Crypt and Villus Enterochromaffin Cells are Distinct Stress Sensors in the Gut.

The crypt-villus structure of the small intestine serves as an essential protective barrier, with its integrity monitored by the gut's sensory system. Enterochromaffin (EC) cells, which are rare sensory epithelial cells that release serotonin (5-HT), surveil the mucosal environment and signal both within and outside the gut. However, it remains unclear whether EC cells in intestinal crypts and villi respond to different stimuli and elicit distinct responses. In this study, we introduce a new reporter mouse model to observe the release and propagation of serotonin in live intestines. Using this system, we show that crypt EC cells exhibit two modes of serotonin release: transient receptor potential A1 (TRPA1)-dependent tonic serotonin release that controls basal ionic secretion, and irritant-evoked serotonin release that activates gut sensory neurons. Furthermore, we find that a thick protective mucus layer prevents TRPA1 receptors on crypt EC cells from responding to luminal irritants such as reactive electrophiles; if this mucus layer is compromised, then crypt EC cells become susceptible to activation by luminal irritants. On the other hand, villus EC cells detect oxidative stress through TRPM2 channels and co-release serotonin and ATP to activate nearby gut sensory fibers. Our work highlights the physiological importance of intestinal architecture and differential TRP channel expression in sensing noxious stimuli that elicit nausea and/or pain sensations in the gut.

Gut enterochromaffin cells drive visceral pain and anxiety.

Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain1. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved2. Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men1. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.

Pruritogenic mechanisms and gut sensation: putting the "irritant" into irritable bowel syndrome.

Chronic abdominal pain is a common clinical condition experienced by patients with irritable bowel syndrome (IBS). A general lack of suitable treatment options for the management of visceral pain is the major contributing factor to the debilitating nature of the disease. Understanding the underlying causes of chronic visceral pain is pivotal to identifying new effective therapies for IBS. This review provides the current evidence, demonstrating that mediators and receptors that induce itch in the skin also act as "gut irritants" in the gastrointestinal tract. Activation of these receptors triggers specific changes in the neuronal excitability of sensory pathways responsible for the transmission of nociceptive information from the periphery to the central nervous system leading to visceral hypersensitivity and visceral pain. Accumulating evidence points to significant roles of irritant mediators and their receptors in visceral hypersensitivity and thus constitutes potential targets for the development of more effective therapeutic options for IBS.

Inhibition of the dorsomedial hypothalamus substantially decreases brown adipose tissue sympathetic discharge induced by activation of the lateral habenula.

The lateral habenula (LHb) is an evolutionarily ancient nucleus that plays an important role in the detection of salient/adverse environmental events. We have previously shown that the LHb is involved in brown adipose tissue (BAT) thermogenesis elicited by stressful situations, and that the medullary raphé, a key lower brainstem sympathetic control centre, mediates BAT thermogenesis elicited by stimulating the LHb. Since there are no direct projections from the LHb to the medullary raphé, it is plausible that the dorsomedial hypothalamus (DMH), a brain region known to be important for thermoregulatory responses to stress, is involved in this thermogenic pathway. In this study we aimed to test this possibility. In anaesthetized Sprague-Dawley rats, we recorded electrical discharges directly from sympathetic fibres that innervate BAT, as well as BAT temperature. Injections of bicuculline (1 nmol in 100 nl), a neuronal activator by disinhibition, into the LHb increased BAT sympathetic nerve discharge by 4.9 ± 1.4dBµV (n = 7, P < 0.05) and BAT temperature by 1.0 ± 0.1 °C (n = 7, P < 0.01). Subsequent injections of muscimol (0.25 nmol in 100 nl), a neuronal inhibitor, into the DMH promptly reduced BAT sympathetic nerve discharge by 4.7 ± 1.3 dBµV (n = 7, P < 0.05) and BAT temperature by 0.3 ± 0.1 °C (n = 7, P < 0.05). Injections of a mixture of the ionotropic glutamate receptor antagonists, DL-2-Amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dioneis (CNQX) into the DMH, after activation of the LHb, also significantly decreased BAT sympathetic nerve discharge and BAT temperature. These results suggest that, for sympathetically-mediated BAT thermogenesis, the DMH is part of the neural circuitry linking the LHb with the medullary raphé.

Alpha2-adrenergic receptor agonists prevent emotional hyperthermia.

Emotionally significant stimuli, including potential threats from the external environment, trigger an increase in body temperature, a response known as emotional hyperthermia. Sympathetically-mediated brown adipose tissue (BAT) thermogenesis contributes substantially to this hyperthermic response. The systemic administration of α2-adrenergic agonists is known to inhibit both febrile and shivering responses. In the present study, we investigated whether systemic administration of clonidine, a α2-adrenoceptor agonist, attenuates the emotional hyperthermia evoked in conscious unrestrained rats suddenly confronted with a second (intruder) rat, itself confined to a small cage. Pre-implanted thermistors were used to measure BAT and body temperature in conscious, freely moving, male Sprague-Dawley rats. The rats were pre-treated with intraperitoneally administered vehicle (Ringer solution) or clonidine (1, 10 and 100 µg/kg). Clonidine, in a dose-dependent manner, reduced the intruder-elicited increases in BAT (log-dose linear regression F(1,16) = 9.52, R2 = 0.37, P < 0.01) and body temperature (F(1,16) = 6.48, R2 = 0.29, P < 0.05). We also investigated, in anesthetized rats, whether systemic clonidine administration inhibits BAT sympathetic nerve discharge evoked via activation of neurons in the lateral habenula (LHb) - a nucleus involved in the regulation of emotional hyperthermia. In anesthetized rats, clonidine abolished the BAT sympathetic nerve discharges elicited via bicuculline-mediated disinhibition of the LHb. These results suggest that activation of central α2-adrenergic receptors attenuates the process of emotional hyperthermia by reduction of BAT thermogenesis.

Activating dopamine D2 receptors reduces brown adipose tissue thermogenesis induced by psychological stress and by activation of the lateral habenula.

Emotional hyperthermia is the increase in body temperature that occurs as a response to an animal detecting a salient, survival-relevant stimulus. Brown adipose tissue (BAT) thermogenesis, controlled via its sympathetic innervation, contributes to this temperature increase. Here, we have used an intruder rat experimental model to determine whether quinpirole-mediated activation of dopamine D2 receptors attenuates emotional hyperthermia in conscious rats. In anesthetized rats, we determined whether systemic quinpirole reduces BAT nerve discharge induced by activation of the medullary raphé and the lateral habenula (LHb). We measured BAT and body temperature with chronically implanted thermistors in conscious, freely moving, individually housed, male rats (resident rats). Either vehicle or quinpirole was administered, intraperitoneally, to the resident rat 30 min before introduction of a caged intruder rat. Quinpirole, in a dose-dependent manner, reduced intruder-elicited increases in BAT and body temperature. Pre-treatment with the D2 antagonist spiperone, but not the selective D1 antagonist SCH-23390, prevented this quinpirole-elicited decrease. In anesthetized rats, quinpirole abolished BAT sympathetic nerve discharge elicited by bicuculline-mediated activation of the LHb, but not the medullary raphé. Thus, activation of dopamine D2 receptors reduces the BAT thermogenesis that contributes to emotional hyperthermia. We provide evidence that these dopamine D2 receptors are located in the thermogenic pathway between the LHb and the lower brainstem pre-sympathetic control centre in the medullary raphé.

Amyotrophic lateral sclerosis mutant TDP-43 may cause synaptic dysfunction through altered dendritic spine function.

Altered cortical excitability and synapse dysfunction are early pathogenic events in amyotrophic lateral sclerosis (ALS) patients and animal models. Recent studies propose an important role for TAR DNA-binding protein 43 (TDP-43), the mislocalization and aggregation of which are key pathological features of ALS. However, the relationship between ALS-linked TDP-43 mutations, excitability and synaptic function is not fully understood. Here, we investigate the role of ALS-linked mutant TDP-43 in synapse formation by examining the morphological, immunocytochemical and excitability profile of transgenic mouse primary cortical pyramidal neurons that over-express human TDP-43A315T In TDP-43A315T cortical neurons, dendritic spine density was significantly reduced compared to wild-type controls. TDP-43A315T over-expression increased the total levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropinionic acid (AMPA) glutamate receptor subunit GluR1, yet the localization of GluR1 to the dendritic spine was reduced. These postsynaptic changes were coupled with a decrease in the amount of the presynaptic marker synaptophysin that colocalized with dendritic spines. Interestingly, action potential generation was reduced in TDP-43A315T pyramidal neurons. This work reveals a crucial effect of the over-expression mutation TDP-43A315T on the formation of synaptic structures and the recruitment of GluR1 to the synaptic membrane. This pathogenic effect may be mediated by cytoplasmic mislocalization of TDP-43A315T Loss of synaptic GluR1, and reduced excitability within pyramidal neurons, implicates hypoexcitability and attenuated synaptic function in the pathogenic decline of neuronal function in TDP-43-associated ALS. Further studies into the mechanisms underlying AMPA receptor-mediated excitability changes within the ALS cortical circuitry may yield novel therapeutic targets for treatment of this devastating disease.

Neurons in ventral tegmental area tonically inhibit sympathetic outflow to brown adipose tissue: possible mediation of thermogenic signals from lateral habenula.

The lateral habenula (LHb), a nucleus involved in the response to salient, especially adverse, environmental events, is implicated in brown adipose tissue (BAT) thermogenesis caused by these events. LHb-elicited thermogenesis involves a neural pathway to the lower brain stem sympathetic control center in the medullary raphé. There are no direct connections from the LHb to the medullary raphé. LHb-mediated behavioral responses involve inhibitory control over the dopamine neurons in the ventral tegmental area (VTA), mediated via an excitatory drive from the LHb to GABAergic neurons in the tail of the VTA. We hypothesized that inhibition of the VTA is also involved in LHb-mediated BAT thermogenesis. To test this hypothesis, inhibition of neurons in the VTA with muscimol increased BAT sympathetic nerve discharge by 22.0 ± 9.2 dBµV ( n = 24, P < 0.0001) and BAT temperature by 1.2 ± 0.1°C ( P < 0.001). This response was abolished by inhibition of the medullary raphé neurons with muscimol. BAT thermogenesis initiated with focal injections of bicuculline in the LHb was reversed by subsequent blockade of GABAA receptors in the VTA with bicuculline. These results suggest that, at least in anesthetized rats, neurons in the VTA tonically inhibit BAT thermogenesis via a link, presently unknown, to the medullary raphé. Removal of this VTA-initiated inhibition is an important mechanism whereby LHb neurons activate BAT thermogenesis.

Reduced Excitability and Increased Neurite Complexity of Cortical Interneurons in a Familial Mouse Model of Amyotrophic Lateral Sclerosis.

Cortical interneurons play a crucial role in regulating inhibitory-excitatory balance in brain circuits, filtering synaptic information and dictating the activity of pyramidal cells through the release of GABA. In the fatal motor neuron (MN) disease, amyotrophic lateral sclerosis (ALS), an imbalance between excitation and inhibition is an early event in the motor cortex, preceding the development of overt clinical symptoms. Patients with both sporadic and familial forms of the disease exhibit reduced cortical inhibition, including patients with mutations in the copper/zinc superoxide-dismutase-1 (SOD1) gene. In this study, we investigated the influence of the familial disease-causing hSOD1-G93A ALS mutation on cortical interneurons in neuronal networks. We performed whole-cell patch-clamp recordings and neurobiotin tracing from GFP positive interneurons in primary cortical cultures derived from Gad67-GFP::hSOD1G93A mouse embryos. Targeted recordings revealed no overt differences in the passive properties of Gad67-GFP::hSOD1G93A interneurons, however the peak outward current was significantly diminished and cells were less excitable compared to Gad67-GFP::WT controls. Post hoc neurite reconstruction identified a significantly increased morphological complexity of the Gad67-GFP::hSOD1G93A interneuron neurite arbor compared to Gad67-GFP::WT controls. Our results from the SOD1 model suggest that cortical interneurons have electrophysiological and morphological alterations that could contribute to attenuated inhibitory function in the disease. Determining if these phenomena are driven by the network or represent intrinsic alteration of the interneuron may help explain the emergence of inhibitory susceptibility and ultimately disrupted excitability, in ALS.

The Microtubule-Modulating Drug Epothilone D Alters Dendritic Spine Morphology in a Mouse Model of Mild Traumatic Brain Injury.

Microtubule dynamics underpin a plethora of roles involved in the intricate development, structure, function, and maintenance of the central nervous system. Within the injured brain, microtubules are vulnerable to misalignment and dissolution in neurons and have been implicated in injury-induced glial responses and adaptive neuroplasticity in the aftermath of injury. Unfortunately, there is a current lack of therapeutic options for treating traumatic brain injury (TBI). Thus, using a clinically relevant model of mild TBI, lateral fluid percussion injury (FPI) in adult male Thy1-YFPH mice, we investigated the potential therapeutic effects of the brain-penetrant microtubule-stabilizing agent, epothilone D. At 7 days following a single mild lateral FPI the ipsilateral hemisphere was characterized by mild astroglial activation and a stereotypical and widespread pattern of axonal damage in the internal and external capsule white matter tracts. These alterations occurred in the absence of other overt signs of trauma: there were no alterations in cortical thickness or in the number of cortical projection neurons, axons or dendrites expressing YFP. Interestingly, a single low dose of epothilone D administered immediately following FPI (and sham-operation) caused significant alterations in the dendritic spines of layer 5 cortical projection neurons, while the astroglial response and axonal pathology were unaffected. Specifically, spine length was significantly decreased, whereas the density of mushroom spines was significantly increased following epothilone D treatment. Together, these findings have implications for the use of microtubule stabilizing agents in manipulating injury-induced synaptic plasticity and indicate that further study into the viability of microtubule stabilization as a therapeutic strategy in combating TBI is warranted.

Mild Traumatic Brain Injury Leads to Decreased Inhibition and a Differential Response of Calretinin Positive Interneurons in the Injured Cortex.

It is clear that even mild forms of traumatic brain injury (TBI) can have lasting cognitive effects; however, the specific cellular changes responsible for the functional deficits remain poorly understood. Previous studies suggest that not all neurons respond in the same way and that changes to neuronal architecture may be subtype specific. The current study aimed to characterize the response of interneurons to TBI. To model TBI in vitro, the neurites of primary cortical neurons were transected at 15 days in vitro. In response, calretinin+ interneurons underwent significant neurite remodeling around the injury site. By examining the response of pyramidal neurons, GAD67-GFP+ interneurons, and calretinin+ interneurons to the injury, we found that this response was specific to the calretinin+ cells. To determine whether calretinin+ interneurons respond in this way to a clinically relevant in vivo model of mild diffuse and focal injury, we subjected mice to the lateral fluid percussion injury model. We found that calretinin+ interneuron density was unaltered by this mild injury, but consistent with our in vitro data, these neurons underwent morphological alterations in their dendrites. These alterations evolved over a 28-day period, and calretinin+ interneurons in the injured mice had a reduction in mean dendrite length and reduced number of secondary dendrites than those in the sham-injured controls by 7 days post-injury. Further, these structural alterations were accompanied by a reduction in the frequency of miniature inhibitory post-synaptic currents in layer V pyramidal neurons. These data suggest that even a mild TBI can lead to an overall change in the excitatory/inhibitory balance of the cortex that may play an important role in the longer-term behavioral pathology associated with mild TBI.

The microtubule-stabilizing drug Epothilone D increases axonal sprouting following transection injury in vitro.

Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are considered a hallmark feature of the degeneration that occurs after traumatic brain injury (TBI). Therefore, microtubule-stabilizing drugs are attractive potential therapeutics for use following TBI. The best-known drug in this category is Paclitaxel, a widely used anti-cancer drug that has produced promising outcomes when employed in the treatment of various animal models of nervous system trauma. However, Paclitaxel is not ideal for the treatment of patients with TBI due to its limited blood-brain barrier (BBB) permeability. Herein we have characterized the effect of the brain penetrant microtubule-stabilizing agent Epothilone D (Epo D) on post-injury axonal sprouting in an in vitro model of CNS trauma. Epo D was found to modulate axonal sprout number in a dose dependent manner, increasing the number of axonal sprouts generated post-injury. Elevated sprouting was observed when analyzing the total population of injured neurons, as well as in selective analysis of Thy1-YFP-labeled excitatory neurons. However, we found no effect of Epo D on axonal sprout length or outgrowth speed. These findings indicate that Epo D specifically affects injury-induced axonal sprout generation, but not net growth. Our investigation demonstrates that primary cultures of cortical neurons are tolerant of Epo D exposure, and that Epo D significantly increases their regenerative response following structural injury. Therefore Epo D may be a potent therapeutic for enhancing regeneration following CNS injury. This article is part of a Special Issue entitled 'Traumatic Brain Injury'.

Treatment of erythrocytes with the 2-cys peroxiredoxin inhibitor, Conoidin A, prevents the growth of Plasmodium falciparum and enhances parasite sensitivity to chloroquine.

The human erythrocyte contains an abundance of the thiol-dependant peroxidase Peroxiredoxin-2 (Prx2), which protects the cell from the pro-oxidant environment it encounters during its 120 days of life in the blood stream. In malarial infections, the Plasmodium parasite invades red cells and imports Prx2 during intraerythrocytic development, presumably to supplement in its own degradation of peroxides generated during cell metabolism, especially hemoglobin (Hb) digestion. Here we demonstrate that an irreversible Prx2 inhibitor, Conoidin A (2,3-bis(bromomethyl)-1,4-dioxide-quinoxaline; BBMQ), has potent cytocidal activity against cultured P. falciparum. Parasite growth was also inhibited in red cells that were treated with BBMQ and then washed prior to parasite infection. These cells remained susceptible to merozoite invasion, but failed to support normal intraerythrocytic development. In addition the potency of chloroquine (CQ), an antimalarial drug that prevents the detoxification of Hb-derived heme, was significantly enhanced in the presence of BBMQ. CQ IC50 values decreased an order of magnitude when parasites were either co-incubated with BBMQ, or introduced into BBMQ-pretreated cells; these effects were equivalent for both drug-resistant and drug-sensitive parasite lines. Together these results indicate that treatment of red cells with BBMQ renders them incapable of supporting parasite growth and increases parasite sensitivity to CQ. We also propose that molecules such as BBMQ that target host cell proteins may constitute a novel host-directed therapeutic approach for treating malaria.