Phyllis C. Pugh

GABA depolarization is required for experience-dependent synapse unsilencing in adult born neurons

Neural activity enhances adult neurogenesis, enabling experience to influence the construction of new circuits. GABAA receptor-mediated depolarization of newborn neurons in the adult and developing brain promotes glutamatergic synaptic integration since chronic reduction of GABA depolarization impairs morphological maturation and formation of glutamatergic synapses. Here we demonstrate an acute role of GABA depolarization in glutamatergic synaptic integration. Using proopiomelanocortin enhanced–green fluorescent protein reporter mice, we identify a developmental stage when adult-generated neurons have glutamatergic synaptic transmission mediated solely by NMDA receptors (NMDARs), representing the initial silent synapses before AMPA receptor (AMPAR)-mediated functional transmission. We show that pairing synaptic stimulation with postsynaptic depolarization results in synapse unsilencing that requires NMDAR activation. GABA synaptic depolarization enables activation of NMDARs in the absence of AMPAR-mediated transmission and is required for synapse unsilencing induced by synaptic activity in vitro as well as a brief exposure to an enriched environment in vivo. The rapid appearance of AMPAR-mediated EPSCs and the lack of maturational changes show that GABA depolarization acutely allows NMDAR activation required for initial synapse unsilencing. Together, these results also reveal that adult-generated neurons in a critical period for survival use GABA signaling to rapidly initiate functional glutamate-mediated transmission in response to experience.

Nicotinic acetylcholine receptor agonists promote survival and reduce apoptosis of chick ciliary ganglion neurons.

The abundance, diversity, and ubiquitous expression of neuronal nicotinic acetylcholine receptors (AChRs) suggest that many are involved in functions other than synaptic transmission. We now report that a major AChR class promotes neuronal survival. The 10-day survival of ciliary ganglion neurons in basal culture medium (MEM) was approximately 35%, but increased to approximately 75% in MEM containing nicotine (MEM/Nic) or carbachol, an effect similar to that achieved by chronic depolarization with KCl. Pharmacological experiments revealed that agonist-enhanced survival requires activation of AChRs sensitive to alpha-bungarotoxin (alphaBgt). alphaBgt-AChRs partly support neuronal survival by limiting apoptosis since fewer apoptotic neurons were observed in MEM/Nic compared to MEM. Moreover, nicotinic survival support was not further enhanced by fibroblast growth factor, as seen for KCl, but increased to 100% by adding PACAP, a trophic neuropeptide present in the ganglion. These results indicate that alphaBgt-AChR activation regulates neuronal survival and suggest a mechanism involving reduced apoptosis and interaction with an endogenous neuropeptide growth factor.

Inborn stress reactivity shapes adult behavioral consequences of early-life maternal separation stress.

Early-life experience strongly impacts neurodevelopment and stress susceptibility in adulthood. Maternal separation (MS), an established model of early-life adversity, has been shown to negatively impact behavioral and endocrine responses to stress in adulthood. However, the impact of MS in rats with heightened inborn stress susceptibility has not been fully explored. To address this issue we conducted MS in Wistar-Kyoto (WKY) rats, an animal model of comorbid depression and anxiety, and Wistar rats, which share a similar genetic background with WKYs. WKY and Wistar pups experienced either 180-min daily MS or 15-min separation (neonatal handling) during the first two postnatal weeks, and were tested for depressive- and anxiety- like behaviors in adulthood. Exposure to early-life MS in WKY rats decreased anxiety- and depressive- like behaviors, leading to increased exploration on the open field test (OFT), enhanced social interaction, and diminished immobility on the forced swim test. MS had an opposite effect in Wistar offspring, leading to enhanced anxiety-like behaviors, such as reduced OFT exploration and decreased social interaction. These findings are consistent with the match/mismatch theory of disease and the predictive adaptive response, which suggests that early life stress exposure can confer adaptive value in later life within certain individuals. Our data supports this theory, showing that early-life MS has positive and perhaps adaptive effects within stress-vulnerable WKY offspring. Future studies will be required to elucidate the neurobiological underpinnings of contrasting behavioral effects of MS on WKY vs. Wistar offspring.

PACAP support of neuronal survival requires MAPK- and activity-generated signals

Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed in the parasympathetic ciliary ganglion (CG) and modulates nicotinic acetylcholine receptor function. PACAP also provides trophic support, promoting partial survival of CG neurons in culture and full survival when accompanied by membrane depolarization. We probed the adenylate cyclase (AC) and phospholipase-C (PLC) transduction cascades stimulated by PACAP to determine their respective roles in supporting neuronal survival and examined their interaction with signals generated by membrane activity. While PLC-dependent signaling was dispensable, AC-generated signals proved critical for PACAP to support neuronal survival. Specifically, PACAP-supported survival was mimicked by 8Br-cAMP and blocked by inhibiting either PKA or the phosphorylation of mitogen-activated protein kinase (MAPK). The ability of PACAP to promote survival was additionally dependent on spontaneous activity as blocking Na+ or Ca2+ channel currents completely abrogated trophic effects. Our results underscore the importance of coordinated MAPK- and activity-generated signals in transducing neuropeptide-mediated parasympathetic neuronal survival.

Enhanced Integration of Newborn Neurons after Neonatal Insults

The production and integration of adult-generated neurons in the dentate gyrus is dramatically perturbed by a variety of pathological insults, including repetitive seizures and hypoxia/ischemia. Less is known about how insults affect early postnatal neurogenesis, during the developmental period when the majority of dentate neurons are produced. Here we tested how single episodes of hypoxia or chemically induced seizure activity in postnatal day 10 mice alter granule cell production and integration. Although neither insult was sufficient to alter the number of newborn neurons nor the population of proliferating cells, both treatments increased the dendritic complexity of newborn granule cells that were born around the time of the insult. Surprisingly, only the dendritic enhancement caused by hypoxia was associated with increased synaptic integration. These results suggest that alterations in dendritic integration can be dissociated from altered neural production and that integration appears to have a lower threshold for perturbation. Furthermore, newborn neurons in adult mice that experienced neonatal hypoxia had reduced dendritic length while having no alterations in number. Together these results suggest that single insults during the neonatal period can have both long- and short-term consequences for neuronal maturation.

PACAP/PAC1R signaling modulates acetylcholine release at neuronal nicotinic synapses.

Neuropeptides collaborate with conventional neurotransmitters to regulate synaptic output. Pituitary adenylate cyclase-activating polypeptide (PACAP) co-localizes with acetylcholine in presynaptic nerve terminals, is released by stimulation, and enhances nicotinic acetylcholine receptor- (nAChR-) mediated responses. Such findings implicate PACAP in modulating nicotinic neurotransmission, but relevant synaptic mechanisms have not been explored. We show here that PACAP acts via selective high-affinity G-protein coupled receptors (PAC(1)Rs) to enhance transmission at nicotinic synapses on parasympathetic ciliary ganglion (CG) neurons by rapidly and persistently increasing the frequency and amplitude of spontaneous, impulse-dependent nicotinic excitatory postsynaptic currents (sEPSCs). Of the canonical adenylate cyclase (AC) and phospholipase-C (PLC) transduction cascades stimulated by PACAP/PAC(1)R signaling, only AC-generated signals are critical for synaptic modulation since the increases in sEPSC frequency and amplitude were mimicked by 8-Bromo-cAMP, blocked by inhibiting AC or cAMP-dependent protein kinase (PKA), and unaffected by inhibiting PLC. Despite its ability to increase agonist-induced nAChR currents, PACAP failed to influence nAChR-mediated impulse-independent miniature EPSC amplitudes (quantal size). Instead, evoked transmission assays reveal that PACAP/PAC(1)R signaling increased quantal content, indicating that it modulates synaptic function by increasing vesicular ACh release from presynaptic terminals. Lastly, signals generated by the retrograde messenger, nitric oxide- (NO-) are critical for the synaptic modulation since the PACAP-induced increases in spontaneous EPSC frequency, amplitude and quantal content were mimicked by NO donor and absent after inhibiting NO synthase (NOS). These results indicate that PACAP/PAC(1)R activation recruits AC-dependent signaling that stimulates NOS to increase NO production and control presynaptic transmitter output at neuronal nicotinic synapses.

Maternal Style Selectively Shapes Amygdalar Development and Social Behavior in Rats Genetically Prone to High Anxiety.

The early-life environment critically influences neurodevelopment and later psychological health. To elucidate neural and environmental elements that shape emotional behavior, we developed a rat model of individual differences in temperament and environmental reactivity. We selectively bred rats for high versus low behavioral response to novelty and found that high-reactive (bred high-responder, bHR) rats displayed greater risk-taking, impulsivity and aggression relative to low-reactive (bred low-responder, bLR) rats, which showed high levels of anxiety/depression-like behavior and certain stress vulnerability. The bHR/bLR traits are heritable, but prior work revealed bHR/bLR maternal style differences, with bLR dams showing more maternal attention than bHRs. The present study implemented a cross-fostering paradigm to examine the contribution of maternal behavior to the brain development and emotional behavior of bLR offspring. bLR offspring were reared by biological bLR mothers or fostered to a bLR or bHR mother and then evaluated to determine the effects on the following: (1) developmental gene expression in the hippocampus and amygdala and (2) adult anxiety/depression-like behavior. Genome-wide expression profiling showed that cross-fostering bLR rats to bHR mothers shifted developmental gene expression in the amygdala (but not hippocampus), reduced adult anxiety and enhanced social interaction. Our findings illustrate how an early-life manipulation such as cross-fostering changes the brains developmental trajectory and ultimately impacts adult behavior. Moreover, while earlier studies highlighted hippocampal differences contributing to the bHR/bLR phenotypes, our results point to a role of the amygdala as well. Future work will pursue genetic and cellular mechanisms within the amygdala that contribute to bHR/bLR behavior either at baseline or following environmental manipulations.

Nicotinic acetylcholine receptors in the nervous system

Publisher Summary This chapter discusses about nicotine acetylcholine receptors (AChRs) in the nervous system. AChRs channels are widely expressed in the nervous system where they are involved in signaling, neurological disease, and motivational/addictive behaviors. Like their relatives in muscle and electric organ, neuronal AChRs are pentameric transmembrane proteins belonging to a superfamily of ligand-gated ion channels that also includes GABAA, glycine and serotonin (5-HT 3 ) receptors. In each case, the occupation of the receptors ligand binding domain by agonist induces an allosteric interaction among its constituent subunits causing the channel domain to make a transition from closed to open state. Despite these similarities, there are important differences among individual members of the ligand-gated ion channel superfamily, specifically between AChRs on neurons and those on muscle fibers. In particular, when compared with muscle AChRs, neuronal AChRs are assembled from a broader palette of homologous yet distinct subunits, resulting in a much wider array of receptor subtypes. This chapter provides an introduction to basic structural and functional features of AChRs. It describes the structural elements, and functions associated with neuronal AChRs by summarizing and updating information on these topics from previous treatments. It also addresses more recent findings that implicate AChRs in novel functional processes.

PACAP induces plasticity at autonomic synapses by nAChR-dependent NOS1 activation and AKAP-mediated PKA targeting

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide found at synapses throughout the central and autonomic nervous system. We previously found that PACAP engages a selective G-protein coupled receptor (PAC1R) on ciliary ganglion neurons to rapidly enhance quantal acetylcholine (ACh) release from presynaptic terminals via neuronal nitric oxide synthase (NOS1) and cyclic AMP/protein kinase A (PKA) dependent processes. Here, we examined how PACAP stimulates NO production and targets resultant outcomes to synapses. Scavenging extracellular NO blocked PACAP-induced plasticity supporting a retrograde (post- to presynaptic) NO action on ACh release. Live-cell imaging revealed that PACAP stimulates NO production by mechanisms requiring NOS1, PKA and Ca(2+) influx. Ca(2+)-permeable nicotinic ACh receptors composed of α7 subunits (α7-nAChRs) are potentiated by PKA-dependent PACAP/PAC1R signaling and were required for PACAP-induced NO production and synaptic plasticity since both outcomes were drastically reduced following their selective inhibition. Co-precipitation experiments showed that NOS1 associates with α7-nAChRs, many of which are perisynaptic, as well as with heteromeric α3*-nAChRs that generate the bulk of synaptic activity. NOS1-nAChR physical association could facilitate NO production at perisynaptic and adjacent postsynaptic sites to enhance focal ACh release from juxtaposed presynaptic terminals. The synaptic outcomes of PACAP/PAC1R signaling are localized by PKA anchoring proteins (AKAPs). PKA regulatory-subunit overlay assays identified five AKAPs in ganglion lysates, including a prominent neuronal subtype. Moreover, PACAP-induced synaptic plasticity was selectively blocked when PKA regulatory-subunit binding to AKAPs was inhibited. Taken together, our findings indicate that PACAP/PAC1R signaling coordinates nAChR, NOS1 and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses. Such coordination has broad relevance for understanding the control of autonomic synapses and consequent visceral functions.

A subset of presympathetic-premotor neurons within the centrally projecting Edinger-Westphal nucleus expresses urocortin-1

Numerous motivated behaviors require simultaneous activation of somatomotor and autonomic functions. We have previously characterized the organization of brain circuits that may mediate this integration. Presympathetic premotor neurons (PSPMNs) that are part of such circuits are distributed across multiple brain regions, which mediate stress-elicited behavioral and physiological responses, including the Edinger-Westphal nucleus (EW). Based on its connectivity and function, EW has recently been re-classified into a preganglionic (EWpg) and a centrally projecting (EWcp) population. Neurons within EWcp are the major source of urocortin 1 (Ucn-1), an analog of the corticotropin-releasing factor that binds the CRFR1 and CRFR2 receptors and has been implicated in mediating homeostatic responses to stress. We hypothesized that a subset of EWcp PSPMNs expresses Ucn-1. Utilizing dual-label immunofluorescence, we initially mapped the distribution of Ucn-1 and cholinergic neurons within EW in colchicine pre-treated rats. Based on this labeling we divided EWcp into three neuroanatomical levels. To examine connections of EWcp neurons to the gastrocnemius muscle and the adrenal gland, we next employed trans-synaptic tract-tracing in a second group of rats, utilizing two pseudorabies virus (PRV) recombinants that express unique reporter proteins. Using multi-label immunofluorescent staining, we identified the presence of Ucn-1-positive PSPMNs, dually labeled with PRV and present throughout the entire extent of EWcp and intermingled with Ucn-1 neurons infected with one or neither of the viral recombinants. Compared to rats pretreated with colchicine, we observed significantly fewer Ucn-1 neurons in animals that received PRV injections. Post hoc analyses revealed significantly fewer Ucn-1 neurons at the rostral level as compared to the caudal and middle levels. These data suggest functional and anatomic heterogeneity within EWcp; this organization may coordinate various aspects of stress-elicited and emotionally salient behaviors.