Cholecystokinin-expressing GABAergic (CCK-GABA) neurons are perisomatic inhibitory cells that have been argued to regulate emotion and sculpt the network oscillations associated with cognition. However, no study has selectively manipulated CCK-GABA neuron activity during behavior in freely-moving animals. To explore the behavioral effects of activating CCK-GABA neurons on emotion and cognition, we utilized a novel intersectional genetic mouse model coupled with a chemogenetic approach. Specifically, we generated triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FL-hM3Dq (CCK-GABA/hM3Dq) mice that expressed the synthetic excitatory hM3Dq receptor in CCK-GABA neurons. Results showed that clozapine-N-oxide (CNO)-mediated activation of CCK-GABA neurons did not alter open field (OF) or tail suspension (TS) performance and only slightly increased anxiety in the elevated plus maze (EPM). Although CNO treatment had only modestly affected emotional behavior, it significantly enhanced multiple cognitive and memory behaviors including social recognition, contextual fear conditioning, contextual discrimination, object recognition, and problem-solving in the puzzle box. Collectively, these findings suggest that systemic activation of CCK-GABA neurons minimally affects emotion but significantly enhances cognition and memory. Our results imply that CCK-GABA neurons are more functionally diverse than originally expected and could serve as a potential therapeutic target for the treatment of cognitive/memory disorders.

There is growing evidence that interneurons (INs) orchestrate neural activity and plasticity in corticoamygdala circuits to regulate fear behaviors. However, defining the precise role of cholecystokinin-expressing INs (CCK INs) remains elusive due to the technical challenge of parsing this population from CCK-expressing principal neurons (CCK PNs). Here, we used an intersectional genetic strategy in CCK-Cre;Dlx5/6-Flpe double-transgenic mice to study the anatomical, molecular and electrophysiological properties of CCK INs in the basal amygdala (BA) and optogenetically manipulate these cells during fear extinction. Electrophysiological recordings confirmed that this strategy targeted GABAergic cells and that a significant proportion expressed functional cannabinoid CB1 receptors; a defining characteristic of CCK-expressing basket cells. However, immunostaining showed that subsets of the genetically-targeted cells expressed either neuropeptide Y (NPY; 29%) or parvalbumin (PV; 17%), but not somatostatin (SOM) or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-α. Further morphological and electrophysiological analyses showed that four IN types could be identified among the EYFP-expressing cells: CCK/cannabinoid receptor type 1 (CB1R)-expressing basket cells, neurogliaform cells, PV+ basket cells, and PV+ axo-axonic cells. At the behavioral level, in vivo optogenetic photostimulation of the targeted population during extinction acquisition led to reduced freezing on a light-free extinction retrieval test, indicating extinction memory facilitation; whereas photosilencing was without effect. Conversely, non-selective (i.e., inclusive of INs and PNs) photostimulation or photosilencing of CCK-targeted cells, using CCK-Cre single-transgenic mice, impaired extinction. These data reveal an unexpectedly high degree of phenotypic complexity in a unique population of extinction-modulating BA INs.

Topography in the avian cochlear nucleus magnocellularis (NM) is represented as gradually increasing characteristic frequency (CF) along the caudolateral-to-rostromedial axis. In this study, we characterized the organization and cell biophysics of the caudolateral NM (NMc) in chickens (Gallus gallus). Examination of cellular and dendritic architecture first revealed that NMc contains small neurons and extensive dendritic processes, in contrast to adendritic, large neurons located more rostromedially. Individual dye-filling study further demonstrated that NMc is divided into two subregions, with NMc2 neurons having larger and more complex dendritic fields than NMc1. Axonal tract tracing studies confirmed that NMc1 and NMc2 neurons receive afferent inputs from the auditory nerve and the superior olivary nucleus, similar to the adendritic NM. However, the auditory axons synapse with NMc neurons via small bouton-like terminals, unlike the large end bulb synapses on adendritic NM neurons. Immunocytochemistry demonstrated that most NMc2 neurons express cholecystokinin but not calretinin, distinct from NMc1 and adendritic NM neurons that are cholecystokinin negative and mostly calretinin positive. Finally, whole-cell current clamp recordings revealed that NMc neurons require significantly lower threshold current for action potential generation than adendritic NM neurons. Moreover, in contrast to adendritic NM neurons that generate a single-onset action potential, NMc neurons generate multiple action potentials to suprathreshold sustained depolarization. Taken together, our data indicate that NMc contains multiple neuron types that are structurally, connectively, molecularly, and physiologically different from traditionally defined NM neurons, emphasizing specialized neural properties for processing low-frequency sounds.

To understand the hippocampus, it is necessary to understand the subiculum. Unlike other hippocampal subfields, the subiculum projects to almost all distal hippocampal targets, highlighting its critical importance for external networks. The present studies, in male rats and mice, reveal a new category of dorsal subiculum neurons that innervate both the mammillary bodies (MBs) and the retrosplenial cortex (RSP). These bifurcating neurons comprise almost half of the hippocampal cells that project to RSP. The termination of these numerous collateral projections was visualized within the medial mammillary nucleus and the granular RSP (area 29). These collateral projections included subiculum efferents that cross to the contralateral MBs. Within the granular RSP, the collateral projections form a particularly dense plexus in deep Layer II and Layer III. This retrosplenial termination site colocalized with markers for VGluT2 and neurotensin. While efferents from the hippocampal CA fields standardly collateralize, subiculum projections often have only one target site. Consequently, the many collateral projections involving the RSP and the MBs present a relatively unusual pattern for the subiculum, which presumably relates to how both targets have complementary roles in spatial processing. Furthermore, along with the anterior thalamic nuclei, the MBs and RSP are key members of a memory circuit, which is usually described as both starting and finishing in the hippocampus. The present findings reveal how the hippocampus simultaneously engages different parts of this circuit, so forcing an important revision of this network.

The ability of neurons to produce behaviorally relevant activity in the absence of pathology relies on the fine balance of synaptic inhibition to excitation. In the hippocampal CA1 microcircuit, this balance is maintained by a diverse population of inhibitory interneurons that receive largely similar glutamatergic afferents as their target pyramidal cells, with EPSCs generated by both AMPA receptors (AMPARs) and NMDA receptors (NMDARs). In this study, we take advantage of a recently generated GluN2A-null rat model to assess the contribution of GluN2A subunits to glutamatergic synaptic currents in three subclasses of interneuron found in the CA1 region of the hippocampus. For both parvalbumin-positive and somatostatin-positive interneurons, the GluN2A subunit is expressed at glutamatergic synapses and contributes to the EPSC. In contrast, in cholecystokinin (CCK)-positive interneurons, the contribution of GluN2A to the EPSC is negligible. Furthermore, synaptic potentiation at glutamatergic synapses on CCK-positive interneurons does not require the activation of GluN2A-containing NMDARs but does rely on the activation of NMDARs containing GluN2B and GluN2D subunits.

A key assumption in studies of cortical functions is that excitatory principal neurons, but not inhibitory cells express calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) resulting in a widespread use of CaMKIIα promoter-driven protein expression for principal cell manipulation and monitoring their activities. Using neuroanatomical and electrophysiological methods we demonstrate that in addition to pyramidal neurons, multiple types of cortical GABAegic cells are targeted by adeno-associated viral vectors (AAV) driven by the CaMKIIα promoter in both male and female mice. We tested the AAV5 and AAV9 serotype of viruses with either Channelrhodopsin 2 (ChR2)-mCherry or Archaerhodopsin-T-green fluorescent protein (GFP) constructs, with different dilutions. We show that in all cases, the reporter proteins can visualize a large fraction of different interneuron types, including parvalbumin (PV), somatostatin (SST), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and cholecystokinin (CCK)-containing GABAergic cells, which altogether cover around 60% of the whole inhibitory cell population in cortical structures. Importantly, the expression of the excitatory opsin Channelrhodopsin 2 in the interneurons effectively drive spiking of infected GABAergic cells even if the immunodetectability of reporter proteins is ambiguous. Thus, our results challenge the use of CaMKIIα promoter-driven protein expression as a selective tool in targeting cortical glutamatergic neurons using viral vectors.

The local circuitry within olfactory bulb (OB) glomeruli filters, transforms, and facilitates information transfer from olfactory sensory neurons to bulb output neurons. Two key elements of this circuit are glutamatergic tufted cells (TCs) and GABAergic periglomerular (PG) cells, both of which actively shape mitral cell activity and bulb output. A subtype of TCs, the external TCs (eTCs), can synaptically excite PG cells, but there are unresolved questions about other aspects of the glomerular connections, including the extent of connectivity between eTCs and the precise nature of reciprocal interactions between TCs and PG cells. We combined patch-clamp recordings in OB slices and optophysiological tools to investigate local functional connections within glomeruli of mice and rats. When TCs that express cholecystokinin (CCK) were optically suppressed, excitatory inputs to "uniglomerular" PG cells that extend dendrites to one glomerulus were decreased, consistent with TC activation being required for most excitation of these PG cells. However, TC suppression had no effect on EPSCs in eTCs, arguing that TCs make few, if any, direct glutamatergic synaptic connections with eTCs. The absence of synaptic connections among eTCs was also supported by recordings in eTC pairs. Last, we show using similar optical suppression methods that GAD65-expressing PG cells, mainly uniglomerular cells, provide strong inhibition in eTCs. Our results imply that the local network of CCK-expressing TCs form potent reciprocal chemical synaptic connections with GAD65-expressing uniglomerular PG cells but not eTCs. This configuration favors local inhibition over recurrent excitation within a glomerulus, limiting its output.

In mammalian olfaction, inhalation drives the temporal patterning of neural activity that underlies early olfactory processing. It remains poorly understood how the neural circuits that process incoming olfactory information are engaged in the context of inhalation-linked dynamics. Here, we used artificial inhalation and two-photon calcium imaging to compare the dynamics of activity evoked by odorant inhalation across major cell types of the mouse olfactory bulb (OB). We expressed GCaMP6f or jRGECO1a in mitral and tufted cell (MTC) subpopulations, olfactory sensory neurons (OSNs), and two major juxtaglomerular interneuron classes and imaged responses to a single inhalation of odorant. Activity in all cell types was strongly linked to inhalation, and all cell types showed some variance in the latency, rise times, and durations of their inhalation-linked response. Juxtaglomerular interneuron dynamics closely matched that of sensory inputs, while MTCs showed the highest diversity in responses, with a range of latencies and durations that could not be accounted for by heterogeneity in sensory input dynamics. Diversity was apparent even among "sister" tufted cells innervating the same glomerulus. Surprisingly, inhalation-linked responses of MTCs were highly overlapping and could not be distinguished on the basis of their inhalation-linked dynamics, with the exception of a subpopulation of superficial tufted cells expressing cholecystokinin (CCK). Our results are consistent with a model in which diversity in inhalation-linked patterning of OB output arises first at the level of sensory input and is enhanced by feedforward inhibition from juxtaglomerular interneurons which differentially impact different subpopulations of OB output neurons.

Hypofunction of N-methyl-d-aspartate receptors (NMDARs) in inhibitory GABAergic interneurons is implicated in the pathophysiology of schizophrenia (SZ), a heritable disorder with many susceptibility genes. However, it is still unclear how SZ risk genes interfere with NMDAR-mediated synaptic transmission in diverse inhibitory interneuron populations. One putative risk gene is neuregulin 1 (NRG1), which signals via the receptor tyrosine kinase ErbB4, itself a schizophrenia risk gene. The type I isoform of NRG1 shows increased expression in the brain of SZ patients, and ErbB4 is enriched in GABAergic interneurons expressing parvalbumin (PV) or cholecystokinin (CCK). Here, we investigated ErbB4 expression and synaptic transmission in interneuronal populations of the hippocampus of transgenic mice overexpressing NRG1 type I (NRG1tg-type-I mice). Immunohistochemical analyses confirmed that ErbB4 was coexpressed with either PV or CCK in hippocampal interneurons, but we observed a reduced number of ErbB4-immunopositive interneurons in the NRG1tg-type-I mice. NMDAR-mediated currents in interneurons expressing PV (including PV+ basket cells) or CCK were reduced in NRG1tg-type-I mice compared to their littermate controls. We found no difference in AMPA receptor-mediated currents. Optogenetic activation (5 pulses at 20 Hz) of local glutamatergic fibers revealed a decreased NMDAR-mediated contribution to disynaptic GABAergic inhibition of pyramidal cells in the NRG1tg-type-I mice. GABAergic synaptic transmission from either PV+ or CCK+ interneurons, and glutamatergic transmission onto pyramidal cells, did not significantly differ between genotypes. The results indicate that synaptic NMDAR-mediated signaling in hippocampal interneurons is sensitive to chronically elevated NGR1 type I levels. This may contribute to the pathophysiological consequences of increased NRG1 expression in SZ.

A fundamental strategy in sensory coding is parallel processing, whereby unique, distinct features of sensation are computed and projected to the central target in the form of submodal maps. It remains unclear, however, whether such parallel processing strategy is employed in the main olfactory system, which codes the complex hierarchical odor and behavioral scenes. A potential scheme is that distinct subsets of projection neurons in the olfactory bulb (OB) form parallel projections to the targets. Taking advantage of the observation that the distinct projection neurons develop at different times, we developed a Cre-loxP-based method that allows for birthdate-specific labeling of cell bodies and their axon projections in mice. This birthdate tag analysis revealed that the mitral cells (MCs) born in an early developmental stage and the external tufted cells (TCs) born a few days later form segregated parallel projections. Specifically, the latter subset converges the axons onto only two small specific targets, one of which, located at the anterolateral edge of the olfactory tubercle (OT), excludes widespread MC projections. This target is made up of neurons that express dopamine D1 but not D2 receptor and corresponds to the most anterolateral isolation of the CAP compartments (aiCAP) that were defined previously. This finding of segregated projections suggests that olfactory sensing does indeed involve parallel processing of functionally distinct submodalities. Importantly, the birthdate tag method used here may pave the way for deciphering the functional meaning of these individual projection pathways in the future.