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Previous studies indicate that the activity of hypothalamic POMC neurons can be regulated by glucose via intracellular mechanisms, but its regulation by lactate is poorly understood. In addition to its energetic role, lactate acts as a signaling molecule. In this study, we evaluated the function and location of the lactate receptor, hydroxycarboxylic acid receptor 1 (HCAR1). We used a conditional genetic approach to label POMC neurons and evaluated their sensitivity to lactate using patch-clamp recordings. l-Lactate and 3-chloro-5-hydroxybenzoic acid (3Cl-HBA), HCAR1 specific agonist depolarized POMC neurons and the increase in excitability was abolished by pertussis toxin (PTX), indicating the involvement of Gαi/o-protein-coupled receptors. In addition, the depolarization of a subset of POMC neurons was sensitive to α-cyano-4-hydroxycinnamate (4-CIN), a lactate transporter blocker, suggesting that the depolarization induced by l-lactate can also occur by direct intracellular action. Surprisingly, HCAR1 was not detected in POMC neurons, but instead localized in astrocytes. These results suggest a new lactate-mediated mechanism for astrocyte-neuron intercellular communication.

Local interneurons of the olfactory bulb (OB) are densely innervated by long-range GABAergic neurons from the basal forebrain (BF), suggesting that this top-down inhibition regulates early processing in the olfactory system. However, how GABAergic inputs modulate the OB output neurons, the mitral/tufted cells, is unknown. Here, in male and female mice acute brain slices, we show that optogenetic activation of BF GABAergic inputs produced distinct local circuit effects that can influence the activity of mitral/tufted cells in the spatiotemporal domains. Activation of the GABAergic axons produced a fast disinhibition of mitral/tufted cells consistent with a rapid and synchronous release of GABA onto local interneurons in the glomerular and inframitral circuits of the OB, which also reduced the spike precision of mitral/tufted cells in response to simulated stimuli. In addition, BF GABAergic inhibition modulated local oscillations in a layer-specific manner. The intensity of locally evoked θ oscillations was decreased on activation of top-down inhibition in the glomerular circuit, while evoked γ oscillations were reduced by inhibition of granule cells. Furthermore, BF GABAergic input reduced dendrodendritic inhibition in mitral/tufted cells. Together, these results suggest that long-range GABAergic neurons from the BF are well suited to influence temporal and spatial aspects of processing by OB circuits.SIGNIFICANCE STATEMENTDisruption of GABAergic inhibition from the basal forebrain (BF) to the olfactory bulb (OB) impairs the discrimination of similar odors, yet how this centrifugal inhibition influences neuronal circuits in the OB remains unclear. Here, we show that the BF GABAergic neurons exclusively target local inhibitory neurons in the OB, having a functional disinhibitory effect on the output neurons, the mitral cells. Phasic inhibition by BF GABAergic neurons reduces spike precision of mitral cells and lowers the intensity of oscillatory activity in the OB, while directly modulating the extent of dendrodendritic inhibition. These circuit-level effects of this centrifugal inhibition can influence the temporal and spatial dynamics of odor coding in the OB.

Action potentials are the basic unit of information in the nervous system, and their reliable detection and decoding holds the key to understanding how the brain generates complex thought and behavior. Transduction of these signals into microwave signal oscillations can enable wireless sensors that report on brain activity through magnetic induction. In this letter, we demonstrate that action potentials from the lateral giant neurons of crayfish can induce microwave oscillations in nanoscale magnetic tunnel junctions (NMTJs). We show that action potentials activate microwave oscillations in NMTJs with an amplitude that follows the action potential signal, demonstrating that the device has both the sensitivity and temporal resolution to respond to action potentials from a single neuron. The activation of magnetic oscillations by action potentials, together with the small surface area and the high-frequency tunability, makes these devices potential candidates for high-resolution sensing of bioelectric signals from neural tissues. These device attributes may be useful for the design of high-throughput bidirectional brain–machine interfaces.

Magnetic nanoparticles (MNPs) have been used as effective vehicles for targeted delivery of theranostic agents in the brain. The advantage of magnetic targeting lies in the ability to control the concentration and distribution of therapy to a desired target region using external driving magnets. In this study, we investigated the behavior and safety of MNP motion in brain tissue. We found that MNPs move and form nanoparticle chains in the presence of a uniform magnetic field, and that this chaining is influenced by the applied magnetic field intensity and the concentration of MNPs in the tissue. Using electrophysiology recordings, immunohistochemistry and fluorescent imaging we assessed the functional health of neurons and neural circuits and found no adverse effects associated with MNP motion through brain tissue.Much research has been done to test the use of nanocarriers for gaining access across the blood brain barrier (BBB). In this respect, magnetic nanoparticles (MNPs) are one of the most studied candidates. Nonetheless, the behavior and safety of MNP once inside brain tissue remains unknown. In this article, the authors thus studied this very important subject.

Neuromodulation of olfactory circuits by acetylcholine (ACh) plays an important role in odor discrimination and learning. Early processing of chemosensory signals occurs in two functionally and anatomically distinct regions, the main and accessory olfactory bulbs (MOB and AOB), which receive extensive cholinergic input from the basal forebrain. Here, we explore the regulation of AOB and MOB circuits by ACh, and how cholinergic modulation influences olfactory-mediated behaviors in mice. Surprisingly, despite the presence of a conserved circuit, activation of muscarinic ACh receptors revealed marked differences in cholinergic modulation of output neurons: excitation in the AOB and inhibition in the MOB. Granule cells (GCs), the most abundant intrinsic neuron in the OB, also exhibited a complex muscarinic response. While GCs in the AOB were excited, MOB GCs exhibited a dual muscarinic action in the form of a hyperpolarization and an increase in excitability uncovered by cell depolarization. Furthermore, ACh influenced the input–output relationship of mitral cells in the AOB and MOB differently showing a net effect on gain in mitral cells of the MOB, but not in the AOB. Interestingly, despite the striking differences in neuromodulatory actions on output neurons, chemogenetic inhibition of cholinergic neurons produced similar perturbations in olfactory behaviors mediated by these two regions. Decreasing ACh in the OB disrupted the natural discrimination of molecularly related odors and the natural investigation of odors associated with social behaviors. Thus, the distinct neuromodulation by ACh in these circuits could underlie different solutions to the processing of general odors and semiochemicals, and the diverse olfactory behaviors they trigger. SIGNIFICANCE STATEMENT State-dependent cholinergic modulation of brain circuits is critical for several high-level cognitive functions, including attention and memory. Here, we provide new evidence that cholinergic modulation differentially regulates two parallel circuits that process chemosensory information, the accessory and main olfactory bulb (AOB and MOB, respectively). These circuits consist of remarkably similar synaptic arrangement and neuronal types, yet cholinergic regulation produced strikingly opposing effects in output and intrinsic neurons. Despite these differences, the chemogenetic reduction of cholinergic activity in freely behaving animals disrupted odor discrimination of simple odors, and the investigation of social odors associated with behaviors signaled by the Vomeronasal system.

Glucose is a key modulator of feeding behavior. By acting in peripheral tissues and in the central nervous system, it directly controls the secretion of hormones and neuropeptides and modulates the activity of the autonomic nervous system. GLUT2 is required for several glucoregulatory responses in the brain, including feeding behavior, and is localized in the hypothalamus and brainstem, which are the main centers that control this behavior. In the hypothalamus, GLUT2 has been detected in glial cells, known as tanycytes, which line the basal walls of the third ventricle (3V). This study aimed to clarify the role of GLUT2 expression in tanycytes in feeding behavior using 3V injections of an adenovirus encoding a shRNA against GLUT2 and the reporter EGFP (Ad‐shGLUT2). Efficient in vivo GLUT2 knockdown in rat hypothalamic tissue was demonstrated by qPCR and Western blot analyses. Specificity of cell transduction in the hypothalamus and brainstem was evaluated by EGFP‐fluorescence and immunohistochemistry, which showed EGFP expression specifically in ependymal cells, including tanycytes. The altered mRNA levels of both orexigenic and anorexigenic neuropeptides suggested a loss of response to increased glucose in the 3V. Feeding behavior analysis in the fasting‐feeding transition revealed that GLUT2‐knockdown rats had increased food intake and body weight, suggesting an inhibitory effect on satiety. Taken together, suppression of GLUT2 expression in tanycytes disrupted the hypothalamic glucosensing mechanism, which altered the feeding behavior.

VoltageFluor (VF) dyes have the potential to optically measure voltage in excitable membranes with the combination of high spatial and temporal resolution essential to better characterize the voltage dynamics of large groups of excitable cells. VF dyes sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. We show that tuning the driving force for PeT (ΔGPeT + w) through systematic chemical substitution modulates voltage sensitivity, estimate (ΔGPeT + w) values from experimentally measured redox potentials, and validate the voltage sensitivities in patch-clamped HEK cells for 10 new VF dyes. VF2.1(OMe).H, with a 48% ΔF/F per 100 mV, shows approximately 2-fold improvement over previous dyes in HEK cells, dissociated rat cortical neurons, and medicinal leech ganglia. Additionally, VF2.1(OMe).H faithfully reports pharmacological effects and circuit activity in mouse olfactory bulb slices, thus opening a wide range of previously inaccessible applications for voltage sensitive dyes.

The brain is a complex network of interconnected circuits that exchange electrical signals with each other. These electrical signals provide insight on how neural circuits code information, and give rise to sensations, thoughts, emotions and actions. Currents methods to detect and modulate these electrical signals use implanted electrodes or optical fields with light sensitive dyes in the brain. These techniques require complex surgeries or suffer low resolution. In this talk we explore a new method to both image and stimulate single neurons using spintronics. We propose using a Spin Transfer Nano-Oscillators (STNOs) as a nanoscale sensor that converts neuronal action potentials to microwave field oscillations that can be detected wirelessly by magnetic induction. We will describe our recent proof-of-concept demonstration of both detection and wireless modulation of neuronal activity using STNOs. For detection we use electrodes to connect a STNO to a lateral giant crayfish neuron. When we stimulate the neuron, the STNO responds to the neuronal activity with a corresponding microwave signal. For modulation, we stimulate the STNOs wirelessly using an inductively coupled solenoid. The STNO rectifies the induced microwave signal to produce a direct voltage. This direct voltage from the STNO, when applied in the vicinity of a mammalian neuron, changes the frequency of electrical signals produced by the neuron.

Funding for Open Access provided by the UMD Libraries Open Access Publishing Fund., The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1-/- and the M1/M3-/- knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing.

By regulating inhibition at dendrodendritic synapses between mitral and granule cells (GCs), noradrenergic neurons extending from the brainstem provide an input essential for odour processing in the olfactory bulb (OB). In the accessory OB (AOB), we have recently shown that noradrenaline (NA) increases GABA inhibitory input on to mitral cells (MCs) by exciting GCs. Here, we show that GCs in the main OB (MOB) exhibit a similar response to NA, indicating a common mechanism for noradrenergic regulation of GCMC inhibition throughout the OB. In GCs of the MOB, NA (10 μM) produced a robust excitatory effect that included a slow afterdepolarization that followed a train of action potentials evoked by a current stimulus. The depolarization and slow afterdepolarization in GCs were blocked by the α1A-adrenergic receptor (AR) selective antagonist WB 4101 (30 nm) and mimicked by the α(1A)-AR selective agonist A 61603 (1 μM). In recordings from MCs, A 61603 (30 nm-1 μM) produced a sizeable increase in the frequency of spontaneous and miniature IPSCs, an effect completely abolished by the GABAA receptor antagonist gabazine (5 μM). Likewise, activation of β-ARs increased the frequency of spontaneous IPSCs; however, this effect was smaller and confined to the first postnatal weeks. NA enhanced inhibition in MCs across a broad concentration range (0.1-30 μM) and its effects were completely abolished by a mixture of α1- and β-AR antagonists (1 μM prazosin and 10 μM propranolol). Furthermore, the general α2-AR agonist clonidine (10 μM) failed to affect sIPSC frequency. Thus, the NA-mediated increase in GCMC inhibition in the OB results mostly from activation of the α1A-AR subtype.

Funding for Open Access provided by the UMD Libraries Open Access Publishing Fund., An important contribution to neural circuit oscillatory dynamics is the ongoing activation and inactivation of hyperpolarization-activated currents (/h). Network synchrony dynamics play an important role in the initial processing of odor signals by the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). In the mouse olfactory bulb, we show that /h is present in granule cells (GCs), the most prominent inhibitory neuron in the olfactory bulb, and that /h underlies subthreshold resonance in GCs. In accord with the properties of /h, the currents exhibited sensitivity to changes in extracellular K+ concentration and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidin chloride), a blocker of /h. ZD7288 also caused GCs to hyperpolarize and increase their input resistance, suggesting that /h is active at rest in GCs. The inclusion of cAMP in the intracellular solution shifted the activation of /h to less negative potentials in the MOB, but not in the AOB, suggesting that channels with different subunit composition mediate /h in these regions. Furthermore, we show that mature GCs exhibit /h-dependent subthreshold resonance in the theta frequency range (4–12 Hz). Another inhibitory subtype in the MOB, the periglomerular cells, exhibited /h-dependent subthreshold resonance in the delta range (1–4 Hz), while principal neurons, the mitral cells, do not exhibit /h-dependent subthreshold resonance. Importantly, /h size, as well as the strength and frequency of resonance in GCs, exhibited a postnatal developmental progression, suggesting that this development of /h in GCs may differentially contribute to their integration of sensory input and contribution to oscillatory circuit dynamics.

The accessory olfactory bulb (AOB), the first relay of chemosensory information in the Vomeronasal system, receives extensive cholinergic innervation from the basal forebrain. Cholinergic modulation of neuronal activity in the olfactory bulb has been hypothesized to play an important role in olfactory processing; however, little is known about the cellular actions of acetylcholine (ACh) within the AOB. Here using in vitro slice preparation, we show that muscarinic acetylcholine receptor (mAChR) activation increases neuronal excitability of granule and mitral/tufted cells (GCs and MCs) in the AOB. Activation of mAChRs increased excitability of GCs by three distinct mechanisms: induction of a long-lasting depolarization, activation of a slow afterdepolarization (sADP), and an increase in excitatory glutamatergic input due to MC depolarization. The depolarization and sADP were elicited by the selective agonist 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]- N,N,N-trimethyl-2-butyn-1-aminium chloride (100 μM) and blocked by low concentrations of pirenzepine (300 nM), indicating that they result from activation of M1-like mAChRs. In contrast, cholinergic stimulation increased the excitability of MCs via recruitment of nicotinic AChRs (nAChRs) and M1-like mAChRs. Submaximal activation of these receptors, however, decreased the excitability of MCs. Surprisingly, we found that unlike GCs in the main olfactory bulb, GCs in the AOB are excited by mAChR activation in young postnatal neurons, suggesting marked differences in cholinergic regulation of development between these two regions of the olfactory bulb.

Sensors and Actuators B: Chemical The article of record as published may be found at http://dx.doi.org/10.1016/j.snb.2016.05.048. Supplementary material as published may be found at http://dx.doi.org/10.1016/j.snb.2016.05.048. On-chip sensing of physical signals is now relatively straightforward, with on-chip tilt sensors and cell phone cameras now ubiquitous, but sensing of odorants has proven to be far more challenging. A general purpose, mobile electronic nose would address pressing needs in security and food safety. We demonstrate here an approach that combines living olfactory sensory neurons (OSNs) with integrated circuitry. The hybrid bio-electronic chip harnesses the as-yet unparalleled capabilities of olfactory sensory neurons – in variety, selectivity, and ability to function in the real world – and takes advantage of modern silicon chip technology for detecting and processing electrical signals. OSNs were cultured directly over an array of electrodes on the surface of a custom integrated circuit so that the signals, a series of “spikes” upon odorant binding, could be locally detected and processed. Consistent and selective responses to specific odorants were recorded from individual neurons. Funded by Naval Postgraduate School Maryland NanoCenter and its FabLab National Science Foundation

Several lines of evidence suggest that odorants are recognized through a combinatorial process in the olfactory system; a single odorant is recognized by multiple receptors and multiple odorants are recognized by the same receptor. However few details of how this might actually function for any particular odour set or receptor family are available. Approaching the problem from the ligands rather than the receptors, we used the response to a common odorant, octanal, as the basis for defining multiple receptor profiles. Octanal and other aldehydes induce large EOG responses in the rodent olfactory epithelium, suggesting that these compounds activate a large number of odour receptors (ORs). Here, we have determined and compared the pharmacological profile of different octanal receptors using Ca2+ imaging in isolated olfactory sensory neurones (OSNs). It is believed that each OSN expresses only one receptor, thus the response profile of each cell corresponds to the pharmacological profile of one particular receptor. We stimulated the cells with a panel of nine odorants, which included octanal, octanoic acid, octanol and cinnamaldehyde among others (all at 30μm). Cluster analysis revealed several distinct pharmacological profiles for cells that were all sensitive to octanal. Some receptors had a broad molecular range, while others were activated only by octanal. Comparison of the profiles with that of the one identified octanal receptor, OR-I7, indicated several differences. While OR-I7 is activated by low concentrations of octanal and blocked by citral, other receptors were less sensitive to octanal and not blocked by citral. A lower estimate for the maximal number of octanal receptors is between 33 and 55. This large number of receptors for octanal suggests that, although the peripheral olfactory system is endowed with high sensitivity, discrimination among different compounds probably requires further central processing.

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.

It is an open question how ion channel subunits that lack protein–protein binding motifs become targeted and covalently modified by cellular signaling enzymes. Here, we show that Src-family protein tyrosine kinases (PTKs) bind to heteromultimeric Shaker -family voltage-gated potassium (Kv) channels by interactions between the Src homology 3 (SH3) domain and the proline-rich SH3 domain ligand sequence in the Shaker -family subunit Kv1.5. Once bound to Kv1.5, Src-family PTKs phosphorylate adjacent subunits in the Kv channel heteromultimer that lack proline-rich SH3 domain ligand sequences. This SH3-dependent tyrosine phosphorylation contributes to significant suppression of voltage-evoked currents flowing through the heteromultimeric channel. These results demonstrate that Kv1.5 subunits function as SH3-dependent adaptor proteins that marshal Src-family kinases to heteromultimeric potassium channel signaling complexes, and thereby confer functional sensitivity upon coassembled channel subunits that are themselves not bound directly to Src-family kinases by allowing their phosphorylation. This is a mechanism for information transfer between subunits in heteromultimeric ion channels that is likely to underlie the generation of combinatorial signaling diversity in the control of cellular electrical excitability.

Glutamate receptors guide the proliferation, migration, and differentiation of glial cells. Here, we characterize AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA receptor protein expression and function and mRNA expression in hippocampal glial cultures. By immunocytochemistry, GluR2 (the subunit that limits the Ca(2+) permeability of AMPA receptors) exhibited prominent labeling in hippocampal glial cultures. Double-labeling of GluR2 with GFAP and with A2B5 revealed GluR2 subunit expression on type-1 and type-2 astrocyte lineage cells. GluR1 subunit expression was more prominent in type-1 than in type-2 astrocytes. To characterize functional properties of glutamate receptors expressed in cultured hippocampal astrocytes, we performed whole-cell patch clamp recording. Application of L-glutamate, AMPA, and kainate, but not NMDA, to small, rounded cells (morphologically identified as type-2 astrocytes) elicited inward currents which were blocked by the AMPA/kainate antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Cyclothiazide potentiated AMPA- and kainate-elicited currents, indicative of AMPA-preferring receptors. Current voltage analysis indicated that type-2 astrocyte AMPA receptors were electrically linear, indicative of GluR2-containing, Ca(2+)-impermeable AMPA receptors. By Northern blot analysis, GluR1 mRNA was highest in astrocyte cultures from cerebellum and hippocampus and moderate in astrocyte cultures from neocortex and striatum. GluR3 mRNA was detectable in astrocyte cultures from cerebellum and neocortex. GluR2 and NR1 mRNA expression were not detected in astrocytes cultured from any brain region examined. In situ hybridization studies showed wide expression of GluR1 mRNA in cultured astrocytes; GluR2 and GluR3 mRNAs were near background levels. Thus, cultured type-2 astrocytes express functional AMPA receptors in a cell-specific and region-specific manner, consistent with their role in neuronal-glial communication.

Spermine and other endogenous polyamines potentiate, block and permeate the N -methyl- d -aspartate receptor channel. To identify structural determinants of the N -methyl- d -aspartate channel that mediate spermine's actions, we generated mutant receptors with asparagine (N) to glutamine (Q) or arginine (R) substitutions in the selectivity filter of the channel. We demonstrate that mutation of the three critical asparagines in this domain differentially affects block by phencyclidine and both potentiation and block by spermine. N-to-Q and N-to-R mutations in the N site of the NR1 subunit (N598 in NR1 011 , N619 in NR1 100 ) and N-to-Q mutations in the N and N+1 sites (N595 and N596 in NR2A, respectively) of the NR2 subunit (Q/NN, R/NN, N/QN, N/NQ, Q/QN and Q/NQ receptors) reduced affinity for phencyclidine. The Q/NN receptor showed markedly reduced potentiation by spermine, with little or no change in spermine block. The R/NN receptor showed markedly reduced spermine potentiation and affinity for spermine at its block site. The N/QN, N/NQ and Q/QN mutant receptors showed somewhat enhanced spermine block, while the Q/NQ double mutant exhibited significantly more enhanced spermine block. Thus, the asparagine residues critical to Ca 2+ permeability and Mg 2+ block of N -methyl- d -aspartate channels are also critical to block by spermine and phencyclidine. To examine the interaction of spermine and phencyclidine within the channel, we performed competition studies. Spermine appeared to compete with phencyclidine for binding to the receptor; however, blocks by phencyclidine and by spermine were not additive. The findings suggest that spermine can bind to a site in the external vestibule of the channel to impede phencyclidine binding, but allow Na + influx.

Granule cells (GCs) are the most abundant inhibitory neuronal type in the olfactory bulb and play a critical role in olfactory processing. GCs regulate the activity of principal neurons, the mitral cells, through dendrodendritic synapses, shaping the olfactory bulb output to other brain regions. GC excitability is regulated precisely by intrinsic and extrinsic inputs, and this regulation is fundamental for odor discrimination. Here, we used channelrhodopsin to stimulate GABAergic axons from the basal forebrain selectively and show that this stimulation generates reliable inhibitory responses in GCs. Furthermore, selective in vivo inhibition of GABAergic neurons in the basal forebrain by targeted expression of designer receptors exclusively activated by designer drugs produced a reversible impairment in the discrimination of structurally similar odors, indicating an important role of these inhibitory afferents in olfactory processing.

Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ~1,000-different receptors in the rodent. From the perspective of olfactory coding, the availability of such a vast number of chemosensory receptors poses several fascinating questions; in addition, such a large repertoire provides an attractive biological model to study ligand-receptor interactions. The limited functional expression of these receptors in heterologous systems, however, has greatly hampered attempts to deorphanize them. We have employed a successful approach that combines electrophysiological and imaging techniques to analyze the response profiles of single sensory neurons. Our approach has enabled us to characterize the "odor space" of a population of native aldehyde receptors and the molecular range of a genetically engineered receptor, OR-I7.

By regulating inhibition at dendrodendritic synapses between mitral and granule cells (GCs), noradrenergic neurons extending from the brainstem provide an input essential for odour processing in the olfactory bulb (OB). In the accessory OB (AOB), we have recently shown that noradrenaline (NA) increases GABA inhibitory input on to mitral cells (MCs) by exciting GCs. Here, we show that GCs in the main OB (MOB) exhibit a similar response to NA, indicating a common mechanism for noradrenergic regulation of GC→MC inhibition throughout the OB. In GCs of the MOB, NA (10 μm) produced a robust excitatory effect that included a slow afterdepolarization that followed a train of action potentials evoked by a current stimulus. The depolarization and slow afterdepolarization in GCs were blocked by the α1A-adrenergic receptor (AR) selective antagonist WB 4101 (30 nm) and mimicked by the α1A-AR selective agonist A 61603 (1 μm). In recordings from MCs, A 61603 (30 nm–1 μm) produced a sizeable increase in the frequency of spontaneous and miniature IPSCs, an effect completely abolished by the GABAA receptor antagonist gabazine (5 μm). Likewise, activation of β-ARs increased the frequency of spontaneous IPSCs; however, this effect was smaller and confined to the first postnatal weeks. NA enhanced inhibition in MCs across a broad concentration range (0.1–30 μm) and its effects were completely abolished by a mixture of α1- and β-AR antagonists (1 μm prazosin and 10 μm propranolol). Furthermore, the general α2-AR agonist clonidine (10 μm) failed to affect sIPSC frequency. Thus, the NA-mediated increase in GC→MC inhibition in the OB results mostly from activation of the α1A-AR subtype.

Actions of the polyamines spermine and spermidine on NMDA-induced currents were examined in cultured hippocampal neurons from embryonic rat. In whole-cell patch experiments using voltage-clamp, spermine (300 μM) produced about a two-fold potentiation of responses to NMDA (at −70 mV in the presence of saturating glycine); half-maximal potentiation was elicited at 207 μM. The potentiation produced by spermine was somewhat greater at positive potentials. The onset of potentiation was fast ( t 1 2 s ), indicative of an extracellular site of action. Spermidine was of comparable potency but less efficacious than spermine in potentiating NMDA responses. In excised outside-out patches, spermine exhibited two actions on NMDA-induced single-channel responses. In some patches, it increased the channel open probability; both frequency of channel opening and burst length were increased with no significant change in the mean open duration, which accounted for much of the potentiation seen in whole-cell experiments. In all patches, spermine decreased channel conductance at negative voltages, an effect ascribable to fast channel block (with a possible contribution by charge screening). These results are consistent with opposing actions of polyamines mediated at distinct sites on the NMDA receptor.

Mechanisms influencing the development of olfactory bulb glomeruli are poorly understood. While odor receptors (ORs) play an important role in olfactory sensory neuron (OSN) axon targeting/coalescence (Mombaerts et al., 1996; Wang et al., 1998; Feinstein and Mombaerts, 2004), recent work showed that G protein activation alone is sufficient to induce OSN axon coalescence (Imai et al., 2006; Chesler et al., 2007), suggesting an activity-dependent mechanism in glomerular development. Consistent with these data, OSN axon projections and convergence are perturbed in mice deficient for adenylyl cyclase III, which is downstream from the OR and catalyzes the conversion of ATP to cAMP. However, in cyclic nucleotide-gated (CNG) channel knock-out mice OSN axons are only transiently perturbed (Lin et al., 2000), suggesting that the CNG channel may not be the sole target of cAMP. This prompted us to investigate an alternative channel, the hyperpolarization-activated, cyclic nucleotide-gated cation channel (HCN), as a potential developmental target of cAMP in OSNs. Here, we demonstrate that HCN channels are developmentally precocious in OSNs and therefore are plausible candidates for affecting OSN axon development. Inhibition of HCN channels in dissociated OSNs significantly reduced neurite outgrowth. Moreover, in HCN1 knock-out mice the formation of glomeruli was delayed in parallel with perturbations of axon organization in the olfactory nerve. These data support the hypothesis that the outgrowth and coalescence of OSN axons is, at least in part, subject to activity-dependent mechanisms mediated via HCN channels.

Modulation of dendrodendritic synapses by the noradrenergic system in the accessory olfactory bulb (AOB) plays a key role in the formation of memory in olfactory-mediated behaviors. We have recently shown that noradrenaline (NA) inhibits mitral cells by increasing γ-aminobutyric acid inhibitory input onto mitral cells in the AOB, suggesting an excitatory action of NA on granule cells (GCs). Here, we show that NA (10 μM) elicits a long-lasting depolarization of GCs. This effect is mediated by activation of α1-adrenergic receptors as the depolarization is mimicked by phenylephrine (PE, 30 μM) and completely blocked by the α1-adrenergic receptor antagonist prazosin (300 nM). In addition to this depolarization, application of NA induced the appearance of a slow afterdepolarization (sADP) following a stimulus-elicited train of action potentials. Similarly, the group I metabotropic glutamate receptor (mGluR1) agonist DHPG (10–30 μM) also produced a depolarization of GCs and the appearance of a stimulus-induced sADP. The ionic and voltage dependence and sensitivity to blockers of the sADP suggest that it is mediated by the nonselective cationic conductance ICAN. Thus the excitatory action resulting from the activation of these receptors could be mediated by a common transduction target. Surprisingly, the excitatory effect of PE on GCs was completely blocked by the mGluR1 antagonist LY367385 (100 μM). Conversely, the effect of DHPG was not antagonized by the α1-adrenergic receptor antagonist prazosin (300 nM). These results suggest that most of the noradrenergic effect on GCs in the AOB is mediated by potentiation of a basal activity of mGluR1s.

The response of olfactory sensory neurons to TNT and RDX as well as to some volatile organic compounds present in the vapors of antipersonnel landmines has been studied both in the pig and in the rat. GC/MS analyses of different plastic components of six different kinds of landmines were performed in order to identify the components of the "perfume" of mines. Studies on rat olfactory mucosa were carried out with electro-olfactogram and calcium imaging techniques, while changes in the cyclic adenosine monophosphate (cAMP) levels following exposure to odorants and explosives were used as a criterion to evaluate the interaction of TNT and RDX with olfactory receptors in a preparation of isolated pig olfactory cilia. These studies indicate that chemical compounds associated with explosives and explosive devices can activate mammalian olfactory receptors.

Administration of serotonin (5-hydroxytryptamine, 5-HT) to pyramidal cells of the CA1 region of the hippocampus results in a hyperpolarizing response which is followed by a rebound depolarization and a decrease in the calcium-activated afterhyperpolarization (AHP). While the hyperpolarizing response has been previously shown to be mediated by receptors of the 5-HT1A subtype, the identity of the receptor(s) involved in the depolarizing response and decrease of the AHP have not been identified. In the present study the effectiveness of a series of 5-HT receptor antagonists in blocking the membrane depolarization and reduction of the AHP was assessed. While a variety of 5-HT1 and 5-HT2 antagonists were found to be ineffective, the substituted benzamide BRL 24924 was found to be a potent and selective antagonist of the 5-HT-induced depolarization and decrease in the AHP in this region. This effect however appeared unrelated to the ability of this compound to block 5-HT3 receptors, as ICS 205-930 and MDL 72222 were markedly less efficacious in blocking these effects of 5-HT. Upon blockade of 5-HT1A receptors, 5-HT elicits a depolarization which is accompanied by a marked increase in excitability. These effects were also dose-dependently antagonized by BRL 24924. The present results thus suggest the presence in the CA1 region of the hippocampus of a novel 5-HT receptor at which BRL 24924 functions as a selective antagonist and which is capable of mediating slow excitatory responses in central neurons.

Olfactory receptors (ORs) comprise a large gene family (-1200 proteins encoded by OR genes) and thus are excellent candidates for the fabrication of sensitive biosensors for explosives and other chemicals of interest. Our research project aims to address these two fundamental questions: Which are the volatile components of landmines recognized specifically by animals? How do olfactory neurons located in their nasal cavity respond to the "perfume" of mines? As regards the first question, six different plastic components of six different kinds of landmines have been analyzed. To address the second question, the response of olfactory neurons to these volatile organic compounds present in the vapors of mines, as well as to TNT and RDX, has been studied both in the pig and in the rat. Studies on rat olfactory mucosa have been carried out with electro-olfactogram (EOG) and calcium imaging techniques, while changes in the cAMP levels following exposure to odorants and explosives were used as criterion to evaluate the interaction of TNT and RDX with olfactory receptors in a preparation of isolated pig olfactory cilia. Our studies demonstrate, for the first time, that mammalian olfactory receptors can be activated by explosives and chemical given off from explosive devices and represent a deeper understanding of the molecular mechanisms by which landmine volatiles interact with sensory olfactory neurons and their receptors, necessary for the preparation of a successful biosensor.

Noradrenergic modulation of dendrodendritic synapses between the mitral and granule cells in the accessory olfactory bulb (AOB) is postulated to play a key role in the formation of memory in olfactory-mediated behaviors. Current models propose that noradrenaline (NA) increases excitation of mitral/tufted cells (M/TCs) by decreasing the release of GABA from granule cells. However, surprisingly little is known about the actions of NA at the cellular level in the AOB. Here, in recordings from AOB slices, we show that NA decreases the firing frequency of M/TCs in response to stimulation. This effect is attributable to an increase in the GABA inhibitory input to M/TCs. Application of NA (10 μm) produced an ∼20-fold increase in the frequency of GABA-induced miniature IPSCs (mIPSCs) without changing their amplitude. A pharmacological analysis indicated that the increase in mIPSCs frequency results from activation of α1 adrenergic receptors. In addition to increasing the mIPSC frequency, NA also potentiated GABA inhibitory currents induced by direct stimulation of granule cells. Together, our results suggest that NA increases the release of GABA from granule cells by acting on presynaptic receptors. Thus, the role of the noradrenergic activity in the AOB may be opposite than suggested previously: we find that the overall effect of NA in the AOB is inhibition of M/TCs.

Ion channels and ionotropic neurotransmitter receptors have long been investigated as the principle targets of inhaled volatile anesthetics (VAs), but emerging evidence suggests that G-protein coupled receptors (GPCRs) might also directly interact with VAs. To survey the extent of interaction between VAs and diverse GPCRs, we have turned to the 1000+ member family of olfactory receptors (ORs), taking advantage of their unique expression pattern of a single OR per neuron. Through optical imaging and electrophysiological recordings, we show that different VAs trigger the normal transduction cascade in distinct subsets of cells in a dose-dependant manner. Together with evidence of antagonism by odorants, this selective activation strongly implicates a direct action of VAs upon particular olfactory receptors. The finding that VAs stimulate nearly 8% of olfactory GPCRs suggests that probing related Class A GPCRs may reveal a pool of VA targets whose altered signaling contributes to anesthetic effects.

Specific anosmia, the inability to detect a particular odour, has been well documented for decades in human and animal populations. Indeed, the existence of specific anosmias was a favoured argument used to support a receptor-mediated mechanism of odourant detection prior to the molecular identification of a large family of olfactory G-protein-coupled receptors (GPCRs) in the early 1990s (Amoore, 1974; Buck & Axel, 1991). One well known anosmia in humans is the inability to sense 5-α-androst-16-en-3-one (androstenone). Androstenone is variously described as having an unpleasant (urine, sweat) or pleasant odour (sweet, floral), yet a fraction of the population cannot detect its presence. Moreover, androstenone is a pheromone in boars and is found in urine and axillary sweat in humans, making it a prospective candidate for odour-mediated communication in humans. While a role for androstenone as a human pheromone is open to debate, a widely accepted finding is the ability of humans who are initially insensitive to androstenone to acquire sensitivity to it upon continued exposure (Wysocki et al. 1989). Since the 1989 anecdotal discovery of C.J. Wysocki, several other studies have shown that humans and other species can acquire sensitivity to androstenone as well as to other odourants (Wang et al. 1993; Pause et al. 1999; Dalton et al. 2002). However, the mechanism(s) of this increased sensitivity are poorly understood. In this issue of The Journal of PhysiologyWang et al. (2004) provide evidence for a mechanism of increased sensitivity in the olfactory epithelium of humans. The testing of induced sensitivity to androstenone generally involves the selection of an experimental group that shows complete insensitivity or an abnormally high detection threshold. These subjects are repeatedly presented with androstenone over a period of 2–8 weeks, and their detection threshold monitored. As a result of this treatment some individuals show a decrease in their threshold of detection. Recently, it has been shown that the prevalence of true anosmia to androstenone in humans is much lower than originally documented (Bremner et al. 2003). Only ∼2% (versus∼30%) of the population is truly anosmic and Bremner et al. showed that individuals classified a priori as anosmic could under a more rigorous test be classified as hyposmics. This finding highlights the requirement that a careful distinction between anosmia and hyposmia is necessary for fully understanding the mechanisms by which induced sensitization occurs. Two possible mechanisms have been postulated for the increase in androstenone sensitivity, a peripheral mechanism, involving the sensory epithelium and a central mechanism, with a less defined source. In the former, interaction of androstenone with a population of maturing olfactory sensory neurones (OSNs) leads either to an increase of androstenone-specific receptors or to an increase in the number of androstenone-sensitive OSNs (Yee & Wysocki, 2001). Alternatively, a recent study argued that the increased sensitivity involves central components of the olfactory system (Mainland et al. 2002). In this study only one of the subject's nostrils was exposed to androstenone. Nonetheless, subjects showed increased sensitivity to androstenone in both nostrils, as evidenced by an increase in detection accuracy. However, the authors caution that a peripheral mechanism could also contribute to the increased sensitivity. The studies of Wang et al. (2004) presented here are in agreement with a peripheral site for this increased sensitivity to androstenone. They show, for the first time in humans, that both the electroolfactogram (EOG) responses and the olfactory event-related potential (ERP) are increased. An important issue that these studies raise is that of the relationship between the threshold for detection of a particular odourant and the possibility of acquiring sensitivity to it. While the threshold for androstenone decreased as the subjects were repeatedly exposed to this compound, the threshold for another test compound, amyl acetate, was unchanged. However, the threshold of detection for amyl acetate was initially lower than the threshold for androstenone, suggesting the possibility that only compounds with high thresholds can exhibit increases in sensitivity. What is the molecular mechanism by which continuous exposure to a particular odourant results in increased sensitivity? The proposed peripheral mechanism points to an increase in the number of androstenone-sensitive receptors. The availability of microarray chips containing olfactory receptor probes could be useful to examine this possibility. For other anosmias, most notably to isovaleric acid, the genomic locus is known, at least in mice (Griff & Reed, 1995; Zhang & Firestein, 2002), and the androstenone anosmia could be similarly identified. However, it is possible that sensitization of other components of the transduction pathway, beside the receptors themselves, could account for increased sensitivity. Although the present results seem to clearly indicate a peripheral mechanism, the varied perceptions of androstenone in humans, from unpleasant to pleasant, suggest that more than one mechanism is responsible for modulating sensitivity in the olfactory system. A more difficult question to answer is how the repeated exposure to androstenone induces a cellular change in the sensory cell (or basal cells). A possible clue may already exist in the vast literature describing the mechanisms by which neurotransmitters and synthetic compounds decrease or increase the activity of the many other GPCRs in neural and other systems.

An odor perception is the brain's interpretation of the activation pattern of many peripheral sensory neurons that are differentially sensitive to a wide variety of odors. The sensitivity of these neurons is determined by which of the thousand or so odor receptor proteins they express on their surface. Understanding the odor code thus requires mapping the receptive range of odorant receptors. We have adopted a pharmacological approach that uses a large and diverse pool of odorous compounds to characterize the molecular receptive field of an odor receptor. We found a high specificity for certain molecular features, but high tolerance for others—a strategy that enables the olfactory apparatus to be both highly discriminating, and able to recognize several thousand odorous compounds.

Dendrodendritic inhibition between mitral and granule cells in the olfactory bulb is thought to play an important role in olfactory discrimination. In this issue of Neuron , Xiong and Chen (2002) explore the propagation of action potentials along the secondary dendrites of mitral cells and their modulation by dendrodendritic inhibition.

The effects of serotonin on pyramidal cells of layer V of the medial prefrontal cortex were examined using intracellular recording techniques in rat brain slices in vitro. Bath administration of serotonin (0.3-100 microM) produced two distinct responses which could be differentiated physiologically and pharmacologically. The first of these responses was a membrane hyperpolarization. This effect of serotonin was associated with a decrease in input resistance and was independent of the transmembrane chloride gradient, suggesting that it was mediated by an increase in potassium conductance. The ability of serotonin to induce a hyperpolarization was mimicked by (+/-)-8-hydroxy-dipropylaminotetralin hydrobromide and was blocked by BMY 7378 and spiperone but not by ketanserin, indicating that it was mediated by the activation of receptors of the 5-hydroxytryptamine1A subtype. The second response to serotonin involved a membrane depolarization, the replacement of the afterhyperpolarization that follows a burst of spikes in these cells by a slow depolarizing afterpotential, and a decrease in spike frequency accommodation. These effects were mimicked by 4-bromo-2,5-dimethoxyphenyl-isopropylamine and antagonized by ketanserin and by low concentrations of spiperone, indicating that they were mediated by the activation of 5-hydroxytryptamine2 receptors. Interestingly, qualitatively identical responses could be elicited in these cells by activation of muscarinic and alpha 1-adrenergic receptors suggesting that 5-hydroxytryptamine2, muscarinic and alpha 1-adrenergic receptors converge onto a common set of membrane mechanisms to increase cellular excitability. Although 5-hydroxytryptamine1A and 5-hydroxytryptamine2 receptors mediated opposing effects on membrane excitability, most pyramidal neurons appeared to express both receptor subtypes on their membrane surface. The coactivation of both receptor subtypes resulted in a selective enhancement of responsiveness to strong excitatory stimuli with little effect on weaker stimuli. The paradoxical presence of two serotonin receptors mediating opposite effects on membrane excitability in the same cell provides a flexible mechanism by which serotonin might regulate how pyramidal neurons encode incoming excitatory stimuli onto firing activity.

Neurogenesis persists in the olfactory system throughout life. The mechanisms of how new neurons are generated, how they integrate into circuits, and their role in coding remain mysteries. Here we report a technique that will greatly facilitate research into these questions. We found that electroporation can be used to robustly and selectively label progenitors in the Subventicular Zone. The approach was performed postnatally, without surgery, and with near 100% success rates. Labeling was found in all classes of interneurons in the olfactory bulb, persisted to adulthood and had no adverse effects. The broad utility of electroporation was demonstrated by encoding a calcium sensor and markers of intracellular organelles. The approach was found to be effective in wildtype and transgenic mice as well as rats. Given its versatility, robustness, and both time and cost effectiveness, this method offers a powerful new way to use genetic manipulation to understand adult neurogenesis.

A superfusion system was used to study the effects of excitatory amino acids (EAA) on release of [3H]dopamine ([3H]DA) previously taken up by rat substantia nigra (SN) slices. The EAA tested (20-250 microM), with the exception of quisqualate and kainate, markedly evoked [3H]DA release from nigral slices when Mg2+ ions were omitted from the superfusion medium. The EAA receptor agonists exhibited the following relative potency in stimulating [3H]DA release: L-glutamate (L-Glu) greater than N-methyl-D-aspartate (NMDA) greater than NM(D,L)A greater than D-Glu much greater than quisqualate = kainate. D-2-Amino-5-phosphonovalerate (100-200 microM), an antagonist for NMDA receptors, substantially reduced [3H]DA release evoked by L-Glu or NMDA. In contrast, L-Glu diethyl ester (100-200 microM) produced a lesser blocking effect on [3H]DA release evoked by the EAA. Further experiments showed that the NMDA-mediated release of [3H]DA was totally suppressed by the omission of Ca2+ or by the addition of tetrodotoxin (0.1 microM) to the superfusion medium. In addition, strychnine, an antagonist for glycine (Gly) receptors, significantly decreased NMDA (100 microM)-evoked as well as glycine (100 microM)-evoked release of [3H]DA from nigral slices. The results shown support the idea that activation of NMDA subtype receptors in SN may trigger a Ca2+-dependent release of DA from dendrites of nigro-striatal DA-containing neurons. Furthermore, a transsynaptic mechanism that may partially involve Gly-containing interneurons is proposed to account for some of the events mediating NMDA receptor activation and DA release in SN.

Polyamines such as spermine are thought to be endogenous regulators of NMDA (N-methyl-d-aspartate)-type glutamate receptors. Polyamine block of NMDA receptors was studied in excised outside-out patches from rat hippocampal neurons and Xenopus oocytes expressing recombinant receptors. Extracellular spermine and arcaine reduced NMDA single-channel conductance in a voltage-dependent manner, with partial relief of block evident at large inside negative membrane potentials. Reducing extracellular Na+ concentration increased the apparent affinities for spermine and arcaine, indicating strong interaction between spermine and permeant ions. Internal spermine also blocked NMDA channels in a voltage-dependent manner, with relief of block evident at large inside positive potentials. The Woodhull model of channel block by an impermeant ion adequately described the actions of external spermine from −60 to +60mV, but failed for more negative potentials. Eyring rate theory for a permeable blocker with two barriers and one binding site adequately described the voltage-dependent block and relief from block by both external and internal spermine over the range of −120 to +60mV. These findings indicate that polyamines block and permeate neuronal NMDA receptor channels from the extracellular and intracellular sides, although sensitivity to internal spermine is probably too low to be physiologically relevant.