Cells and circuits for amygdala neuroplasticity in the transition to chronic pain.

Maladaptive plasticity is linked to the chronification of diseases such as pain, but the transition from acute to chronic pain is not well understood mechanistically. Neuroplasticity in the central nucleus of the amygdala (CeA) has emerged as a mechanism for sensory and emotional-affective aspects of injury-induced pain, although evidence comes from studies conducted almost exclusively in acute pain conditions and agnostic to cell type specificity. Here, we report time-dependent changes in genetically distinct and projection-specific CeA neurons in neuropathic pain. Hyperexcitability of CRF projection neurons and synaptic plasticity of parabrachial (PB) input at the acute stage shifted to hyperexcitability without synaptic plasticity in non-CRF neurons at the chronic phase. Accordingly, chemogenetic inhibition of the PB→CeA pathway mitigated pain-related behaviors in acute, but not chronic, neuropathic pain. Cell-type-specific temporal changes in neuroplasticity provide neurobiological evidence for the clinical observation that chronic pain is not simply the prolonged persistence of acute pain.

The parabrachial to central amygdala pathway is critical to injury-induced pain sensitization in mice.

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PBN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. However, the functional significance of this pathway in the modulation of the somatosensory component of pain was recently challenged by studies showing that spinal nociceptive neurons do not target CeA-projecting PBN cells and that manipulations of this pathway have no effect on reflexive-defensive somatosensory responses to peripheral noxious stimulation. Here, we showed that activation of CeA-projecting PBN neurons is critical to increase both stimulus-evoked and spontaneous nociceptive responses following an injury in male and female mice. Using optogenetic-assisted circuit mapping, we confirmed a functional excitatory projection from PBN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increased the expression of the neuronal activity marker Fos in CeA-projecting PBN neurons and that chemogenetic inactivation of these cells decreased behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we showed that chemogenetic activation of CeA-projecting PBN neurons is sufficient to induced bilateral hypersensitivity without injury. Together, our results indicate that the PBN→CeA pathway is a key modulator of pain-related behaviors that can increase reflexive-defensive and affective-motivational responses to somatosensory stimulation in injured states without affecting nociception under normal physiological conditions.

The parabrachial to central amygdala circuit is a key mediator of injury-induced pain sensitization.

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PbN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. At the behavioral level, the PbN→CeA pathway has been proposed to serve as a general alarm system to potential threats that modulates pain-related escape behaviors, threat memory, aversion, and affective-motivational (but not somatosensory) responses to painful stimuli. Increased sensitivity to previously innocuous somatosensory stimulation is a hallmark of chronic pain. Whether the PbN→CeA circuit contributes to heightened peripheral sensitivity following an injury, however, remains unknown. Here, we demonstrate that activation of CeA-projecting PbN neurons contributes to injury-induced behavioral hypersensitivity but not baseline nociception in male and female mice. Using optogenetic assisted circuit mapping, we confirmed a functional excitatory projection from PbN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increases the expression of the neuronal activity marker c-Fos in CeA-projecting PbN neurons and chemogenetic inactivation of these cells reduces behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we show that chemogenetic activation of CeA-projecting PbN neurons is sufficient to induce bilateral hypersensitivity without injury. Together, our results demonstrate that the PbN→CeA pathway is a key modulator of pain-related behaviors that can amplify responses to somatosensory stimulation in pathological states without affecting nociception under normal physiological conditions. Significance Statement: Early studies identified the spino-ponto-amygdaloid pathway as a major ascending circuit conveying nociceptive inputs from the spinal cord to the brain. The functional significance of this circuit to injury-induced hypersensitivity, however, remains unknown. Here, we addressed this gap in knowledge using viral-mediated anatomical tracers, ex-vivo electrophysiology and chemogenetic intersectional approaches in rodent models of persistent pain. We found that activation of this pathway contributes to injury-induced hypersensitivity, directly demonstrating a critical function of the PbN→CeA circuit in pain modulation.

An inhibitory circuit from central amygdala to zona incerta drives pain-related behaviors in mice.

Central amygdala neurons expressing protein kinase C-delta (CeA-PKCδ) are sensitized following nerve injury and promote pain-related responses in mice. The neural circuits underlying modulation of pain-related behaviors by CeA-PKCδ neurons, however, remain unknown. In this study, we identified a neural circuit that originates in CeA-PKCδ neurons and terminates in the ventral region of the zona incerta (ZI), a subthalamic structure previously linked to pain processing. Behavioral experiments show that chemogenetic inhibition of GABAergic ZI neurons induced bilateral hypersensitivity in uninjured mice and contralateral hypersensitivity after nerve injury. In contrast, chemogenetic activation of GABAergic ZI neurons reversed nerve injury-induced hypersensitivity. Optogenetic manipulations of CeA-PKCδ axonal terminals in the ZI further showed that inhibition of this pathway reduces nerve injury-induced hypersensitivity whereas activation of the pathway produces hypersensitivity in the uninjured paws. Altogether, our results identify a novel nociceptive inhibitory efferent pathway from CeA-PKCδ neurons to the ZI that bidirectionally modulates pain-related behaviors in mice.

Mechanism of noradrenaline-induced stimulation of Na-K ATPase activity in the rat brain: implications on REM sleep deprivation-induced increase in brain excitability.

Rapid eye movement (REM) sleep is a unique phenomenon expressed in all higher forms of animals. Its quantity varies in different species and with ageing; it is also affected in several psycho-somatic disorders. Several lines of studies showed that after REM sleep loss, the levels of noradrenaline (NA) increase in the brain. The NA in the brain modulates neuronal Na-K ATPase activity, which helps maintaining the brain excitability status. The detailed mechanism of increase in NA level after REM sleep loss and the effect of NA on stimulation of Na-K ATPase in the neurons have been discussed. The findings have been reviewed and discussed with an aim to understand the role of REM sleep in maintaining brain excitability status.

Reciprocal changes in noradrenaline and GABA levels in discrete brain regions upon rapid eye movement sleep deprivation in rats.

Rapid eye movement sleep (REMS) plays important role in maintenance of normal brain functions. Neurons containing various neurotransmitters in different brain regions interact to regulate this complex phenomenon in health and diseases. The number of neuronal projections, their firing rates and neurotransmitter levels vary in different brain regions under various conditions leading to normal or altered patho-physio-behavioral states. In this study using high performance liquid chromatography (HPLC) we quantified noradrenaline (NA) and gamma-amino butyric acid (GABA) levels in locus coeruleus (LC), dorsal raphe (DR), pedunculo-pontine tegmentum (PPT), frontal lobe (FL), cortex and hippocampus (Hippo) in control and after 96 h REMS deprivation (REMSD) rats. Normal free moving control (FMC) rats were taken as standard cage controls. To rule out non-specific effects large platform control (LPC) and post-REMSD recovery (REC) were carried out. The levels of NA and GABA in discrete brain regions upon REMSD were statistically compared with all the controls. Upon REMSD, although NA levels significantly increased and the GABA levels decreased in the LC, PPT and cortex, in Hippo their levels showed opposite responses. Only NA levels increased in FL, while only GABA levels were decreased in the DR after REMSD. Most of the altered neurotransmitter levels returned to normal levels in REC rats. The findings help understanding the neurochemical basis of REMSD and its associated effects.

Heparin binding carboxypeptidase E protein exhibits antibacterial activity in human semen.

Carboxypeptidase E (CPE) cleaves basic amino acid residues at the C-terminal end and involves in the biosynthesis of numerous peptide hormones and neurotransmitters. It was purified from human seminal plasma by ion exchange, heparin affinity and gel filtration chromatography followed by identification through SDS-PAGE and MALDI-TOF/MS analysis, which was further confirmed by western blotting. CPE was characterized as glycoprotein by Periodic Acid Schiff (PAS) staining and treating with deglycosylating enzyme N-glycosidase F. The interaction of CPE with heparin was illustrated by surface plasmon resonance (SPR) and in silico interaction analysis. The association constant (KA) and dissociation constant (KD) of CPE with heparin was determined by SPR and found to be 1.06 × 10(5)M and 9.46 × 10(-6)M, respectively. It was detected in human spermatozoa also by western blotting using mouse anti-CPE primary antibody. 20-100 µg/ml concentration of CPE was observed as highly effective in killing Escherichia coli by colony forming unit (CFU) assay. We suggest that CPE might act not only in the innate immunity of male reproductive tract but also regulate sperm fertilization process by interacting heparin.

Proteomic identification of camel seminal plasma: purification of ß-nerve growth factor.

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, ß-nerve growth factor (ß-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-ß-NGF primary antibody.

Rapid eye movement sleep deprivation modulates synapsinI expression in rat brain.

Rapid eye movement sleep (REMS) deprivation (REMSD) has been reported to elevate neurotransmitter level in the brain; however, intracellular mechanism of its increased release was not studied. Phosphorylation of synapsinI, a synaptic vesicle-associated protein, is involved in the regulation of neurotransmitter release. In this study, rats were REMS deprived by classical flowerpot method; free moving control (FMC), large platform control (LPC) and recovery control (REC) was carried out. In another set REMS deprived rats were intraperitoneally (i.p.) injected with α1-adrenoceptor antagonist, prazosin (PRZ). Effects of REMSD on Na-K ATPase activity and on the total synapsinI as well as phosphorylated synapsinI levels were estimated in synaptosomes prepared from whole brain. It was observed that REMSD significantly increased synaptosomal Na-K ATPase activity, which was prevented by PRZ. Western blotting of the same samples by anti-synapsinI and anti-synapsinI-phosphoSer603 showed that REMSD increased both the total as well as phospho-form of synapsinI as compared to respective levels in FMC and LPC samples. These findings suggest a functional link between REMSD and synaptic vesicular mobilization at the presynaptic terminal, a process that is essential for neurotransmitter release. The findings help explaining the intracellular mechanism of elevated neurotransmitter release associated to REMSD.

Human serum albumin as a new interacting partner of prolactin inducible protein in human seminal plasma.

Prolactin inducible protein (PIP) is a 17 kDa glycoprotein. It binds to many proteins including fibrinogen, actin, keratin, myosin, immunoglobulin G, CD4, and human zinc-alpha-2 glycoprotein. Its ability to bind a large array of proteins indicates its multifaceted role in various biological processes, such as fertility, immunoregulation, antimicrobial activity, apoptosis, and tumor progression. Here, we present the first report of native human serum albumin (HSA)-PIP complex formation in seminal plasma. The complex was purified by chromatographic separation techniques, analyzed by gel electrophoresis, identified by MALDI-TOF mass spectrometry and validated by co-immunoprecipitation coupled with western blotting experiments. Moreover, the behavior of complex in solution was analyzed by dynamic light scattering and interacting residues were identified by in silico protein-protein docking. The purified protein complex shows two bands (67 kDa and 17 kDa) on SDS-PAGE gel and a single band (~85 kDa) on native PAGE gel. The predicted complex structure has 13 intermolecular hydrogen bonds, which may contribute to the overall stability of the complex. As HSA has been known to preserve the motility of sperm, native HSA-PIP complex formation may point towards an important role of PIP, which can directly be correlated with male fertility/infertility.