Minmin Luo

Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function

Optogenetic methods have emerged as powerful tools for dissecting neural circuit connectivity, function and dysfunction. We used a bacterial artificial chromosome (BAC) transgenic strategy to express the H134R variant of channelrhodopsin-2, ChR2(H134R), under the control of cell type–specific promoter elements. We performed an extensive functional characterization of the newly established VGAT-ChR2(H134R)-EYFP, ChAT-ChR2(H134R)-EYFP, Tph2-ChR2(H134R)-EYFP and Pvalb(H134R)-ChR2-EYFP BAC transgenic mouse lines and demonstrate the utility of these lines for precisely controlling action-potential firing of GABAergic, cholinergic, serotonergic and parvalbumin-expressing neuron subsets using blue light. This resource of cell type–specific ChR2(H134R) mouse lines will facilitate the precise mapping of neuronal connectivity and the dissection of the neural basis of behavior.

Response Correlation Maps of Neurons in the Mammalian Olfactory Bulb

To define the relationship between glomerular activation patterns and neuronal olfactory responses in the main olfactory bulb, intracellular recordings were combined with optical imaging of intrinsic signals. Response correlation maps (RCMs) were constructed by correlating the fluctuations in membrane potential and firing rate during odorant presentations with patterns of glomerular activation. The RCMs indicated that mitral/tufted cells were excited by activation of a focal region surrounding their principal glomerulus and generally inhibited by activation of more distant regions. However, the structure of the RCMs and the relative contribution of excitatory and inhibitory glomerular input evolved and even changed sign during and after odorant application. These data suggest a dynamic center-surround organization of mitral/tufted cell receptive fields.

Dorsal Raphe Neurons Signal Reward through 5-HT and Glutamate

The dorsal raphe nucleus (DRN) in the midbrain is a key center for serotonin (5-hydroxytryptamine; 5-HT)-expressing neurons. Serotonergic neurons in the DRN have been theorized to encode punishment by opposing the reward signaling of dopamine neurons. Here, we show that DRN neurons encode reward, but not punishment, through 5-HT and glutamate. Optogenetic stimulation of DRN Pet-1 neurons reinforces mice to explore the stimulation-coupled spatial region, shifts sucrose preference, drives optical self-stimulation, and directs sensory discrimination learning. DRN Pet-1 neurons increase their firing activity during reward tasks, and this activation can be used to rapidly change neuronal activity patterns in the cortex. Although DRN Pet-1 neurons are often associated with 5-HT, they also release glutamate, and both neurotransmitters contribute to reward signaling. These experiments demonstrate the ability of DRN neurons to organize reward behaviors and might provide insights into the underlying mechanisms of learning facilitation and anhedonia treatment.

Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors.

Most sensory systems are primarily specialized to detect one sensory modality. Here we report that olfactory sensory neurons (OSNs) in the mammalian nose can detect two distinct modalities transmitted by chemical and mechanical stimuli. As revealed by patch-clamp recordings, many OSNs respond not only to odorants, but also to mechanical stimuli delivered by pressure ejections of odor-free Ringer solution. The mechanical responses correlate directly with the pressure intensity and show several properties similar to those induced by odorants, including onset latency, reversal potential and adaptation to repeated stimulation. Blocking adenylyl cyclase or knocking out the cyclic nucleotide–gated channel CNGA2 eliminates the odorant and the mechanical responses, suggesting that both are mediated by a shared cAMP cascade. We further show that this mechanosensitivity enhances the firing frequency of individual neurons when they are weakly stimulated by odorants and most likely drives the rhythmic activity (theta oscillation) in the olfactory bulb to synchronize with respiration.

Acute and Long-Term Suppression of Feeding Behavior by POMC Neurons in the Brainstem and Hypothalamus, Respectively

POMC-derived melanocortins inhibit food intake. In the adult rodent brain, POMC-expressing neurons are located in the arcuate nucleus (ARC) and the nucleus tractus solitarius (NTS), but it remains unclear how POMC neurons in these two brain nuclei regulate feeding behavior and metabolism differentially. Using pharmacogenetic methods to activate or deplete neuron groups in separate brain areas, in the present study, we show that POMC neurons in the ARC and NTS suppress feeding behavior at different time scales. Neurons were activated using the DREADD (designer receptors exclusively activated by designer drugs) method. The evolved human M3-muscarinic receptor was expressed in a selective population of POMC neurons by stereotaxic infusion of Cre-recombinase–dependent, adeno-associated virus vectors into the ARC or NTS of POMC-Cre mice. After injection of the human M3-muscarinic receptor ligand clozapine-N-oxide (1 mg/kg, i.p.), acute activation of NTS POMC neurons produced an immediate inhibition of feeding behavior. In contrast, chronic stimulation was required for ARC POMC neurons to suppress food intake. Using adeno-associated virus delivery of the diphtheria toxin receptor gene, we found that diphtheria toxin–induced ablation of POMC neurons in the ARC but not the NTS, increased food intake, reduced energy expenditure, and ultimately resulted in obesity and metabolic and endocrine disorders. Our results reveal different behavioral functions of POMC neurons in the ARC and NTS, suggesting that POMC neurons regulate feeding and energy homeostasis by integrating long-term adiposity signals from the hypothalamus and short-term satiety signals from the brainstem.

Long-range GABAergic projection in a circuit essential for vocal learning

The anterior forebrain pathway (AFP) in the passerine song system is essential for song learning but not for song production. Several lines of evidence suggest that area X, a major nucleus in the AFP, forms part of the avian striatum. A key feature of striatal projection neurons is that they use the inhibitory neurotransmitter γ‐aminobutyric acid (GABA). Some area X neurons express GABA‐like immunoreactivity, but the neurotransmitter phenotype of the projection neurons is largely unknown.

Odor Information Processing by the Olfactory Bulb Analyzed in Gene-Targeted Mice

In mammals, olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) gene project with precise stereotypy onto mitral/tufted (M/T) cells in the main olfactory bulb (MOB). It remains challenging to understand how incoming olfactory signals are transformed into outputs of M/T cells. By recording from OSNs expressing mouse I7 receptor and their postsynaptic neurons in the bulb, we found that I7 OSNs and their corresponding M/T cells exhibit similarly selective tuning profiles at low concentrations. Increasing the concentration significantly reduces response selectivity for both OSNs and M/T cells, although the tuning curve of M/T cells remains comparatively narrow. By contrast, interneurons in the MOB are broadly tuned, and blocking GABAergic neurotransmission reduces selectivity of M/T cells at high odorant concentrations. Our results indicate that olfactory information carried by an OR is channeled to its corresponding M/T cells and support the role of lateral inhibition via interneurons in sharpening the tuning of M/T cells.

Guanylyl cyclase-D in the olfactory CO2 neurons is activated by bicarbonate

Atmospheric CO2 is an important environmental cue that regulates several types of animal behavior. In mice, CO2 responses of the olfactory sensory neurons (OSNs) require the activity of carbonic anhydrase to catalyze the conversion of CO2 to bicarbonate and the opening of cGMP-sensitive ion channels. However, it remains unknown how the enhancement of bicarbonate levels results in cGMP production. Here, we show that bicarbonate activates cGMP-producing ability of guanylyl cyclase-D (GC-D), a membrane GC exclusively expressed in the CO2-responsive OSNs, by directly acting on the intracellular cyclase domain of GC-D. Also, the molecular mechanism for GC-D activation is distinct from the commonly believed model of “release from repression” for other membrane GCs. Our results contribute to our understanding of the molecular mechanisms of CO2 sensing and suggest diverse mechanisms of molecular activation among membrane GCs.

Optogenetic Activation of Basal Forebrain Cholinergic Neurons Modulates Neuronal Excitability and Sensory Responses in the Main Olfactory Bulb

The main olfactory bulb (MOB) in mammals receives massive centrifugal input from cholinergic neurons in the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, the activity of which is thought to be correlated with animal behaving states, such as attention. Cholinergic signals in the bulb facilitate olfactory discrimination and learning, but it has remained controversial how the activity of HDB cholinergic neurons modulates neuronal excitability and olfactory responses in the MOB. In this study, we used an optogenetic approach to selectively activate HDB cholinergic neurons and recorded the effect of this activation on the spontaneous firing activity and odor-evoked responses of mouse MOB neurons. Cells were juxtacellularly labeled and their specific types were morphologically determined. We find that light stimulation of HDB cholinergic neurons inhibits the spontaneous firing activity of all major cell types, including mitral/tufted (M/T) cells, periglomerular (PG) cells, and GABAergic granule cells. Inhibitions are significantly produced by stimulation at 10 Hz and further enhanced at higher frequencies. In addition, cholinergic activation sharpens the olfactory tuning curves of a majority of M/T cells but broadly potentiates odor-evoked responses of PG cells and granule cells. These results demonstrate strong effects of the basal forebrain cholinergic system on modulating neuronal excitability in the MOB and support the hypothesis that cholinergic activity increases olfactory discrimination capability.

Role for the Membrane Receptor Guanylyl Cyclase-C in Attention Deficiency and Hyperactive Behavior

A receptor for gut hormones also functions in the brain, where its loss affects attention. Midbrain dopamine neurons regulate many important behavioral processes, and their dysfunctions are associated with several human neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD) and schizophrenia. Here, we report that these neurons in mice selectively express guanylyl cyclase-C (GC-C), a membrane receptor previously thought to be expressed mainly in the intestine. GC-C activation potentiates the excitatory responses mediated by glutamate and acetylcholine receptors via the activity of guanosine 3′,5′-monophosphate–dependent protein kinase (PKG). Mice in which GC-C has been knocked out exhibit hyperactivity and attention deficits. Moreover, their behavioral phenotypes are reversed by ADHD therapeutics and a PKG activator. These results indicate important behavioral and physiological functions for the GC-C/PKG signaling pathway within the brain and suggest new therapeutic targets for neuropsychiatric disorders related to the malfunctions of midbrain dopamine neurons.