AB5060 Sigma-AldrichAnti-Substance P Receptor Antibody, pain

Various GABAergic neuron types of the amygdala cooperate to control principal cell firing during fear-related and other behaviors, and understanding their specialized roles is important. Among GABAergic neurons, the so-called intercalated cells (ITCcs) are critically involved in the expression and extinction of fear memory. Tightly clustered small-sized spiny neurons constitute the majority of ITCcs, but they are surrounded by sparse, larger neurons (L-ITCcs) for which very little information is known. We report here a detailed neurochemical, structural and physiological characterization of rat L-ITCcs, as identified with juxtacellular recording/labeling in vivo. We supplement these data with anatomical and neurochemical analyses of nonrecorded L-ITCcs. We demonstrate that L-ITCcs are GABAergic, and strongly express metabotropic glutamate receptor 1α and GABAA receptor α1 subunit, together with moderate levels of parvalbumin. Furthermore, L-ITCcs are innervated by fibers enriched with metabotropic glutamate receptors 7a and/or 8a. In contrast to small-sized spiny ITCcs, L-ITCcs possess thick, aspiny dendrites, have highly branched, long-range axonal projections, and innervate interneurons in the basolateral amygdaloid complex. The axons of L-ITCcs also project to distant brain areas, such as the perirhinal, entorhinal, and endopiriform cortices. In vivo recorded L-ITCcs are strongly activated by noxious stimuli, such as hindpaw pinches or electrical footshocks. Consistent with this, we observed synaptic contacts on L-ITCc dendrites from nociceptive intralaminar thalamic nuclei. We propose that, during salient sensory stimulation, L-ITCcs disinhibit local and distant principal neurons, acting as "hub cells," to orchestrate the activity of a distributed network.

Sensory and sympathetic nerve fibers (SNF) innervate bone and epiphyseal growth plate. The role of neuronal signals for proper endochondral ossification during skeletal growth is mostly unknown. Here, we investigated the impact of the absence of sensory neurotransmitter substance P (SP) and the removal of SNF on callus differentiation, a model for endochondral ossification in adult animals, and on bone formation.In order to generate callus, tibia fractures were set in the left hind leg of wild type (WT), tachykinin 1-deficient (Tac1-/-) mice (no SP) and animals without SNF. Locomotion was tested in healthy animals and touch sensibility was determined early after fracture. Callus tissue was prepared for immunofluorescence staining for SP, neurokinin1-receptor (NK1R), tyrosine-hydroxylase (TH) and adrenergic receptors α1, α2 and β2. At the fracture site, osteoclasts were stained for TRAP, osteoblasts were stained for RUNX2, and histomorphometric analysis of callus tissue composition was performed. Primary murine bone marrow derived macrophages (BMM), osteoclasts, and osteoblasts were tested for differentiation, activity, proliferation and apoptosis in vitro. Femoral fractures were set in the left hind leg of all the three groups for mechanical testing and μCT-analysis.Callus cells stained positive for SP, NK1R, α1d- and α2b adrenoceptors and remained β2-adrenoceptor and TH-negative. Absence of SP and SNF did not change the general locomotion but reduces touch sensitivity after fracture. In mice without SNF, we detected more mesenchymal callus tissue and less cartilaginous tissue 5 days after fracture. At day 13 past fracture, we observed a decrease of the area covered by hypertrophic chondrocytes in Tac1-/- mice and mice without SNF, a lower number of osteoblasts in Tac1-/- mice and an increase of osteoclasts in mineralized callus tissue in mice without SNF. Apoptosis rate and activity of osteoclasts and osteoblasts isolated from Tac1-/- and sympathectomized mice were partly altered in vitro. Mechanical testing of fractured- and contralateral legs 21 days after fracture, revealed an overall reduced mechanical bone quality in Tac1-/- mice and mice without SNF. μCT-analysis revealed clear structural alteration in contralateral and fractured legs proximal of the fracture site with respect to trabecular parameters, bone mass and connectivity density. Notably, structural parameters are altered in fractured legs when related to unfractured legs in WT but not in mice without SP and SNF.The absence of SP and SNF reduces pain sensitivity and mechanical stability of the bone in general. The micro-architecture of the bone is profoundly impaired in the absence of intact SNF with a less drastic effect in SP-deficient mice. Both sympathetic and sensory neurotransmitters are indispensable for proper callus differentiation. Importantly, the absence of SP reduces bone formation rate whereas the absence of SNF induces bone resorption rate. Notably, fracture chondrocytes produce SP and its receptor NK1 and are positive for α-adrenoceptors indicating an endogenous callus signaling loop. We propose that sensory and sympathetic neurotransmitters have crucial trophic effects which are essential for proper bone formation in addition to their classical neurological actions.

We previously showed that gastrin-releasing peptide receptor (GRPR) in the spinal cord is important for mediating nonhistaminergic itch. Neuromedin B receptor (NMBR), the second member of the mammalian bombesin receptor family, is expressed in a largely nonoverlapping pattern with GRPR in the superficial spinal cord, and its role in itch transmission remains unclear. Here, we report that Nmbr knock-out (KO) mice exhibited normal scratching behavior in response to intradermal injection of pruritogens. However, mice lacking both Nmbr and Grpr (DKO mice) showed significant deficits in histaminergic itch. In contrast, the chloroquine (CQ)-evoked scratching behavior of DKO mice is not further reduced compared with Grpr KO mice. These results suggest that NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for CQ-evoked scratching behavior. Interestingly, GRPR activity is enhanced in Nmbr KO mice despite the lack of upregulation of Grpr expression; so is NMBR in Grpr KO mice. We found that NMB acts exclusively through NMBR for itch transmission, whereas GRP can signal through both receptors, albeit to NMBR to a much lesser extent. Although NMBR and NMBR(+) neurons are dispensable for histaminergic itch, GRPR(+) neurons are likely to act downstream of NMBR(+) neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling.

The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+)-imaging on isolated taste cells, it was observed that SP induces Ca(2+) -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+)-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+)-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+)-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+) responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.

The substance P/NK1 receptor system plays an important role in the regulation of stress and emotional responding and as such had been implicated in the pathophysiology of anxiety and depression. The present study investigated whether alterations in the substance P/NK1 receptor system in brain areas which regulate emotional responding accompany the depressive behavioural phenotype observed in the olfactory bulbectomised (OB) mouse. The effect of NK1 receptor deletion on behavioural responding and monoamine levels in discrete brain regions of the OB model, were also examined. Substance P levels in the frontal cortex and NK1 receptor expression in the amygdala and hippocampus were enhanced following olfactory bulbectomy. Although NK1 receptor knockout (NK1-/-) mice did not exhibit altered behavioural responding in the open field test, noradrenaline levels were enhanced in the frontal cortex, amygdala and hippocampus, as were serotonin levels in the frontal cortex. Locomotor activity and exploratory behaviour were enhanced in wild type OB mice, indicative of a depressive-like phenotype, an effect attenuated in NK1-/- mice. Bulbectomy induced a decrease in noradrenaline and 5-HIAA in the frontal cortex and an increase in serotonin in the amygdala, effects attenuated in OB NK1-/- mice. The present studies indicate that alterations in substance P/NK1 receptor system underlie, at least in part, the behavioural and monoaminergic changes in this animal model of depression.

Hippocampal oscillations reflect coordinated neuronal activity on many timescales. Distinct types of GABAergic interneuron participate in the coordination of pyramidal cells over different oscillatory cycle phases. In the CA3 area, which generates sharp waves and gamma oscillations, the contribution of identified GABAergic neurons remains to be defined. We have examined the firing of a family of cholecystokinin-expressing interneurons during network oscillations in urethane-anesthetized rats and compared them with firing of CA3 pyramidal cells. The position of the terminals of individual visualized interneurons was highly diverse, selective, and often spatially coaligned with either the entorhinal or the associational inputs to area CA3. The spike timing in relation to theta and gamma oscillations and sharp waves was correlated with the innervated pyramidal cell domain. Basket and dendritic-layer-innervating interneurons receive entorhinal and associational inputs and preferentially fire on the ascending theta phase, when pyramidal cell assemblies emerge. Perforant-path-associated cells, driven by recurrent collaterals of pyramidal cells fire on theta troughs, when established pyramidal cell assemblies are most active. In the CA3 area, slow and fast gamma oscillations occurred on opposite theta oscillation phases. Perforant-path-associated and some COUP-TFII-positive interneurons are strongly coupled to both fast and slow gamma oscillations, but basket and dendritic-layer-innervating cells are weakly coupled to fast gamma oscillations only. During sharp waves, different interneuron types are activated, inhibited, or remain unaffected. We suggest that specialization in pyramidal cell domain and glutamatergic input-specific operations, reflected in the position of GABAergic terminals, is the evolutionary drive underlying the diversity of cholecystokinin-expressing interneurons.

Although extinction-based therapies are among the most effective treatments for anxiety disorders, the neural bases of fear extinction remain still essentially unclear. Recent evidence suggests that the intercalated cell masses of the amygdala (ITCs) are critical structures for fear extinction. However, the neuronal organization of ITCs and how distinct clusters contribute to different fear states are still entirely unknown. Here, by combining whole-cell patch-clamp recordings and biocytin labeling with full anatomical reconstruction of the filled neurons and ultrastructural analysis of their synaptic contacts, we have elucidated the cellular organization and efferent connections of one of the main ITC clusters in mice. Our data showed an unexpected heterogeneity in the axonal pattern of medial paracapsular ITC (Imp) neurons and the presence of three distinct neuronal subtypes. Functionally, we observed that the Imp was preferentially activated during fear expression, whereas extinction training and extinction retrieval activated the main ITC nucleus (IN), as measured by quantifying Zif268 expression. This can be explained by the IPSPs evoked in the IN after Imp stimulation, most likely through the GABAergic monosynaptic innervation of IN neurons by one subtype of Imp cells, namely the medial capsular-projecting (MCp)-Imp neurons. MCp-Imp neurons also target large ITC cells that surround ITC clusters and express the metabotropic glutamate receptor 1α. These findings reveal a distinctive participation of ITC clusters to different fear states and the underlying anatomical circuitries, hence shedding new light on ITC networks and providing a novel framework to elucidate their role in fear expression and extinction.

In the mammalian retina, information concerning various aspects of an image is transferred in parallel, and cone bipolar cells are thought to play a major role in this parallel processing. We have examined the synaptic connections of calbindin-immunoreactive (IR) ON cone bipolar cells in the inner plexiform layer (IPL) of rabbit retina and have compared these synaptic connections with those that we have previously described for neurokinin 1 (NK1) receptor-IR cone bipolar cells. A total of 325 synapses made by calbindin-IR bipolar axon terminals have been identified in sublamina b of the IPL. The axons of calbindin-IR bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and are coupled to putative AII amacrine cells via gap junctions. The major output from calbindin-IR bipolar cells is to amacrine cell processes. These data resemble our findings for NK1 receptor-IR bipolar cells. However, the incidences of output synapses to ganglion cell dendrites of calbindin-IR bipolar cells are higher compared with the NK1-receptor-IR bipolar cells. On the basis of stratification level and synaptic connections, calbindin-IR ON cone bipolar cells might thus play an important role in the processing of various visual aspects, such as contrast, orientation, and approach sensing, and in transferring rod signals to the ON cone pathway.

Musculoskeletal injury and inflammation is associated with performance of repetitive and forceful tasks. In this study, we examined the effects of performing a voluntary, highly repetitive, negligible force (HRNF) reaching task on spinal cord neurochemicals involved in nociception. To our knowledge, no other laboratories are examining spinal cord nociceptive neurochemicals in response to repetitive motion-induced injury and inflammation. The purpose of this study was to extend our earlier findings related to central neurotransmitters from a low demand task to a higher demand task. Specifically, this study determined immunoreactivity of a peptidergic pro-nociceptive transmitter (substance P) and one of its receptors, neurokinin-1 (NK-1) receptor, in spinal cord dorsal horns in rats performing a HRNF reaching task for 6-10 weeks. The relationship of these spinal cord changes with the number of TNFalpha immunopositive cells in flexor forelimb muscles and with previously observed forearm grip strength changes from these same rats were examined. Performance of the HRNF task resulted in significantly increased substance P and NK-1 receptor immunoreactivity in the superficial lamina of spinal cord dorsal horns at 6 and 10 weeks compared to trained controls (p<0.01). The increased substance P and NK-1 receptor immmunoreactivity were positively correlated with declines in forearm grip strength, an assay of movement-related hyperalgesia (r=0.70, p<0.01 and r=0.64, p<0.05, respectively). The increased substance P and NK-1 receptor immmunoreactivity were also positively correlated with increased TNF immunopositive cells in forelimb flexor muscles (r=0.85, p<0.001 and r=0.88, p<0.001, respectively). Thus, our highly repetitive task leads to increased spinal cord pro-nociceptive neurochemicals that are most likely directed by forelimb muscle inflammation and pain.

The synthesis enzyme glutamic acid decarboxylase (GAD65 or GAD67) identifies neurons as GABAergic. Recent studies have characterized the physiological properties of spinal cord GABAergic interneurons using lines of GAD67-green fluorescent protein (GFP) transgenic mice. A more complete characterization of their phenotype is required to better understand the role of this population of inhibitory neurons in spinal cord function. Here, we characterize the distribution of lumbar spinal cord GAD67-GFP neurons at postnatal days (P) 0, 7, and 14, and adult based on their co-expression with GABA and determine the molecular phenotype of GAD67-GFP neurons at P14 based on the expression of various neuropeptides, calcium binding proteins, and other markers. At all ages greater than 67% of GFP(+) neurons were also GABA(+). With increasing age; (i) GFP(+) and GABA(+) cell numbers declined, (ii) ventral horn GFP(+) and GABA(+) neurons vanished, and (iii) somatic labeling was reduced while terminal labeling increased. At P14, vasoactive intestinal peptide and bombesin were expressed in approximately 63% and approximately 35% of GFP(+) cells, respectively. Somatostatin was found in a small number of neurons, whereas calcitonin gene-related peptide never co-localized with GFP. Moderate co-expression was found for all the Ca(2+) binding proteins examined. Notably, most laminae I-II parvalbumin(+) neurons were also GFP(+). Neurogranin, a protein kinase C substrate, was found in approximately 1/2 of GFP(+) cells. Lastly, while only 7% of GFP(+) cells contain nitric oxide synthase (NOS), these cells represent a large fraction of all NOS(+) cells. We conclude that GAD67-GFP neurons represent the majority of spinal GABAergic neurons and that mouse dorsal horn GAD67-GFP(+) neurons comprise a phenotypically diverse population.