Vectors as Neuroscience Tools.

Recombinant viruses are highly efficient vehicles for in vivo gene delivery. Viral vectors expand the neurobiology toolbox to include direct and rapid anterograde, retrograde, and trans-synaptic delivery of tracers, sensors, and actuators to the mammalian brain. Each viral type offers unique advantages and limitations. To establish strategies for selecting a suitable viral type, this article aims to provide readers with an overview of viral recombinant technology, viral structure, tropism, and differences between serotypes and pseudotypes for three of the most commonly used vectors in neurobiology research: adeno-associated viruses, retro/lentiviruses, and glycoprotein-deleted rabies viruses. © 2019 by John Wiley & Sons, Inc.

Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics.

Advances in design and use of light-sensitive and light-emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long-term, and cell-specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno-associated virus (AAV), retrovirus/lentivirus, and glycoprotein-deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high-quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.

Lactation induces increased IPSC bursting in oxytocinergic neurons.

Hypothalamic magnocellular neurosecretory cells (MNCs) undergo dramatic structural reorganization during lactation in female rats that is thought to contribute to the pulsatile secretion of oxytocin critical for milk ejection. MNCs from male rats generate robust bursts of GABAergic synaptic currents, a subset of which are onset-synchronized between MNC pairs, but the functional role of the IPSC bursts is not known. To determine the physiological relevance of IPSC bursts, we compared MNCs from lactating and non-lactating female rats using whole-cell recordings in brain slices. We recorded a sixfold increase in the incidence of IPSC bursts in oxytocin (OT)-MNCs from lactating rats compared to non-lactating rats, whereas there was no change in IPSC bursts in vasopressin (VP)-MNCs. Synchronized bursts of IPSCs were observed in pairs of MNCs in slices from lactating rats. Our data indicate, therefore, that IPSC bursts are upregulated specifically in OT-MNCs during lactation, and may, therefore, contribute via rebound depolarization to the spike trains in OT neurons that lead to reflex milk ejection.

Reduction of mint-1, mint-2, and APP overexpression in okadaic acid-treated neurons.

Treatment of neurons with okadaic acid, a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neuronal death, and to create a research model of Alzheimer's disease. Amyloid precursor protein (APP) is the precursor protein of the beta-amyloid peptide that accumulates in extracellular plaques in Alzheimer's disease. Several studies have shown that mint-1 (munc18-interacting protein 1) and mint-2 bind to the YENPTY motif in the cytoplasmic domain of APP and inhibit processing of APP to beta-amyloid peptide. Here, we report that, upon neurodegeneration with okadaic acid, mint-1 and mint-2 levels were reduced by proteolytic cleavage, and that these changes were followed by increases in APP levels. We also show that the mint-1 and mint-2 cleavage and APP overexpression were prevented by calpain inhibitor-I and inhibitor-II. These results indicate that mint cleavage might play a role in the pathophysiology of Alzheimer's disease.

Intermuscular adipose tissue is associated with monocyte chemoattractant protein-1, independent of visceral adipose tissue.

OBJECTIVES: Emerging evidence suggests that intermuscular adipose tissue is a risk factor for insulin resistance, but the underlying mechanism still remains unclear. We investigated whether the levels of leptin, adiponectin, and monocyte chemoattractant protein-1 are associated with intermuscular adipose tissue in obese subjects. DESIGN AND METHODS: A cross-sectional study was performed on 77 obese Korean women. Areas of visceral adipose tissue, subcutaneous adipose tissue, and intermuscular adipose tissue were measured by computed tomography scan, and serum concentrations of adipokines were measured by enzyme-linked immunosorbent assays. Correlation between the levels of adipokines and the fat areas was assessed using Pearson correlation and covariate-adjusted multivariable regression. RESULTS: Leptin was positively correlated with subcutaneous adipose tissue (r=0.452, P<0.001), fasting insulin (r=0.403, P<0.001), and homeostasis model assessment of insulin resistance (r=0.360, P=0.001), whereas monocyte chemoattractant protein-1 was positively correlated with intermuscular adipose tissue (r=0.483, P<0.001). After adjustment for age, height, and other body composition metrics, leptin was still related to subcutaneous adipose tissue (ß=0.390, P=0.001). Monocyte chemoattractant protein-1 was associated with intermuscular adipose tissue (ß=0.433, P=0.001) after adjustment for visceral adipose tissue. CONCLUSIONS: Intermuscular adipose tissue was correlated with monocyte chemoattractant protein-1, suggesting its role in the development of insulin resistance.

Regulation of Neuronal Activity in Hypothalamic Vasopressin Neurons.

Vasopressin is a peptide hormone secreted from the posterior pituitary gland in response to various physiological and/or pathological stimuli, including changes in body fluid volume and osmolality and stress exposure. Vasopressin secretion is controlled by the electrical activity of the vasopressinergic magnocellular neurosecretory cells located in the hypothalamic supraoptic nucleus and paraventricular nucleus. Vasopressin release can occur somatodendritically in the hypothalamus or at the level of pituitary axon terminals. The electrical activity of the vasopressin neurons assumes specific patterns of electrical discharge that are under the control of several factors, including the intrinsic properties of the neuronal membrane and synaptic and hormonal inputs. It is increasingly clear that glial cells perform critical signaling functions that contribute to signal transmission in neural circuits. Astrocytes contribute to neuronal signaling by regulating synaptic and extrasynaptic neurotransmission, as well as by mediating bidirectional neuronal-glial transmission. We recently discovered a novel form of neuronal-glial signaling that exploits the full spatial domain of astrocytes to transmit dendritic retrograde signals from vasopressin neurons to distal upstream neuronal targets. This retrograde trans-neuronal-glial transmission allows the vasopressin neurons to regulate their synaptic inputs by controlling upstream presynaptic neuron firing, thus providing a powerful means of controlling hormonal output.

Rapid Nongenomic Glucocorticoid Actions in Male Mouse Hypothalamic Neuroendocrine Cells Are Dependent on the Nuclear Glucocorticoid Receptor.

Corticosteroids act classically via cognate nuclear receptors to regulate gene transcription; however, increasing evidence supports rapid, nontranscriptional corticosteroid actions via activation of membrane receptors. Using whole-cell patch clamp recordings in hypothalamic slices from male mouse genetic models, we tested for nongenomic glucocorticoid actions at glutamate and gamma aminobutyric acid (GABA) synapses in hypothalamic neuroendocrine cells, and for their dependence on the nuclear glucocorticoid receptor (GR). In enhanced green fluorescent protein-expressing CRH neurons of the paraventricular nucleus (PVN) and in magnocellular neurons of the PVN and supraoptic nucleus (SON), dexamethasone activated postsynaptic membrane-associated receptors and G protein signaling to elicit a rapid suppression of excitatory postsynaptic inputs, which was blocked by genetic deletion of type I cannabinoid receptors and a type I cannabinoid receptor antagonist. In magnocellular neurons, dexamethasone also elicited a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs. These data indicate a rapid, synapse-specific glucocorticoid-induced retrograde endocannabinoid signaling at glutamate synapses and nitric oxide signaling at GABA synapses. Unexpectedly, the rapid glucocorticoid effects on both excitatory and inhibitory synaptic transmission were lost with conditional deletion of GR in the PVN and SON in slices from a single minded-1-cre-directed conditional GR knockout mouse. Thus, the nongenomic glucocorticoid actions at glutamate and GABA synapses on PVN and SON neuroendocrine cells are dependent on the nuclear GR. The nuclear GR, therefore, is responsible for transducing the rapid steroid response at the membrane, or is either a critical component in the signaling cascade or regulates a critical component of the signaling cascade of a distinct membrane GR.