Kathryn G. Commons

A neurochemically distinct dorsal raphe-limbic circuit with a potential role in affective disorders.

The serotonergic system arising from the dorsal raphe nucleus (DR) has long been implicated in psychiatric disorders, and is considered one site of action of classical anxiolytic and antidepressant agents. Recent studies implicate the DR as a site of action of novel anxiolytic and antidepressant agents that target neuropeptide systems, such as corticotropin-releasing factor (CRF) and neurokinin 1 (NK1) antagonists. The present study identified unique characteristics of the dorsomedial DR that implicate this particular subregion as a key component of a circuit, which may be targeted by these diverse psychotherapeutic agents. First, it was observed that a cluster of CRF-containing cell bodies was present in the dorsomedial DR of colchicine-treated rats. Dual-labeling immunohistochemistry revealed that almost all CRF-containing neurons were serotonergic, implicating CRF as a cotransmitter with serotonin in this subpopulation of DR neurons. Moreover, dendrites laden with immunoreactivity for NK1 had a striking topographic distribution surrounding and extending into the dorsomedial subregion of the DR, suggesting that NK1 receptor ligands may selectively impact the dorsomedial DR. Finally, anterograde tract tracing from the dorsomedial DR combined with CRF immunohistochemistry revealed that CRF-containing axons from this subregion project to CRF-containing neurons of the central nucleus of the amygdala. Taken together, the present results reveal a circuit whereby NK1 receptor activation in the dorsomedial DR can impact on limbic sources of CRF that have been implicated in emotional responses. This circuit may be relevant for understanding the mechanism of action of novel psychotherapeutic agents that act through NK1 or CRF receptors.

Peptides that fine-tune the serotonin system.

The dorsal raphe nucleus (DR) contains serotonin (5-HT) neurons that innervate the cortex and limbic system and through these projections is thought to regulate cognition and behavior. Clinical and pharmacological findings implicate dysfunctions in the DR-5-HT system in affective disorders, including anxiety, depression and suicide. Although the DR is often considered in light of its 5-HT neurons, recent studies underscore the complexity of this nucleus and its heterogeneous nature. Of particular interest, are peptides that are either present within neurons in the DR, innervate DR-5-HT neurons or act upon local circuitry within the DR to indirectly impact on this 5-HT system. These peptides are positioned to fine-tune the activity of selective groups of serotonergic neurons within the DR and thereby 5-HT release in different terminal fields. This review will focus on substance P and corticotropin-releasing factor as two peptides that act independently and interdependently to influence DR-5-HT function. The role of non-serotonergic components of the DR in translating the effect of each of these peptides is discussed. This synthesis refines our views on the regulation of the DR-5-HT system and importantly, gives insight into mechanisms of endogenous control of DR function, the dysregulation of which may contribute to pathophysiology.

Projections and interconnections of genetically defined serotonin neurons in mice

Brain serotonin neurons are heterogeneous and can be distinguished by several anatomical and physiological characteristics. Toward resolving this heterogeneity into classes of functional relevance, subtypes of mature serotonin neurons were previously identified based on gene expression differences initiated during development in different rhombomeric (r) segments of the hindbrain. This redefinition of mature serotonin neuron subtypes based on the criteria of genetic lineage, along with the enabling genetic fate mapping tools, now allows various functional properties, such as axonal projections, to be allocated onto these identified subtypes. Furthermore, our approach uniquely enables interconnections between the different serotonin neuron subtypes to be determined; this is especially relevant because serotonin neuron activity is regulated by several feedback mechanisms. We used intersectional and subtractive genetic fate mapping tools to generate three independent lines of mice in which serotonin neurons arising in different rhombomeric segments, either r1, r2 or both r3 and r5, were uniquely distinguished from all other serotonin neurons by their expression of enhanced green fluorescent protein. Each of these subgroups of serotonergic neurons had a unique combination of forebrain projection targets. Typically more than one subgroup innervated an individual target area. Unique patterns of interconnections between the different groups of serotonin neurons were also observed and these pathways could subserve feedback regulatory circuits. Overall, the current findings suggest that activation of subsets of serotonin neurons could result in topographic serotonin release in the forebrain coupled with feedback inhibition of serotonin neurons with alternative projection targets.

Cellular basis for the effects of substance P in the periaqueductal gray and dorsal raphe nucleus

Substance P (SP) is known to act at supraspinal sites to influence pain sensitivity as well as to promote anxiety. The effects of SP could be mediated in part by actions in the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN), adjoining mesencephalic cell groups that are strategically positioned to influence both nociception and mood. Previous studies have indicated that SP regulates both enkephalin and serotonin neurotransmission in these brain regions. To determine the mechanism underlying the effects of SP in the PAG and DRN, the distribution of the principal receptor for SP, the neurokinin 1 (NK1) receptor, was examined with respect to other neurotransmitter markers. PAG neurons that had NK1 receptor immunolabeling were interdigitated with and received contacts from enkephalin‐containing neurons. However, only a few (16/144; 11%) neurons with NK1 receptor also contained enkephalin immunoreactivity after colchicine treatment. In the DRN, dendrites containing NK1 receptor were selectively distributed in the dorsomedial subdivision. The majority (132/137; 96%) of these dendrites did not contain immunoreactivity for the serotonin‐synthesizing enzyme tryptophan hydroxylase. In contrast, neuronal profiles with NK1 receptor in both the PAG and the DRN often contained immunolabeling for glutamate. Light and electron microscopic examination revealed that 48–65% of cell bodies and dendrites with NK1 receptor were dually immunolabeled for glutamate. These data suggest that SP directly acts primarily on glutamatergic neurons in the PAG and DRN. To a lesser extent, enkephalin‐containing neurons may be targeted. Through these actions, it may subsequently influence activity of larger populations of neurons containing enkephalin as well as serotonin. This circuitry could contribute to, as well as coordinate, effects of SP on pain perception and mood. J. Comp. Neurol. 447:82–97, 2002.

FREQUENT COLOCALIZATION OF MU OPIOID AND NMDA-TYPE GLUTAMATE RECEPTORS AT POSTSYNAPTIC SITES IN PERIAQUEDUCTAL GRAY NEURONS

In the ventrolateral periaqueductal gray (PAG), endogenous pathways which dampen pain transmission can be activated by either opioids or excitatory amino acids such as N‐methyl D‐aspartate (NMDA). The effects of these ligands may converge, because morphine‐produced analgesia in the PAG can be blocked by NMDA receptor antagonists. To determine the relationship between the subcellular sites where opioid ligands of the mu opioid receptor (MOR) and NMDA receptor ligands may act, we studied the ultrastructural distribution of immunolabeling for MOR and the R1 subunit of the NMDA receptor (NR1) in the ventrolateral PAG. MOR labeling was most commonly distributed along extrasynaptic regions of the plasma membrane of neuronal dendrites (80% or 245/306). In addition, MOR labeling was found presynaptically in axon terminals (13% or 39/306) which preferentially formed symmetric (inhibitory‐type) synapses. NR1 immunoreactivity was also prevalent in dendrites (72% or 242/335), but in contrast to MOR, was usually associated with a subset of postsynaptic densities. Axon terminals (5%, 17/335) and glial processes (18%, 61/335) comprised the remainder of NR1‐labeled profiles. There was a striking colocalization of MOR and NR1 labeling within dendrites. The majority of NR1‐labeled dendrites contained MOR labeling (72%, 176/242) and likewise, the majority of MOR‐labeled dendrites contained NR1 labeling (72%, 176/245). Thus, mu opioid and NMDA receptor ligands may act at several overlapping subcellular sites to modulate behaviors subserved by the ventrolateral PAG, such as antinociception. J. Comp. Neurol. 408:549–559, 1999.

Presynaptic and postsynaptic relations of μ-opioid receptors to γ- aminobutyric acid-immunoreactive and medullary-projecting periaqueductal gray neurons

The ventrolateral portion of the periaqueductal gray (PAG) is one brain region in which ligands of the μ‐opioid receptor (MOR) produce analgesia. In the PAG, MOR ligands are thought to act primarily on inhibitory [e.g., γ‐aminobutyric acidergic (GABAergic)] neurons to disinhibit PAG output rather than directly on medullary‐projecting PAG neurons. In this study, the ultrastructural localization of MOR immunolabeling was examined with respect to either GABAergic PAG neurons or PAG projection neurons that were labeled retrogradely from the rostral ventromedial medulla. Immunoreactivity for MOR and GABA often coexisted within dendrites. Dual‐labeled profiles accounted for subpopulations of dendrites containing immunoreactivity for either MOR (65 of 145 dendrites; 45%) or GABA (65 of 183 dendrites; 35%). In addition, nearly half of PAG neuronal profiles (148 of 344 profiles) that were labeled retrogradely from the ventromedial medulla contained MOR immunoreactivity. MOR was distributed equally among retrogradely labeled neuronal profiles in the lateral and ventrolateral columns of the caudal PAG. With respect to the presynaptic distribution of MOR, approximately half of MOR‐immunolabeled axon terminals (35 of 69 terminals) also contained GABA. Some MOR and GABA dual‐immunolabeled axon terminals contacted unlabeled dendrites (11 of 35 terminals), whereas others contacted GABA‐immunoreactive dendrites (15 of 35 terminals). Furthermore, axon terminals synapsing on medullary‐projecting PAG neurons sometimes contained immunoreactivity for MOR. These data support the model that MOR ligands can act by inhibiting GABAergic neurons, but they also provide evidence that MOR ligands may act directly on PAG output neurons. In addition, MOR at presynaptic sites could affect both GABAergic neurons and output neurons. Thus, the disinhibitory model represents only partially the potential mechanisms by which MOR ligands can modulate output of the PAG. J. Comp. Neurol. 419:532–542, 2000.

Translocation of presynaptic delta opioid receptors in the ventrolateral periaqueductal gray after swim stress

Immunolabeling for the delta opioid receptor (DOR) is localized primarily to axon terminals in the ventrolateral periaqueductal gray (vlPAG). However, rather than on the plasma membrane, DOR immunoreactivity is usually located within the cytoplasmic compartment, often associated with dense‐core vesicles. In this study, the hypothesis that a behavioral stimulus, a cold water swim stress (3 minutes at 4°C; CWSS), could initiate the translocation of the DOR was tested. The subcellular distribution of DOR was examined using a preembedding immunogold‐labeling method and ultrastructural analysis in control rats and in rats that had a CWSS. In both cases, dense‐core vesicles associated with DOR labeling were often within 100 nm of the plasma membrane. When the dense‐core vesicles were near the plasma membrane, sometimes electron‐dense “tethers” appeared between the vesicle and the plasma membrane. However, in rats exposed to CWSS, there was a decrease in immunolabeling associated with dense‐core vesicles that were near the plasma membrane and a significant increase in DOR immunoreactivity associated with the plasma membrane. In addition, there was a significant increase in the fraction of DOR immunoreactivity associated with large clear‐core vesicles; possibly early endosomes. Moreover, after a CWSS, dense‐core vesicles containing DOR immunoreactivity could be visualized fusing with the plasma membrane of synaptic boutons. These data suggest the involvement of DOR in the vlPAG in the behavioral response to CWSS. Furthermore, the results support the hypothesis that the cell surface distribution of presynaptic receptors can be regulated in an activity‐dependent manner by virtue of transport via dense‐core vesicles. J. Comp. Neurol. 464:197–207, 2003.

Failed heart rate recovery at a critical age in 5-HT-deficient mice exposed to episodic anoxia: implications for SIDS

Mice deficient in the transcription factor Pet-1⁻/⁻ have a ∼70% deficiency of brainstem serotonin [5-hydroxytryptamine (5-HT)] neurons and exhibit spontaneous bradycardias in room air at postnatal day (P)5 and P12 and delayed gasping in response to a single episode of anoxia at P4.5 and P9.5 (Cummings KJ, Li A, Deneris ES, Nattie EE. Am J Physiol Regul Integr Comp Physiol 298: R1333-R1342, 2010; and Erickson JT, Sposato BC. J Appl Physiol 106: 1785-1792, 2009). We hypothesized that at a critical age Pet-1⁻/⁻ mice will fail to autoresuscitate during episodic anoxia, ultimately dying from a failure of gasping to restore heart rate (HR). We exposed P5, P8, and P12 Pet-1⁻/⁻ mice and wild-type littermates (WT) to four 30-s episodes of anoxia (97% N₂-3% CO₂), separated by 5 min of room air. We observed excess mortality in Pet-1⁻/⁻ only at P8: 43% of Pet-1⁻/⁻ animals survived past the third episode of anoxia while ∼95% of WT survived all four episodes (P = 0.004). No deaths occurred at P5 and at P12, and one of six Pet-1⁻/⁻ mice died after the fourth episode, while all WT animals survived. At P8, dying Pet-1⁻/⁻ animals had delayed gasping, recovery of HR, and eupnea after the first two episodes of anoxia (P < 0.001 for each); death ultimately occurred when gasping failed to restore HR. Both high- and low-frequency components of HR variability were abnormally elevated in dying Pet-1⁻/⁻ animals following the first episode of anoxia. Dying P8 Pet-1⁻/⁻ animals had significantly fewer 5-HT neurons in the raphe magnus than surviving animals (P < 0.001). Our data indicate a critical developmental window at which a brainstem 5-HT deficiency increases the risk of death during episodes of anoxia. They may apply to the sudden infant death syndrome, which occurs at a critical age and is associated with 5-HT deficiency.

Cellular and subcellular localization of δ opioid receptor immunoreactivity in the rat dentate gyrus

Abstract To study a potential locus of action of opioids in the rat dentate gyrus, we examined the localization of the δ opioid receptor (DOR) by immunocytochemistry. Two antisera raised to unique, non-overlapping peptide sequences located within the extracellular N-terminal sequence of DOR were used. By light microscopy, numerous neurons in the central hilar region were intensely labeled for DOR, while the granule cell layer contained light DOR immunoreactivity. To further characterize hilar neuron cell types which contained DOR, sections through the dentate gyrus were double labeled using immunofluorescence with antisera to DOR and either γ-aminobutyfc acid (GABA), neuropeptide Y (NPY), or somatostatin-28 antisera. Most DOR-labeled perikarya also contained GABA and NPY, while a subpopulation contained somatostatin. Electron microscopic examination of sections labeled for DOR revealed that the immunoreactivity was common in profiles which exhibited the morphological characteristics of granule cells, as well as those of non-granule cells. DOR immunoreactivity was located at postsynaptic sites within neuronal perikarya (2%), dendrites (27%), and dendritic spines (22%); as well as in presynaptic axon terminals (25%) and glia (23%) (n = 279). In dendrites and dendritic spines, DOR immunoreactivity was most often associated with the plasmalemmal surface near asymmetric synapses. In axon terminals, DOR immunoreactivity primarily surrounded small, clear vesicles, and was less consistently found on the plasmalemmal surface. The distribution of DOR-labeled profiles overlapped with, but was not restricted to regions known to contain enkephalin. These data suggest that opiates acting at the DOR can modulate both hilar neurons and granule cells both pre- and postsynaptically.

Morphine-enhanced apoptosis in selective brain regions of neonatal rats

Prolonged neonatal opioid exposure has been associated with: antinociceptive tolerance, long‐term neurodevelopmental delay, cognitive, and motor impairment. Morphine has also been shown to induce apoptotic cell death in vitro studies, but its in vivo effect in developing rat brain is unknown. Thus, we hypothesized that prolongued morphine administration in neonatal rats in a model of antinociceptive tolerance and dependence is associated with increased neuroapoptosis. We analyzed neonatal rats from the following groups (1) naïve group (n = 6); (2) control group (normal saline (NS), n = 5), and (3) morphine group (n = 8). Morphine sulfate or equal volume of NS was injected subcutaneously twice daily for 6½ days starting on postnatal day (PD) 1. Development of antinociceptive tolerance was previously confirmed by Hot Plate test on the 7th day. Evidence of neuronal and glial apoptosis was determined by cleaved caspase‐3 immunofluorescence combined with specific markers. At PD7, morphine administration after 6½ days significantly increased the density of apoptotic cells in the cortex and amygdala, but not in the hippocampus, hypothalamus, or periaqueductal gray. Apoptotic cells exhibited morphology analogous to neurons. Irrespective of the treatment, only a very few individual microglia but not astrocytes were caspase‐3 positive. In summary, repeated morphine administration in neonatal rats (PD1–7) is associated with increased supraspinal apoptosis in distinct anatomical regions known to be important for sensory (cortex) and emotional memory processing (amygdala). Brain regions important for learning (hippocampus), and autonomic and nociceptive processing (hypothalamus and periaqueductal gray) were not affected. Lack of widespread glial apoptosis or robust glial activation following repeated morphine administration suggests that glia might not be affected by chronic morphine at this early age. Future studies should investigate long‐term behavioral sequelae of demonstrated enhanced apoptosis associated with prolonged morphine administration in a neonatal rat model.