Immunoreactive Terminals Research Articles

Role of the central renin‑angiotensin system in hypertension (Review).

Present in more than one billion adults, hypertension is the most significant modifiable risk factor for mortality resulting from cardiovascular disease. Although its pathogenesis is not yet fully understood, the disruption of the renin-angiotensin system (RAS), consisting of the systemic and brain RAS, has been recognized as one of the primary reasons for several types of hypertension. Therefore, acquiring sound knowledge of the basic science of RAS and the under- lying mechanisms of the signaling pathways associated with RAS may facilitate the discovery of novel therapeutic targets with which to promote the management of patients with cardiovascular and kidney disease. In total, 4 types of angiotensin II receptors have been identified (AT1R-AT4R), of which AT1R plays the most important role in vasoconstriction and has been most extensively studied. It has been found in several regions of the brain, and its distribution is highly associated with that of angiotensin-like immunoreactivity in nerve terminals. The effect of AT1R involves the activation of multiple media and signaling pathways, among which the most important signaling pathways are considered to be AT1R/JAK/STAT and Ras/Raf/MAPK pathways. In addition, the regulation of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and cyclic AMP response element-binding (CREB) pathways is also closely related to the effect of ATR1. Their mechanisms of action are related to pro-inflammatory and sympathetic excitatory effects. Central AT1R is involved in almost all types of hypertension, including spontaneous hypertension, salt-sensitive hypertension, obesity-induced hypertension, renovascular hypertension, diabetic hypertension, L-NAME-induced hypertension, stress-induced hypertension, angiotensin II-induced hyper- tension and aldosterone-induced hypertension. There are 2 types of central AT1R blockade, acute blockade and chronic blockade. The latter can be achieved by chemical blockade or genetic engineering. The present review article aimed to high- light the prevalence, functions, interactions and modulation means of central AT-1R in an effort to assist in the treatment of several pathological conditions. The identification of angiotensin-derived peptides and the development of AT-2R agonists may provide a wider perspective on RAS, as well as novel therapeutic strategies.

Partial Activation of TrkB Receptors Corrects Interneuronal Calcium Channel Dysfunction and Reduces Epileptogenic Activity in Neocortex following Injury.

Decreased GABAergic inhibition due to dysfunction of inhibitory interneurons plays an important role in post-traumatic epileptogenesis. Reduced N-current Ca2+ channel function in GABAergic terminals contributes to interneuronal abnormalities and neural circuit hyperexcitability in the partial neocortical isolation (undercut, UC) model of post-traumatic epileptogenesis. Because brain-derived neurotrophic factor (BDNF) supports the development and maintenance of interneurons, we hypothesized that the activation of BDNF tropomyosin kinase B (TrkB) receptors by a small molecule, TrkB partial agonist, PTX BD4-3 (BD), would correct N channel abnormalities and enhance inhibitory synaptic transmission in UC cortex. Immunocytochemistry (ICC) and western blots were used to quantify N- and P/Q-type channels. We recorded evoked (e)IPSCs and responses to N and P/Q channel blockers to determine the effects of BD on channel function. Field potential recordings were used to determine the effects of BD on circuit hyperexcitability. Chronic BD treatment 1) upregulated N and P/Q channel immunoreactivity in GABAergic terminals; 2) increased the effects of N or P/Q channel blockade on evoked inhibitory postsynaptic currents (eIPSCs); 3) increased GABA release probability and the frequency of sIPSCs; and 4) reduced the incidence of epileptiform discharges in UC cortex. The results suggest that chronic TrkB activation is a promising approach for rescuing injury-induced calcium channel abnormalities in inhibitory terminals, thereby improving interneuronal function and suppressing circuit hyperexcitability.

Age-related loss of VGLUT1 excitatory, but not VGAT inhibitory, immunoreactive terminals on motor neurons in spinal cords of old sarcopenic male mice

Age-related changes in ventral lumbar spinal cord (L3-L5) were compared in young [3month, (M)] and old (27M) C57BL/6J male mice. The aged mice had previously been shown to exhibit sarcopenia and changes to peripheral nerve morphology. The putative connectivity of β-III tubulin positive α-motor neurons was compared in immunostained transverse sections using excitatory and inhibitory terminal markers vesicular glutamate transporter-1 (VGLUT1) and vesicular GABA transporter (VGAT). Glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) immunostaining was used to monitor changes in astrocyte and microglial phenotype respectively. For a given motor neuron, the neuronal perimeter was outlined and terminals immunoreactive for VGLUT1 or VGAT in close apposition to the soma were identified. By 27M, the percentage coverage and total number of VGLUT1 immunoreactive terminals immediately adjacent to the soma of α-motor neurons was significantly decreased compared with young mice. However, percentage coverage of immunoreactive VGAT inhibitory terminals did not change significantly with age. The gray matter of 27M spinal cords showed increased astrocytic and microglial activity. The loss of VGLUT1 terminals on α-motor neurons, terminals known to be derived from proprioceptive muscle afferents, may further impair sensorimotor control of hind limb skeletal muscle function in old mice.

Homer1 (VesL-1) in the rat esophagus: focus on myenteric plexus and neuromuscular junction.

Homer1, a scaffolding protein of the postsynaptic density (PSD), enriched at excitatory synapses is known to anchor and modulate group I metabotropic glutamate receptors (mGluRs) and different channel- and receptor-proteins. Homer proteins are expressed in neurons of different brain regions, but also in non-neuronal tissues like skeletal muscle. Occurrence and location of Homer1 and mGluR5 in myenteric plexus and neuromuscular junctions (NMJ) of rat esophagus have yet not been characterized. We located Homer1 and mGluR5 immunoreactivity (-iry) in rat esophagus and focused on myenteric neurons, intraganglionic laminar endings (IGLEs) and NMJs, using double- and triple-label immunohistochemistry and confocal laser scanning microscopy. Homer1-iry was found in a subpopulation of vesicular glutamate transporter 2 (VGLUT2) positive IGLEs and cholinergic varicosities within myenteric ganglia, but neither in nitrergic nor cholinergic myenteric neuronal cell bodies. Homer1-iry was detected in 63% of esophageal and, for comparison, in 35% of sternomastoid NMJs. Besides the location in the PSD, Homer1-iry colocalized with cholinergic markers, indicating a presynaptic location in coarse VAChT/CGRP/NF200- immunoreactive (-ir) terminals of nucleus ambiguus neurons supplying striated esophageal muscle. mGluR5-iry was found in subpopulations of myenteric neuronal cell bodies, VGLUT2-ir IGLEs and cholinergic varicosities within the myenteric neuropil and NMJs of esophagus and sternomastoid muscles. Thus, Homer1 may anchor mGluR5 at presynaptic sites of cholinergic boutons at esophageal motor endplates, in a small subpopulation of VGLUT2-ir IGLEs and cholinergic varicosities within myenteric ganglia possibly modulating Ca2+-currents and neurotransmitter release.

Serotonin 5-HT4 receptors modulate the development of glutamatergic input to the dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is a major serotonin (5-hydroxytryptamine, 5-HT)-producing region in the central nervous system. It receives glutamatergic inputs from several brain regions, which are reciprocally modulated by serotonergic signals. We investigated whether serotonin 5-HT4 receptors (5-HT4Rs) play a role in the development of glutamatergic control of the DRN, with an emphasis on cortical inputs. Double-label immunohistochemistry and confocal microscopy were used to quantify vesicular glutamate transporter 1 (vGluT1)-immunoreactive terminals in the DRN of mice with a null-mutation in the 5-HT4R gene. We found no significant change in the overall density of vGluT1-positive terminals in homozygous and heterozygous mice, but heterozygous mice had a significantly higher density of vGluT1-positive terminals contacting serotonergic neurons. These results suggest that altered 5-HT4R expression may affect the development of cortical glutamatergic control of the DRN.

IGF-1 protects dopamine neurons against oxidative stress: association with changes in phosphokinases.

Insulin-like growth factor-1 (IGF-1) is an endogenous peptide transported across the blood brain barrier that is protective in several brain injury models, including an acute animal model of Parkinson's disease (PD). Motor deficits in PD are due largely to the progressive loss of nigrostriatal dopaminergic neurons. Thus, we examined the neuroprotective potential of IGF-1 in a progressive model of dopamine deficiency in which 6-hydroxydopamine (6-OHDA) is infused into the striatum. Rats received intrastriatal IGF-1 (5 or 50µg) 6h prior to infusion of 4µg 6-OHDA into the same site and were euthanized 1 or 4weeks later. Both concentrations of IGF-1 protected tyrosine hydroxylase (TH) immunoreactive terminals in striatum at 4weeks but not at 1week, indicating that IGF-induced restoration of the dopaminergic phenotype occurred over several weeks. TH-immunoreactive cell loss was only attenuated with 50µg IGF-1. We then examined the effect of striatal IGF-1 on the Ras/ERK1/2 and PI3K/Akt pathways to ascertain whether their activation correlated with IGF-1-induced protection. Striatal and nigral levels of phospho-ERK1/2 were maximal 6h after IGF-1 infusion and, with the exception of an increase in nigral pERK2 at 48h, returned to basal levels by 7days. Phospho-Akt (Ser473) was elevated 6-24h post-IGF-1 infusion in both striatum and substantia nigra concomitant with inhibition of pro-death GSK-3β, a downstream target of Akt. These results suggest that IGF-1 can protect the nigrostriatal pathway in a progressive PD model and that this protection is preceded by activation of key pro-survival signaling cascades.

Partial neurorescue effects of DHA following a 6-OHDA lesion of the mouse dopaminergic system

Pre-clinical data collected in mouse models of Parkinson's disease (PD) support the neuroprotective potential of omega-3 polyunsaturated fatty acids (n-3 PUFA)-enriched diet on the dopaminergic (DAergic) system. In this study, we investigated the effects of an n-3 PUFA-rich diet using a neurorescue/neurorestorative paradigm. C57BL/6 adult mice were submitted to a striatal stereotaxic injection of the neurotoxin 6-hydroxydopamine (6-OHDA) to induce striatal DAergic denervation and subsequent nigral DAergic cell loss. Three weeks post-lesion, mice received either a docosahexaenoic acid (DHA)-enriched or a control diet for a period of 6 weeks. HPLC analyses revealed a 111% post-lesion increase in striatal dopamine levels in the DHA-fed animals compared to controls (ctrl, P<0.05), although no improvement in the motor behavior was observed. DHA treatment led to a 89% rise in tyrosine-hydroxylase (TH)-immunoreactive terminals within the striatum (P<0.05) in lesioned animals. Despite the fact that DHA did not change the number of TH+ neurons in the substantia nigra pars compacta (SNpc), morphological analyses revealed an increased in perimeters (+7%) and areas (+21%) of DAergic cell bodies in treated animals. Collectively, our results suggest that DHA induces a partial neurorescue/neurorestoration of the DAergic system and support further studies to investigate the potential of a diet-based intervention, or at least the combination of such approach, to current treatments in PD.

Annexin A2 in primary afferents contributes to neuropathic pain associated with tissue type plasminogen activator

Annexin A2 (ANX2) is a calcium (Ca2+)-binding protein that binds to acidic phospholipids and is known to play a crucial role in many cellular regulatory processes. In particular, ANX2 has been described as a crucial receptor for thrombolysis by the tissue-type plasminogen activator (tPA) and plasmin system. In the nervous system, tPA is involved in processes of neuronal plasticity such as hippocampal long-term potentiation (LTP) and in the dorsal horn pain in several pain models. We investigated detailed changes in expression of ANX2 after nerve injury and evaluated the interaction with tPA using the rat spared nerve injury (SNI) model. SNI-induced the expression of ANX2 in L4/5 dorsal root ganglia (DRG) neurons. In the spinal cord, constitutive ANX2-immunoreactivity was expressed in laminae I–II. Peripheral nerve injury increased the ANX2 immunoreactive terminals mainly in laminae I–V of the dorsal horn on the side ipsilateral to the nerve injury. Double-labeling analysis revealed the co-localization of ANX2 with tPA in the axons of primary afferents in the dorsal horn. Experimental inhibition of ANX2 and tPA interaction by intrathecal administration of homocysteine significantly prevented and reversed SNI-induced mechanical allodynia. Thus, alterations of ANX2 may be involved in tPA-dependent plasticity after peripheral nerve injury and have an important role in neuropathic pain.

Inhibitory effects of endomorphin-2 on excitatory synaptic transmission and the neuronal excitability of sacral parasympathetic preganglionic neurons in young rats.

The function of the urinary bladder is partly controlled by parasympathetic preganglionic neurons (PPNs) of the sacral parasympathetic nucleus (SPN). Our recent work demonstrated that endomorphin-2 (EM-2)-immunoreactive (IR) terminals form synapses with μ-opioid receptor (MOR)-expressing PPNs in the rat SPN. Here, we examined the effects of EM-2 on excitatory synaptic transmission and the neuronal excitability of the PPNs in young rats (24–30 days old) using a whole-cell patch-clamp approach. PPNs were identified by retrograde labeling with the fluorescent tracer tetramethylrhodamine-dextran (TMR). EM-2 (3 μM) markedly decreased both the amplitude and the frequency of the spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) of PPNs. EM-2 not only decreased the resting membrane potentials (RMPs) in 61.1% of the examined PPNs with half-maximal response at the concentration of 0.282 μM, but also increased the rheobase current and reduced the repetitive action potential firing of PPNs. Analysis of the current–voltage relationship revealed that the EM-2-induced current was reversed at −95 ± 2.5 mV and was suppressed by perfusion of the potassium channel blockers 4-aminopyridine (4-AP) or BaCl2 or by the addition of guanosine 5′-[β-thio]diphosphate trilithium salt (GDP-β-S) to the pipette solution, suggesting the involvement of the G-protein-coupled inwardly rectifying potassium (GIRK) channel. The above EM-2-invoked inhibitory effects were abolished by the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), indicating that the effects of EM-2 on PPNs were mediated by MOR via pre- and/or post-synaptic mechanisms. EM-2 activated pre- and post-synaptic MORs, inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to hyperpolarization of their membrane potentials, respectively. These inhibitory effects of EM-2 on PPNs at the spinal cord level may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic.

Inhibitory and multisynaptic spines, and hemispherical synaptic specialization in the posterodorsal medial amygdala of male and female rats

The density of dendritic spines is sexually dimorphic and variable throughout the female estrous cycle in the rat posterodorsal medial amygdala (MePD), a relevant area for the modulation of reproductive behavior in rats. The local synaptic activity differs between hemispheres in prepubertal animals. Here we used serial section transmission electron microscopy to produce 3D reconstructions of dendritic shafts and spines to characterize synaptic contacts on MePD neurons of both hemispheres in adult males and in females along the estrous cycle. Pleomorphic spines and nonsynaptic filopodia occur in the MePD. On average, 8.6% of dendritic spines received inputs from symmetric gamma-aminobutyric acid (GABA)-immunoreactive terminals, whereas 3.6% received two synaptic contacts on the spine head, neck, or base. Presynaptic terminals in female right MePD had a higher density of synaptic vesicles and docked vesicles than the left MePD, suggesting a higher rate of synaptic vesicle release in the right MePD of female rats. In contrast, males did not show laterality in any of those parameters. The proportion of putative inhibitory synapses on dendritic shafts in the right MePD of females in proestrus was higher than in the left MePD, and higher than in the right MePD in males, or in females in diestrus or estrus. This work shows synaptic laterality depending on sex and estrous cycle phase in mature MePD neurons. Most likely, sexual hormone effects are lateralized in this brain region, leading to higher synaptic activity in the right than in the left hemisphere of females, mediating timely neuroendocrine and social/reproductive behavior.

Neurovascular Changes in Acute, sub-Acute and Chronic Mouse Models of Parkinson’s Disease

Although selective neurodegeneration of nigro-striatal dopaminergic neurons is widely accepted as a cause of Parkinson's disease (PD), the role of vascular components in the brain in PD pathology is not well understood. However, the neurodegeneration seen in PD is known to be associated with neuroinflammatory-like changes that can affect or be associated with brain vascular function. Thus, dysfunction of the capillary endothelial cell component of neurovascular units present in the brain may contribute to the damage to dopaminergic neurons that occurs in PD. An animal model of PD employing acute, sub-acute and chronic exposures of mice to methyl-phenyl-tetrahydropyridine (MPTP) was used to determine the extent to which brain vasculature may be damaged in PD. Fluoro-Turquoise gelatin labeling of microvessels and endothelial cells was used to determine the extent of vascular damage produced by MPTP. In addition, tyrosine hydroxylase (TH) and NeuN were employed to detect and quantify dopaminergic neuron damage in the striatum (CPu) and substantia nigra (SNc). Gliosis was evaluated through GFAP immunohistochemistry. MPTP treatment drastically reduced TH immunoreactive neurons in the SNc (20.68 ± 2.83 in acute; 22.98 ± 2.14 in sub-acute; 10.20 ± 2.24 in chronic vs 34.88 ± 2.91 in controls; p<0.001). Similarly, TH immunoreactive terminals were dramatically reduced in the CPu of MPTP treated mice. Additionally, all three MPTP exposures resulted in a decrease in the intensity, length, and number of vessels in both CPu and SNc. Degenerative vascular changes such as endothelial cell 'clusters' were also observed after MPTP suggesting that vasculature damage may be modifying the availability of nutrients and exposing blood cells and/or toxic substances to neurons and glia. In summary, vascular damage and degeneration could be an additional exacerbating factor in the progression of PD, and therapeutics that protect and insure vascular integrity may be novel treatments for PD.

Neuropeptide Y in the adult and fetal human pineal gland.

Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland. In a series of human fetuses, neuropeptide Y-containing nerve fibers was present and could be detected as early as in the pineal of four- to five-month-old fetuses. This early innervation of the human pineal is different from most rodents, where the innervation starts postnatally.

Spatial and temporal pattern of changes in the number of GAD65-immunoreactive inhibitory terminals in the rat superficial dorsal horn following peripheral nerve injury.

Inhibitory interneurons are an important component of dorsal horn circuitry where they serve to modulate spinal nociception. There is now considerable evidence indicating that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain. A loss of these inhibitory neurons after nerve injury is one of the mechanisms being proposed to account for reduced inhibition; however, this remains controversial. This is in part because previous studies have focused on global measurements of inhibitory neurons without assessing the number of inhibitory synapses. To address this, we conducted a quantitative analysis of the spatial and temporal changes in the number of inhibitory terminals, as detected by glutamic acid decarboxylase 65 (GAD65) immunoreactivity, in the superficial dorsal horn of the spinal cord following a chronic constriction injury (CCI) to the sciatic nerve in rats. Isolectin B4 (IB4) labelling was used to define the location within the dorsal horn directly affected by the injury to the peripheral nerve. The density of GAD65 inhibitory terminals was reduced in lamina I (LI) and lamina II (LII) of the spinal cord after injury. The loss of GAD65 terminals was greatest in LII with the highest drop occurring around 3–4 weeks and a partial recovery by 56 days. The time course of changes in the number of GAD65 terminals correlated well with both the loss of IB4 labeling and with the altered thresholds to mechanical and thermal stimuli. Our detailed analysis of GAD65+ inhibitory terminals clearly revealed that nerve injury induced a transient loss of GAD65 immunoreactive terminals and suggests a potential involvement for these alterations in the development and amelioration of pain behaviour.

Synaptic Connections between Endomorphin 2-Immunoreactive Terminals and μ-Opioid Receptor-Expressing Neurons in the Sacral Parasympathetic Nucleus of the Rat

The urinary bladder is innervated by parasympathetic preganglionic neurons (PPNs) that express μ-opioid receptors (MOR) in the sacral parasympathetic nucleus (SPN) at lumbosacral segments L6-S1. The SPN also contains endomorphin 2 (EM2)-immunoreactive (IR) fibers and terminals. EM2 is the endogenous ligand of MOR. In the present study, retrograde tract-tracing with cholera toxin subunit b (CTb) or wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) via the pelvic nerve combined with immunohistochemical staining for EM2 and MOR to identify PPNs within the SPN as well as synaptic connections between the EM2-IR terminals and MOR-expressing PPNs in the SPN of the rat. After CTb was injected into the pelvic nerve, CTb retrogradely labeled neurons were almost exclusively located in the lateral part of the intermediolateral gray matter at L6-S1 of the lumbosacral spinal cord. All of the them also expressed MOR. EM2-IR terminals formed symmetric synapses with MOR-IR, WGA-HRP-labeled and WGA-HRP/MOR double-labeled neuronal cell bodies and dendrites within the SPN. These results provided morphological evidence that EM2-containing axon terminals formed symmetric synapses with MOR-expressing PPNs in the SPN. The present results also show that EM2 and MOR might be involved in both the homeostatic control and information transmission of micturition.

Acute Colitis Induces Neurokinin 1 Receptor Internalization in the Rat Lumbosacral Spinal Cord

Substance P (SP) and its receptor, the neurokinin 1 receptor (NK1R), play important roles in transmitting and regulating somatosensory nociceptive information. However, their roles in visceral nociceptive transmission and regulation remain to be elucidated. In the previous study, moderate SP immunoreactive (SP-ir) terminals and NK1R-ir neurons were observed in the dorsal commissural nucleus (DCN) of the lumbosacral spinal cord. Thus we hypothesized that the SP-NK1R system is involved in visceral pain transmission and control within the DCN. The acute visceral pain behaviors, the colon histological changes and the temporal and spatial changes of NK1R-ir structures and Fos expression in the neurons of the DCN were observed in rats following lower colon instillation with 5% formalin. The formalin instillation induced significant acute colitis as revealed by the histological changes in the colon. NK1R internalization in the DCN was obvious at 8 min. It reached a peak (75.3%) at 30 min, began to decrease at 90 min (58.1%) and finally reached the minimum (19.7%) at 3 h after instillation. Meanwhile, formalin instillation induced a biphasic visceral pain response as well as a strong expression of Fos protein in the nuclei of neurons in the DCN. Finally, intrathecal treatment with the NK1R antagonist L732138 attenuated the NK1R internalization, Fos expression and visceral nociceptive responses. The present results suggest that the visceral nociceptive information arising from inflamed pelvic organs, such as the lower colon, might be mediated by the NK1R-ir neurons in the DCN of the lumbosacral spinal cord.

Innervation of papilla vallata in diabetic rats

Introduction: Abnormal sensations such as pain and impairment of taste are symptoms of approximately 10% of patients having diabetes mellitus. Aim: The aim of the study was to investigate and quantify the different neuropeptide containing nerve fibres in the vallate papilla of the diabetic rat. Methods: Immunohistochemical methods were used to study the changes of the number of different neuropeptide containing nerve terminals located in the vallate papillae in diabetic rats. Diabetes was induced in the rats with streptozotocin. Results: Two weeks after streptozotocin treatment the number of the substance P, galanin, vasoactive intestinal polypeptide and neuropeptide Y immunoreactive nerve terminals was significantly increased (p&lt;0.05) in the tunica mucosa of the tongue. The number of the lymphocytes and mast cells was also increased significantly. Some of the immunoreactive nerve terminals were located in the lingual epithelium both intragemmally and extragemmally and were seen to comprise dense bundles in the lamina propria just beneath the epithelium. No taste cells were immunoreactive for any of the investigated peptides. Vasoactive intestinal polypeptide and neuropeptide Y immunoreactive nerve fibres were not detected in the taste buds. For weeks after streptozotocin administration the number of the substance P, calcitonin gene related peptide and galanin immunoreactive nerve terminals was decreased both intragemmally and intergemmally. In case of immediate insulin treatment, the number of the immunoreactive nerve terminals was similar to that of the controls, however, insulin treatment given 1 week later to diabetic rats produced a decreased number of nerve fibers. Morphometry revealed no significant difference in papilla size between the control and diabetic groups, but there were fewer taste buds (per papilla). Conclusions: Increased number of immunoreactive nerve terminals and mast cells 2 weeks after the development of diabetes was the consequence of neurogenic inflammation which might cause vasoconstriction and lesions of the oral mucosa. Taste impairment, which developed 4 weeks after streptozotocin treatment could be caused by neuropathic defects and degeneration or morphological changes in the taste buds and nerve fibres. Orv. Hetil., 2013, 154, 443–448.

Understanding the Direct Synaptic Effects of Estradiol

More than 35 years ago, Kelly and coworkers (1) demonstrated that iontophoretic application of an estradiol ester altered the firing rates of a subset of hypothalamic neurons in a matter of milliseconds, an effect that appeared to be stereospecific in that responses were observed to esters of 17 -estradiol, but not its 17 isomer. These observations accompanied the almost simultaneous demonstration by Pietras and Szego (2) that endometrial cells had estradiol recognition sites on their plasma membranes. Kelly et al (1) proposed that neurons might also express stereospecific membrane receptor sites, capable of mediating rapid electrophysiological responses to changes in ovarian estradiol secretion. Progress in characterizing these putative membrane receptors was initially slow, because most work continued to focus on estrogen receptor (ER) actions in the cell nucleus and the regulation of transcriptional response mechanisms. This situation changed dramatically in the early 1990s with the demonstration of rapid effects of estradiol on a number of intracellular signaling pathways (3, 4) as well as direct evidence of ER-like immunoreactivity in dendrites, axon terminals, and postsynaptic densities in the brain (5–7) (reviewed in Blaustein, Ref. 8). Over the ensuing 20 years, multiple potential receptor systems for estradiol have been identified in the central nervous system, signaling via a number of different intracellular pathways (reviewed in Micevych and Christensen, Ref. 9). Studies in cells transfected with vectors expressing ER or ER have shown that whereas newly synthesized receptors concentrate in the cell nucleus, they also incorporate into the plasma membrane (10) where they can initiate estradiol-dependent changes in ERK phosphorylation (11), suggesting that plasma membrane and nuclear ER or ER may be derived from a common source. The demonstration that ER immunoreactivity is present in multiple neuronal cellular compartments raises the question of how the distribution of extranuclear ER is controlled. Are ERs associated with specific cellular organelles or do synaptic and dendritic ERs represent receptors en passant to the plasma membrane? Studies in nonneuronal systems have demonstrated that that ER is trafficked to the plasma membrane via a caveolin-dependent process (12) where it can be anchored in place by palmitoylation (13). Is the same true for ER in neurons? Is the density of ER in the plasma membrane regulated independently of nuclear receptor concentrations? Is ER distributed evenly throughout neuronal cell membranes or is it concentrated specifically in target sites where it can both respond to locally synthesized steroids and exert rapid effects on neurotransmission? The paper by Tabatadze et al (14) in this issue of Endocrinology begins to answer some of these questions. Using tissue fractionationtechniques, theauthorsdemonstrate that ER is concentrated in synaptosomes, particularly in association with synaptic vesicles and neuropeptide-containing large dense-core vesicles. Immunoisolation experiments showthatER is associatedwithbothgammaaminobutryic acid (GABA)and glutamate-containing vesicles. On Western blots, synaptosomal ER immunoreactivity migrates as two bands at approximately 66 and 70 kDa, the ratio of the higher to lower molecular mass forms being greatly decreased by phosphatase treatment, suggesting that the 70kDa band represents a phosphorylated form of the receptor. Finally, treatment of hippocampal slices in vitro was found to decrease the quantity of ER associated with synaptosomal membranes, via a depalmitoylation-dependent mechanism. Overall, the results suggest that ER associates with synaptosomal membranes via a palmitoyla-

Corticotropin-releasing factor in the mouse central nucleus of the amygdala: Ultrastructural distribution in NMDA-NR1 receptor subunit expressing neurons as well as projection neurons to the bed nucleus of the stria terminalis

Corticotropin-releasing factor (CRF) and glutamate are critical signaling molecules in the central nucleus of the amygdala (CeA). Central amygdala CRF, acting via the CRF type 1 receptor (CRF-R1), plays an integral role in stress responses and emotional learning, processes that are generally known to involve functional NMDA-type glutamate receptors. There is also evidence that CRF expressing CeA projection neurons to the bed nucleus of the stria terminalis (BNST) play an important role in stress related behaviors. Despite the potentially significant interactions between CRF and NMDA receptors in the CeA, the synaptic organization of these systems is largely unknown. Using dual labeling high resolution immunocytochemical electron microscopy, it was found that individual somata and dendrites displayed immunoreactivity for CRF and the NMDA-NR1 (NR1) subunit in the mouse CeA. In addition, CRF-containing axon terminals contacted postsynaptic targets in the CeA, some of which also expressed NR1. Neuronal profiles expressing the CRF type 1 receptor (CRF-R1), identified by the expression of green fluorescent protein (GFP) in bacterial artificial chromosome (BAC) transgenic mice, also contained NR1, and GFP immunoreactive terminals formed synapses with NR1 containing dendrites. Although CRF and GFP were only occasionally co-expressed in individual somata and dendritic profiles, contacts between labeled axon terminals and dendrites were frequently observed. A combination of tract tracing and immunocytochemistry revealed that a population of CeA CRF neurons projected to the BNST. It was also found that CRF, or GFP expressing terminals directly contacted CeA-BNST projection neurons. These results indicate that the NMDA receptor is positioned for the postsynaptic regulation of CRF expressing CeA neurons and the modulation of signals conveyed by CRF inputs. Interactions between CRF and NMDA receptor mediated signaling in CeA neurons, including those projecting to the BNST, may provide the synaptic basis for integrating the experience of stress and relevant environmental stimuli with behaviors that may be of particular relevance to stress-related learning and the emergence of psychiatric disorders, including drug addiction.

Cellular and subcellular localization of CXCL12 and CXCR4 in rat nociceptive structures: physiological relevance

Initial studies implicated the chemokine CXC motif ligand 12 (CXCL12) and its cognate CXC motif receptor 4 (CXCR4) in pain modulation. However, there has been no description of the distribution, transport and axonal sorting of CXCL12 and CXCR4 in rat nociceptive structures, and their direct participation in nociception modulation has not been demonstrated. Here, we report that acute intrathecal administration of CXCL12 induced mechanical hypersensitivity in naive rats. This effect was prevented by a CXCR4-neutralizing antibody. To determine the morphological basis of this behavioural response, we used light and electron microscopic immunohistochemistry to map CXCL12- and CXCR4-immunoreactive elements in dorsal root ganglia, lumbar spinal cord, sciatic nerve and skin. Light microscopy analysis revealed CXCL12 and CXCR4 immunoreactivity in calcitonin gene related peptide-containing peptidergic primary sensory neurons, which were both conveyed to central and peripheral sensory nerve terminals. Electron microscopy clearly demonstrated CXCL12 and CXCR4 immunoreactivity in primary sensory nerve terminals in the dorsal horn; both were sorted into small clear vesicles and large dense-core vesicles. This suggests that CXCL12 and CXCR4 are trafficked from nerve cell bodies to the dorsal horn. Double immunogold labelling for CXCL12 and calcitonin gene related peptide revealed partial vesicular colocalization in axonal terminals. We report, for the first time, that CXCR4 receptors are mainly located on the neuronal plasma membrane, where they are present at pre-synaptic and post-synaptic sites of central terminals. Receptor inactivation experiments, behavioural studies and morphological analyses provide strong evidence that the CXCL12/CXCR4 system is involved in modulation of nociceptive signalling.

Excitatory inputs to four types of spinocerebellar tract neurons in the cat and the rat thoraco-lumbar spinal cord.

The cerebellum receives information from the hindlimbs through several populations of spinocerebellar tract neurons. Although the role of these neurons has been established in electrophysiological experiments, the relative contribution of afferent fibres and central neurons to their excitatory input has only been estimated approximately so far. Taking advantage of differences in the immunohistochemistry of glutamatergic terminals of peripheral afferents and of central neurons (with vesicular glutamate transporters VGLUT1 or VGLUT2, respectively), we compared sources of excitatory input to four populations of spinocerebellar neurons in the thoraco-lumbar spinal cord: dorsal spinocerebellar tract neurons located in Clarke's column (ccDSCT) and in the dorsal horn (dhDSCT) and ventral spinocerebellar tract (VSCT) neurons including spinal border (SB) neurons. This was done on 22 electrophysiologically identified intracellularly labelled neurons in cats and on 80 neurons labelled by retrograde transport of cholera toxin b subunit injected into the cerebellum of rats. In both species distribution of antibodies against VGLUT1 and VGLUT2 on SB neurons (which have dominating inhibitory input from limb muscles), revealed very few VGLUT1 contacts and remarkably high numbers of VGLUT2 contacts. In VSCT neurons with excitatory afferent input, the number of VGLUT1 contacts was relatively high although VGLUT2 contacts likewise dominated, while the proportions of VGLUT1 and VGLUT2 immunoreactive terminals were the reverse on the two populations of DSCT neurons. These findings provide morphological evidence that SB neurons principally receive excitatory inputs from central neurons and provide the cerebellum with information regarding central neuronal activity.