Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

Sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli to the central nervous system. Single-cell RNA sequencing has provided insights into the diversity of sensory ganglia cell types in rodents, nonhuman primates, and humans, but it remains difficult to compare cell types across studies and species. We thus constructed harmonized atlases of the DRG and TG that describe and facilitate comparison of 18 neuronal and 11 non-neuronal cell types across six species and 31 datasets. We then performed single-cell/nucleus RNA sequencing of DRG from both human and the highly regenerative axolotl and found that the harmonized atlas also improves cell type annotation, particularly of sparse neuronal subtypes. We observed that the transcriptomes of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The resources presented here can guide future studies in comparative transcriptomics, simplify cell-type nomenclature differences across studies, and help prioritize targets for future analgesic development.

Dorsal hippocampus to nucleus accumbens projections drive reinforcement via activation of accumbal dynorphin neurons.

The hippocampus is pivotal in integrating emotional processing, learning, memory, and reward-related behaviors. The dorsal hippocampus (dHPC) is particularly crucial for episodic, spatial, and associative memory, and has been shown to be necessary for context- and cue-associated reward behaviors. The nucleus accumbens (NAc), a central structure in the mesolimbic reward pathway, integrates the salience of aversive and rewarding stimuli. Despite extensive research on dHPC→NAc direct projections, their sufficiency in driving reinforcement and reward-related behavior remains to be determined. Our study establishes that activating excitatory neurons in the dHPC is sufficient to induce reinforcing behaviors through its direct projections to the dorso-medial subregion of the NAc shell (dmNAcSh). Notably, dynorphin-containing neurons specifically contribute to dHPC-driven reinforcing behavior, even though both dmNAcSh dynorphin- and enkephalin-containing neurons are activated with dHPC stimulation. Our findings unveil a pathway governing reinforcement, advancing our understanding of the hippocampal circuity's role in reward-seeking behaviors.

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons.

ABSTRACT: Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether bradykinin receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, whereas prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor's history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute bradykinin-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting bradykinin signaling as a potential therapeutic target for treating pain in humans.

Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

Peripheral sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli including touch, temperature, and pain to the central nervous system. Recent advances in single-cell RNA-sequencing (scRNA-seq) have provided new insights into the diversity of sensory ganglia cell types in rodents, non-human primates, and humans, but it remains difficult to compare transcriptomically defined cell types across studies and species. Here, we built cross-species harmonized atlases of DRG and TG cell types that describe 18 neuronal and 11 non-neuronal cell types across 6 species and 19 studies. We then demonstrate the utility of this harmonized reference atlas by using it to annotate newly profiled DRG nuclei/cells from both human and the highly regenerative axolotl. We observe that the transcriptomic profiles of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The new resources and data presented here can guide future studies in comparative transcriptomics, simplify cell type nomenclature differences across studies, and help prioritize targets for future pain therapy development.

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons.

Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether BK receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, while prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor’s history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute BK-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting BK signaling as a potential therapeutic target for treating pain in humans.

Effects of ketamine and other rapidly acting antidepressants on hippocampal excitatory and inhibitory transmission.

A single sub-anesthetic intravascular dose of the use-dependent NMDAR antagonist, ketamine, improves mood in patients with treatment resistant depression within hours that can last for days, creating an entirely new treatment strategy for the most seriously ill patients. However, the psychomimetic effects and abuse potential of ketamine require that new therapies be developed that maintain the rapid antidepressant effects of ketamine without the unwanted side effects. This necessitates a detailed understanding of what cellular and synaptic mechanisms are immediately activated once ketamine reaches the brain that triggers the needed changes to elicit the improved behavior. Intense research has centered on the effects of ketamine, and the other rapidly acting antidepressants, on excitatory and inhibitory circuits in hippocampus and medial prefrontal cortex to determine common mechanisms, including key modifications in synaptic transmission and the precise location of the NMDARs that mediate the rapid and sustained antidepressant response. We review data comparing the effects of ketamine with other NMDAR receptor modulators and the muscarinic M1 acetylcholine receptor antagonist, scopolamine, together with evidence supporting the disinhibition hypothesis and the direct inhibition hypothesis of ketamine's mechanism of action on synaptic circuits using preclinical models.

Rats bred for high anxiety exhibit distinct fear-related coping behavior, hippocampal physiology, and synaptic plasticity-related gene expression.

The hippocampus is essential for learning and memory but also regulates emotional behavior. We previously identified the hippocampus as a major brain region that differs in rats bred for emotionality differences. Rats bred for low novelty response (LRs) exhibit high levels of anxiety- and depression-like behavior compared to high novelty responder (HR) rats. Manipulating the hippocampus of high-anxiety LR rats improves their behavior, although no work to date has examined possible HR/LR differences in hippocampal synaptic physiology. Thus, the current study examined hippocampal slice electrophysiology, dendritic spine density, and transcriptome profiling in HR/LR hippocampus, and compared performance on three hippocampus-dependent tasks: The Morris water maze, contextual fear conditioning, and active avoidance. Our physiology experiments revealed increased long-term potentiation (LTP) at CA3-CA1 synapses in HR versus LR hippocampus, and Golgi analysis found an increased number of dendritic spines in basal layer of CA1 pyramidal cells in HR versus LR rats. Transcriptome data revealed glutamate neurotransmission as the top functional pathway differing in the HR/LR hippocampus. Our behavioral experiments showed that HR/LR rats exhibit similar learning and memory capability in the Morris water maze, although the groups differed in fear-related tasks. LR rats displayed greater freezing behavior in the fear-conditioning task, and HR/LR rats adopted distinct behavioral strategies in the active avoidance task. In the active avoidance task, HRs avoided footshock stress by pressing a lever when presented with a warning cue; LR rats, on the other hand, waited until footshocks began before pressing the lever to stop them. Taken together, these findings concur with prior observations of HR rats generally exhibiting active stress coping behavior while LRs exhibit reactive coping. Overall, our current findings coupled with previous work suggest that HR/LR differences in stress reactivity and stress coping may derive, at least in part, from differences in the developing and adult hippocampus.

Intravascular Ketamine Increases Theta-Burst but Not High Frequency Tetanus Induced LTP at CA3-CA1 Synapses Within Three Hours and Devoid of an Increase in Spine Density.

In the past 20 years, ketamine has become a promising treatment for Major Depressive Disorder (MDD) due to its rapid and sustain antidepressant effects in patients. A single ketamine treatment causes improvement in depressive symptoms within hours and can last weeks, long after it is eliminated. Previous studies have demonstrated increased synaptic plasticity at CA3-CA1 synapses in hippocampus (HPC) 24 h post ketamine treatment suggesting increased activity-dependent hippocampal function may underlie the antidepressant effects of ketamine. If true, these changes should also occur within hours of treatment, a time when symptoms are first alleviated in patients. To determine if augmented synaptic plasticity is observed at an earlier time point, we measured theta-burst and high frequency tetanus induced long-term potentiation (LTP) at CA3-CA1 synapses 3 h following intravenous (IV) ketamine administration. Additionally, we measured basal hippocampal function and spine density to investigate whether connectivity was increased with ketamine treatment. We report that theta-burst but not high frequency tetanus induced LTP is significantly increased 3 h after in vivo ketamine with no changes in basal synaptic function or morphology. Our finding supports increased activity-dependent hippocampal function underlying the antidepressant effects of ketamine as it occurs at a time point that correlates with initial improvements of depressive symptoms in patients.

Disinhibition of CA1 pyramidal cells by low-dose ketamine and other antagonists with rapid antidepressant efficacy.

Low-dose ketamine, an open-channel N-methyl d-aspartate receptor (NMDAR) antagonist, mediates rapid antidepressant effects in humans that are mimicked in preclinical rodent models. Disinhibition of pyramidal cells via decreased output of fast-spiking GABAergic interneurons has been proposed as a key mechanism that triggers the antidepressant response. Unfortunately, to date, disinhibition has not been directly demonstrated. Furthermore, whether disinhibition is a common mechanism shared among other antagonists with rapid antidepressant properties in humans has not been investigated. Using in vitro electrophysiology in acute slices of dorsal hippocampus from adult male Sprague-Dawley rats, we examined the immediate effects of a clinically relevant concentration of ketamine to directly test the disinhibition hypothesis. As a mechanistic comparison, we also tested the effects of the glycine site NMDAR partial agonist/antagonist GLYX-13 (rapastinel), the GluN2B subunit-selective NMDAR antagonist Ro 25-6981, and the muscarinic acetylcholine receptor (mAChR) antagonist scopolamine. Low-dose ketamine, GLYX-13, and scopolamine reduced inhibitory input onto pyramidal cells and increased synaptically driven pyramidal cell excitability measured at the single-cell and population levels. Conversely, Ro 25-6981 increased the strength of inhibitory transmission and did not change pyramidal cell excitability. These results show a decrease in the inhibition/excitation balance that supports disinhibition as a common mechanism shared among those antagonists with rapid antidepressant properties. These data suggest that pyramidal cell disinhibition downstream of NMDAR antagonism could serve as a possible biomarker for the efficacy of rapid antidepressant therapy.

PTEN knockdown alters dendritic spine/protrusion morphology, not density.

Mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are implicated in neuropsychiatric disorders including autism. Previous studies report that PTEN knockdown in neurons in vivo leads to increased spine density and synaptic activity. To better characterize synaptic changes in neurons lacking PTEN, we examined the effects of shRNA knockdown of PTEN in basolateral amygdala neurons on synaptic spine density and morphology by using fluorescent dye confocal imaging. Contrary to previous studies in the dentate gyrus, we find that knockdown of PTEN in basolateral amygdala leads to a significant decrease in total spine density in distal dendrites. Curiously, this decreased spine density is associated with increased miniature excitatory postsynaptic current frequency and amplitude, suggesting an increase in number and function of mature spines. These seemingly contradictory findings were reconciled by spine morphology analysis demonstrating increased mushroom spine density and size with correspondingly decreased thin protrusion density at more distal segments. The same analysis of PTEN conditional deletion in the dentate gyrus demonstrated that loss of PTEN does not significantly alter total density of dendritic protrusions in the dentate gyrus, but does decrease thin protrusion density and increases density of more mature mushroom spines. These findings suggest that, contrary to previous reports, PTEN knockdown may not induce de novo spinogenesis, but instead may increase synaptic activity by inducing morphological and functional maturation of spines. Furthermore, behavioral analysis of basolateral amygdala PTEN knockdown suggests that these changes limited only to the basolateral amygdala complex may not be sufficient to induce increased anxiety-related behaviors.

Loss of predominant Shank3 isoforms results in hippocampus-dependent impairments in behavior and synaptic transmission.

The Shank3 gene encodes a scaffolding protein that anchors multiple elements of the postsynaptic density at the synapse. Previous attempts to delete the Shank3 gene have not resulted in a complete loss of the predominant naturally occurring Shank3 isoforms. We have now characterized a homozygous Shank3 mutation in mice that deletes exon 21, including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major naturally occurring Shank3 protein bands detected by C-terminal and N-terminal antibodies, allowing us to more definitively examine the role of Shank3 in synaptic function and behavior. This loss of Shank3 leads to an increased localization of mGluR5 to both synaptosome and postsynaptic density-enriched fractions in the hippocampus. These mice exhibit a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of the hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. These data suggest that Shank3 isoforms are required for normal synaptic transmission/plasticity in the hippocampus, as well as hippocampus-dependent spatial learning and memory.