A brief history of somatostatin interneuron taxonomy or: how many somatostatin subtypes are there, really?

We provide a brief (and unabashedly biased) overview of the pre-transcriptomic history of somatostatin interneuron taxonomy, followed by a chronological summary of the large-scale, NIH-supported effort over the last ten years to generate a comprehensive, single-cell RNA-seq-based taxonomy of cortical neurons. Focusing on somatostatin interneurons, we present the perspective of experimental neuroscientists trying to incorporate the new classification schemes into their own research while struggling to keep up with the ever-increasing number of proposed cell types, which seems to double every two years. We suggest that for experimental analysis, the most useful taxonomic level is the subdivision of somatostatin interneurons into ten or so "supertypes," which closely agrees with their more traditional classification by morphological, electrophysiological and neurochemical features. We argue that finer subdivisions ("t-types" or "clusters"), based on slight variations in gene expression profiles but lacking clear phenotypic differences, are less useful to researchers and may actually defeat the purpose of classifying neurons to begin with. We end by stressing the need for generating novel tools (mouse lines, viral vectors) for genetically targeting distinct supertypes for expression of fluorescent reporters, calcium sensors and excitatory or inhibitory opsins, allowing neuroscientists to chart the input and output synaptic connections of each proposed subtype, reveal the position they occupy in the cortical network and examine experimentally their roles in sensorimotor behaviors and cognitive brain functions.

Flexible gating between subspaces in a neural network model of internally guided task switching.

Behavioral flexibility relies on the brain's ability to switch rapidly between multiple tasks, even when the task rule is not explicitly cued but must be inferred through trial and error. The underlying neural circuit mechanism remains poorly understood. We investigated recurrent neural networks (RNNs) trained to perform an analog of the classic Wisconsin Card Sorting Test. The networks consist of two modules responsible for rule representation and sensorimotor mapping, respectively, where each module is comprised of a circuit with excitatory neurons and three major types of inhibitory neurons. We found that rule representation by self-sustained persistent activity across trials, error monitoring and gated sensorimotor mapping emerged from training. Systematic dissection of trained RNNs revealed a detailed circuit mechanism that is consistent across networks trained with different hyperparameters. The networks' dynamical trajectories for different rules resided in separate subspaces of population activity; the subspaces collapsed and performance was reduced to chance level when dendrite-targeting somatostatin-expressing interneurons were silenced, illustrating how a phenomenological description of representational subspaces is explained by a specific circuit mechanism.

Cell type- and layer-specific plasticity of olfactory bulb interneurons following olfactory sensory neuron ablation.

Lifelong neurogenesis endows the mouse olfactory system with a capacity for regeneration that is unique in the mammalian nervous system. Throughout life, olfactory sensory neurons (OSNs) are generated from olfactory epithelium (OE) stem cells in the nose, while the subventricular zone generates neuroblasts that migrate to the olfactory bulb (OB) and differentiate into multiple populations of inhibitory interneurons. Methimazole (MMZ) selectively ablates OSNs, but OE neurogenesis enables OSN repopulation and gradual recovery of OSN input to the OB within 6 weeks. However, it is not known how OB interneurons are affected by this loss and subsequent regeneration of OSN input following MMZ treatment. We found that dopaminergic neuron density was significantly reduced 7-14 days post-MMZ but recovered substantially at 35 days. The density of parvalbumin-expressing interneurons was unaffected by MMZ; however, their soma size was significantly reduced at 7-14 days post-MMZ, recovering by 35 days. Surprisingly, we found a transient increase in the density of calretinin-expressing neurons in the glomerular and external plexiform layers, but not the granule cell layer, 7 days post-MMZ. This could not be accounted for by increased neurogenesis but may result from increased calretinin expression. Together, our data demonstrate cell type- and layer-specific changes in OB interneuron density and morphology after MMZ treatment, providing new insight into the range of plasticity mechanisms employed by OB circuits during loss and regeneration of sensory input.

Modulation of large rhythmic depolarizations in human large basket cells by norepinephrine and acetylcholine.

Rhythmic brain activity is critical to many brain functions and is sensitive to neuromodulation, but so far very few studies have investigated this activity on the cellular level in vitro in human brain tissue samples. This study reveals and characterizes a novel rhythmic network activity in the human neocortex. Using intracellular patch-clamp recordings of human cortical neurons, we identify large rhythmic depolarizations (LRDs) driven by glutamate release but not by GABA. These LRDs are intricate events made up of multiple depolarizing phases, occurring at ~0.3 Hz, have large amplitudes and long decay times. Unlike human tissue, rat neocortex layers 2/3 exhibit no such activity under identical conditions. LRDs are mainly observed in a subset of L2/3 interneurons that receive substantial excitatory inputs and are likely large basket cells based on their morphology. LRDs are highly sensitive to norepinephrine (NE) and acetylcholine (ACh), two neuromodulators that affect network dynamics. NE increases LRD frequency through ß-adrenergic receptor activity while ACh decreases it via M4 muscarinic receptor activation. Multi-electrode array recordings show that NE enhances and synchronizes oscillatory network activity, whereas ACh causes desynchronization. Thus, NE and ACh distinctly modulate LRDs, exerting specific control over human neocortical activity.

Cholinergic modulation of interhemispheric inhibition in the mouse motor cortex.

Interhemispheric inhibition of the homotopic motor cortex is believed to be effective for accurate unilateral motor function. However, the cellular mechanisms underlying interhemispheric inhibition during unilateral motor behavior remain unclear. Furthermore, the impact of the neuromodulator acetylcholine on interhemispheric inhibition and the associated cellular mechanisms are not well understood. To address this knowledge gap, we conducted recordings of neuronal activity from the bilateral motor cortex of mice during the paw-reaching task. Subsequently, we analyzed interhemispheric spike correlation at the cell-pair level, classifying putative cell types to explore the underlying cellular circuitry mechanisms of interhemispheric inhibition. We found a cell-type pair-specific enhancement of the interhemispheric spike correlation when the mice were engaged in the reaching task. We also found that the interhemispheric spike correlation was modulated by pharmacological acetylcholine manipulation. The local field responses to contralateral excitation differed along the cortical depths, and muscarinic receptor antagonism enhanced the inhibitory component of the field response in deep layers. The muscarinic subtype M2 receptor is predominantly expressed in deep cortical neurons, including GABAergic interneurons. These results suggest that GABAergic interneurons expressing muscarinic receptors in deep layers mediate the neuromodulation of interhemispheric inhibition in the homotopic motor cortex.

Specific and comprehensive genetic targeting reveals brain-wide distribution and synaptic input patterns of GABAergic axo-axonic interneurons.

Axo-axonic cells (AACs), also called chandelier cells (ChCs) in the cerebral cortex, are the most distinctive type of GABAergic interneurons described in the neocortex, hippocampus, and basolateral amygdala (BLA). AACs selectively innervate glutamatergic projection neurons (PNs) at their axon initial segment (AIS), thus may exert decisive control over PN spiking and regulate PN functional ensembles. However, the brain-wide distribution, synaptic connectivity, and circuit function of AACs remain poorly understood, largely due to the lack of specific and reliable experimental tools. Here, we have established an intersectional genetic strategy that achieves specific and comprehensive targeting of AACs throughout the mouse brain based on their lineage (Nkx2.1) and molecular (Unc5b, Pthlh) markers. We discovered that AACs are deployed across essentially all the pallium-derived brain structures, including not only the dorsal pallium-derived neocortex and medial pallium-derived hippocampal formation, but also the lateral pallium-derived claustrum-insular complex, and the ventral pallium-derived extended amygdaloid complex and olfactory centers. AACs are also abundant in anterior olfactory nucleus, taenia tecta, and lateral septum. AACs show characteristic variations in density across neocortical areas and layers and across subregions of the hippocampal formation. Neocortical AACs comprise multiple laminar subtypes with distinct dendritic and axonal arborization patterns. Retrograde monosynaptic tracing from AACs across neocortical, hippocampal, and BLA regions reveal shared as well as distinct patterns of synaptic input. Specific and comprehensive targeting of AACs facilitates the study of their developmental genetic program and circuit function across brain structures, providing a ground truth platform for understanding the conservation and variation of a bona fide cell type across brain regions and species.

Whether we are memorising facts or reacting to a loud noise, nerve cells in different brain areas must be able to communicate with one another through precise, meaningful signals. Specialized nerve cells known as interneurons act as "traffic lights" to precisely regulate when and where this information flows in neural circuits. Axo-axonic cells are a rare type of inhibitory interneuron that are thought to be particularly important for controlling the passage of information between different groups of excitatory neurons. This is because they only connect to one key part of their target cell ­ the axon-initial segment ­ where the electrical signals needed for brain communication (known as action potentials) are initiated. Since axo-axonic cells are inhibitory interneurons, this connection effectively allows them to 'veto' the generation of these signals at their source. Although axo-axonic cells have been identified in three brain regions using traditional anatomical methods, there were no 'tags' readily available that can reliably identify them. Therefore, much about these cells remained unknown, including how widespread they are in the mammalian brain. To solve this problem, Raudales et al. investigated which genes are switched on in axo-axonic cells but not in other cells, identifying a unique molecular signature that could be used to mark, record, and manipulate these cells. Microscopy imaging of brain tissue from mice in which axo-axonic cells had been identified revealed that they are present in many more brain areas than previously thought, including nearly all regions of the broadly defined cerebral cortex and even the hypothalamus, which controls many innate behaviors. Axo-axonic cells were also 'wired up' differently, depending on where they were located; for example, those in brain areas associated with memory and emotions had wider-ranging input connections than other areas. The finding of Raudales et al. provide, for the first time, a method to directly track and manipulate axo-axonic cells in the brain. Since dysfunction in axo-axonic cells is also associated with neurological disorders like epilepsy and schizophrenia, gaining an insight into their distribution and connectivity could help to develop better treatments for these conditions.

Modeling the impact of neuromorphological alterations in Down syndrome on fast neural oscillations.

Cognitive disorders, including Down syndrome (DS), present significant morphological alterations in neuron architectural complexity. However, the relationship between neuromorphological alterations and impaired brain function is not fully understood. To address this gap, we propose a novel computational model that accounts for the observed cell deformations in DS. The model consists of a cross-sectional layer of the mouse motor cortex, composed of 3000 neurons. The network connectivity is obtained by accounting explicitly for two single-neuron morphological parameters: the mean dendritic tree radius and the spine density in excitatory pyramidal cells. We obtained these values by fitting reconstructed neuron data corresponding to three mouse models: wild-type (WT), transgenic (TgDyrk1A), and trisomic (Ts65Dn). Our findings reveal a dynamic interplay between pyramidal and fast-spiking interneurons leading to the emergence of gamma activity (∼40 Hz). In the DS models this gamma activity is diminished, corroborating experimental observations and validating our computational methodology. We further explore the impact of disrupted excitation-inhibition balance by mimicking the reduction recurrent inhibition present in DS. In this case, gamma power exhibits variable responses as a function of the external input to the network. Finally, we perform a numerical exploration of the morphological parameter space, unveiling the direct influence of each structural parameter on gamma frequency and power. Our research demonstrates a clear link between changes in morphology and the disruption of gamma oscillations in DS. This work underscores the potential of computational modeling to elucidate the relationship between neuron architecture and brain function, and ultimately improve our understanding of cognitive disorders.

Thalamic cells and pathway for social memory processing and storage.

In this issue of Neuron, Wang et al.1 demonstrate that parvalbumin interneurons in the sensory thalamic reticular nucleus are necessary and sufficient for regulating social memory in mice, identify a novel cortico-reticular thalamic-parafascicular pathway for social cognition, and highlight an essential role of GABAergic inhibitory neurons in social memory engrams.

Long-range inhibition from prelimbic to cingulate areas of the medial prefrontal cortex enhances network activity and response execution.

It is well established that the medial prefrontal cortex (mPFC) exerts top-down control of many behaviors, but little is known regarding how cross-talk between distinct areas of the mPFC influences top-down signaling. We performed virus-mediated tracing and functional studies in male mice, homing in on GABAergic projections whose axons are located mainly in layer 1 and that connect two areas of the mPFC, namely the prelimbic area (PrL) with the cingulate area 1 and 2 (Cg1/2). We revealed the identity of the targeted neurons that comprise two distinct types of layer 1 GABAergic interneurons, namely single-bouquet cells (SBCs) and neurogliaform cells (NGFs), and propose that this connectivity links GABAergic projection neurons with cortical canonical circuits. In vitro electrophysiological and in vivo calcium imaging studies support the notion that the GABAergic projection neurons from the PrL to the Cg1/2 exert a crucial role in regulating the activity in the target area by disinhibiting layer 5 output neurons. Finally, we demonstrated that recruitment of these projections affects impulsivity and mechanical responsiveness, behaviors which are known to be modulated by Cg1/2 activity.

Diversity of visual inputs to Kenyon cells of the Drosophila mushroom body.

The arthropod mushroom body is well-studied as an expansion layer representing olfactory stimuli and linking them to contingent events. However, 8% of mushroom body Kenyon cells in Drosophila melanogaster receive predominantly visual input, and their function remains unclear. Here, we identify inputs to visual Kenyon cells using the FlyWire adult whole-brain connectome. Input repertoires are similar across hemispheres and connectomes with certain inputs highly overrepresented. Many visual neurons presynaptic to Kenyon cells have large receptive fields, while interneuron inputs receive spatially restricted signals that may be tuned to specific visual features. Individual visual Kenyon cells randomly sample sparse inputs from combinations of visual channels, including multiple optic lobe neuropils. These connectivity patterns suggest that visual coding in the mushroom body, like olfactory coding, is sparse, distributed, and combinatorial. However, the specific input repertoire to the smaller population of visual Kenyon cells suggests a constrained encoding of visual stimuli.

Direct and indirect pathways for heterosynaptic interaction underlying developmental synapse elimination in the mouse cerebellum.

Developmental synapse elimination is crucial for shaping mature neural circuits. In the neonatal mouse cerebellum, Purkinje cells (PCs) receive excitatory synaptic inputs from multiple climbing fibers (CFs) and synapses from all but one CF are eliminated by around postnatal day 20. Heterosynaptic interaction between CFs and parallel fibers (PFs), the axons of cerebellar granule cells (GCs) forming excitatory synapses onto PCs and molecular layer interneurons (MLIs), is crucial for CF synapse elimination. However, mechanisms for this heterosynaptic interaction are largely unknown. Here we show that deletion of AMPA-type glutamate receptor functions in GCs impairs CF synapse elimination mediated by metabotropic glutamate receptor 1 (mGlu1) signaling in PCs. Furthermore, CF synapse elimination is impaired by deleting NMDA-type glutamate receptors from MLIs. We propose that PF activity is crucial for CF synapse elimination by directly activating mGlu1 in PCs and indirectly enhancing the inhibition of PCs through activating NMDA receptors in MLIs.

Electrical Synapses Mediate Embryonic Hyperactivity in a Zebrafish Model of Fragile X Syndrome.

Although hyperactivity is associated with a wide variety of neurodevelopmental disorders, the early embryonic origins of locomotion have hindered investigation of pathogenesis of these debilitating behaviors. The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons (MNs) that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Gap junction electrical synapses drive early spontaneous behavior in zebrafish, prior to the emergence of chemical neurotransmitter networks. We use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show altered sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 34/35. We further show that this hyperexcitable behavior can be rescued by pharmacological inhibition of electrical synapses. We also use functional imaging to examine MN and interneuron (IN) activity in early embryogenesis, finding genetic disruption of electrical gap junctions uncouples activity between mnx1 + MNs and INs. Taken together, our work highlights the importance of electrical synapses in motor development and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initial formation of locomotive circuits.

Inter and intralaminar excitation of parvalbumin interneurons in mouse barrel cortex.

Parvalbumin (PV) interneurons are inhibitory fast-spiking cells with essential roles in directing the flow of information through cortical circuits. These neurons set the balance between excitation and inhibition and control rhythmic activity. PV interneurons differ between cortical layers in their morphology, circuitry, and function, but how their electrophysiological properties vary has received little attention. Here we investigate responses of PV interneurons in different layers of primary somatosensory barrel cortex (BC) to different excitatory inputs. With the genetically-encoded hybrid voltage sensor, hVOS, we recorded voltage changes in many L2/3 and L4 PV interneurons simultaneously, with stimulation applied to either L2/3 or L4. A semi-automated procedure was developed to identify small regions of interest corresponding to single responsive PV interneurons. Amplitude, half-width, and rise-time were greater for PV interneurons residing in L2/3 compared to L4. Stimulation in L2/3 elicited responses in both L2/3 and L4 with longer latency compared to stimulation in L4. These differences in latency between layers could influence their windows for temporal integration. Thus, PV interneurons in different cortical layers of BC respond in a layer specific and input specific manner, and these differences have potential roles in cortical computations.

Dynamics of spike transmission and suppression between principal cells and interneurons in the hippocampus and entorhinal cortex.

Synaptic excitation and inhibition are essential for neuronal communication. However, the variables that regulate synaptic excitation and inhibition in the intact brain remain largely unknown. Here, we examined how spike transmission and suppression between principal cells (PCs) and interneurons (INTs) are modulated by activity history, brain state, cell type, and somatic distance between presynaptic and postsynaptic neurons by applying cross-correlogram analyses to datasets recorded from the dorsal hippocampus and medial entorhinal cortex (MEC) of 11 male behaving and sleeping Long Evans rats. The strength, temporal delay, and brain-state dependency of the spike transmission and suppression depended on the subregions/layers. The spike transmission probability of PC-INT excitatory pairs that showed short-term depression versus short-term facilitation was higher in CA1 and lower in CA3. Likewise, the intersomatic distance affected the proportion of PC-INT excitatory pairs that showed short-term depression and facilitation in the opposite manner in CA1 compared with CA3. The time constant of depression was longer, while that of facilitation was shorter in MEC than in CA1 and CA3. During sharp-wave ripples, spike transmission showed a larger gain in the MEC than in CA1 and CA3. The intersomatic distance affected the spike transmission gain during sharp-wave ripples differently in CA1 versus CA3. A subgroup of MEC layer 3 (EC3) INTs preferentially received excitatory inputs from and inhibited MEC layer 2 (EC2) PCs. The EC2 PC-EC3 INT excitatory pairs, most of which showed short-term depression, exhibited higher spike transmission probabilities than the EC2 PC-EC2 INT and EC3 PC-EC3 INT excitatory pairs. EC2 putative stellate cells exhibited stronger spike transmission to and received weaker spike suppression from EC3 INTs than EC2 putative pyramidal cells. This study provides detailed comparisons of monosynaptic interaction dynamics in the hippocampal-entorhinal loop, which may help to elucidate circuit operations.

Persistent Interruption in Parvalbumin-Positive Inhibitory Interneurons: Biophysical and Mathematical Mechanisms.

Persistent activity in excitatory pyramidal cells (PYRs) is a putative mechanism for maintaining memory traces during working memory. We have recently demonstrated persistent interruption of firing in fast-spiking parvalbumin-expressing interneurons (PV-INs), a phenomenon that could serve as a substrate for persistent activity in PYRs through disinhibition lasting hundreds of milliseconds. Here, we find that hippocampal CA1 PV-INs exhibit type 2 excitability, like striatal and neocortical PV-INs. Modeling and mathematical analysis showed that the slowly inactivating potassium current KV1 contributes to type 2 excitability, enables the multiple firing regimes observed experimentally in PV-INs, and provides a mechanism for robust persistent interruption of firing. Using a fast/slow separation of times scales approach with the KV1 inactivation variable as a bifurcation parameter shows that the initial inhibitory stimulus stops repetitive firing by moving the membrane potential trajectory onto a coexisting stable fixed point corresponding to a nonspiking quiescent state. As KV1 inactivation decays, the trajectory follows the branch of stable fixed points until it crosses a subcritical Hopf bifurcation (HB) and then spirals out into repetitive firing. In a model describing entorhinal cortical PV-INs without KV1, interruption of firing could be achieved by taking advantage of the bistability inherent in type 2 excitability based on a subcritical HB, but the interruption was not robust to noise. Persistent interruption of firing is therefore broadly applicable to PV-INs in different brain regions but is only made robust to noise in the presence of a slow variable, KV1 inactivation.

Neocortical inhibitory imbalance predicts successful sensory detection.

Perceptual success depends on fast-spiking, parvalbumin-positive interneurons (FS/PVs). However, competing theories of optimal rate and correlation in pyramidal (PYR) firing make opposing predictions regarding the underlying FS/PV dynamics. We addressed this with population calcium imaging of FS/PVs and putative PYR neurons during threshold detection. In primary somatosensory and visual neocortex, a distinct PYR subset shows increased rate and spike-count correlations on detected trials ("hits"), while most show no rate change and decreased correlations. A larger fraction of FS/PVs predicts hits with either rate increases or decreases. Using computational modeling, we found that inhibitory imbalance, created by excitatory "feedback" and interactions between FS/PV pools, can account for the data. Rate-decreasing FS/PVs increase rate and correlation in a PYR subset, while rate-increasing FS/PVs reduce correlations and offset enhanced excitation in PYR neurons. These findings indicate that selection of informative PYR ensembles, through transient inhibitory imbalance, is a common motif of optimal neocortical processing.

A specific olfactory bulb interneuron subtype Tpbg/5T4 generated at embryonic and neonatal stages.

Various mammals have shown that sensory stimulation plays a crucial role in regulating the development of diverse structures, such as the olfactory bulb (OB), cerebral cortex, hippocampus, and retina. In the OB, the dendritic development of excitatory projection neurons like mitral/tufted cells is influenced by olfactory experiences. Odor stimulation is also essential for the dendritic development of inhibitory OB interneurons, such as granule and periglomerular cells, which are continuously produced in the ventricular-subventricular zone throughout life. Based on the morphological and molecular features, OB interneurons are classified into several subtypes. The role for each interneuron subtype in the control of olfactory behavior remains poorly understood due to lack of each specific marker. Among the several OB interneuron subtypes, a specific granule cell subtype, which expresses the oncofetal trophoblast glycoprotein (Tpbg or 5T4) gene, has been reported to be required for odor detection and discrimination behavior. This review will primarily focus on elucidating the contribution of different granule cell subtypes, including the Tpbg/5T4 subtype, to olfactory processing and behavior during the embryonic and adult stages.

Preictal dysfunctions of inhibitory interneurons paradoxically lead to their rebound hyperactivity and to low-voltage-fast onset seizures in Dravet syndrome.

Epilepsies have numerous specific mechanisms. The understanding of neural dynamics leading to seizures is important for disclosing pathological mechanisms and developing therapeutic approaches. We investigated electrographic activities and neural dynamics leading to convulsive seizures in patients and mouse models of Dravet syndrome (DS), a developmental and epileptic encephalopathy in which hypoexcitability of GABAergic neurons is considered to be the main dysfunction. We analyzed EEGs from DS patients carrying a SCN1A pathogenic variant, as well as epidural electrocorticograms, hippocampal local field potentials, and hippocampal single-unit neuronal activities in Scn1a+/- and Scn1aRH/+ DS mice. Strikingly, most seizures had low-voltage-fast onset in both patients and mice, which is thought to be generated by hyperactivity of GABAergic interneurons, the opposite of the main pathological mechanism of DS. Analyzing single-unit recordings, we observed that temporal disorganization of the firing of putative interneurons in the period immediately before the seizure (preictal) precedes the increase of their activity at seizure onset, together with the entire neuronal network. Moreover, we found early signatures of the preictal period in the spectral features of hippocampal and cortical field potential of Scn1a mice and of patients' EEG, which are consistent with the dysfunctions that we observed in single neurons and that allowed seizure prediction. Therefore, the perturbed preictal activity of interneurons leads to their hyperactivity at the onset of generalized seizures, which have low-voltage-fast features that are similar to those observed in other epilepsies and are triggered by hyperactivity of GABAergic neurons. Preictal spectral features may be used as predictive seizure biomarkers.

Cholinergic mu-opioid receptor deletion alters reward preference and aversion-resistance.

The endogenous opioid system has been implicated in alcohol consumption and preference in both humans and animals. The mu opioid receptor (MOR) is expressed on multiple cells in the striatum, however little is known about the contributions of specific MOR populations to alcohol drinking behaviors. The current study used mice with a genetic deletion of MOR in cholinergic cells (ChAT-Cre/Oprm1fl/fl) to examine the role of MORs expressed in cholinergic interneurons (CINs) in home cage self-administration paradigms. Male and female ChAT-Cre/Oprm1fl/fl mice were generated and heterozygous Cre+ (knockout) and Cre- (control) mice were tested for alcohol consumption in two drinking paradigms: limited access "Drinking in the Dark" and intermittent access. Quinine was added to the drinking bottles in the DID experiment to test aversion-resistant, "compulsive" drinking. Nicotine and sucrose drinking were also assessed so comparisons could be made with other rewarding substances. Cholinergic MOR deletion did not influence consumption or preference for ethanol (EtOH) in either drinking task. Differences were observed in aversion-resistance in males with Cre + mice tolerating lower concentrations of quinine than Cre-. In contrast to EtOH, preference for nicotine was reduced following cholinergic MOR deletion while sucrose consumption and preference was increased in Cre+ (vs. Cre-) females. Locomotor activity was also greater in females following the deletion. These results suggest that cholinergic MORs participate in preference for rewarding substances. Further, while they are not required for consumption of alcohol alone, cholinergic MORs may influence the tendency to drink despite negative consequences.