Vagal afferent innervation and remodeling in the aortic arch of young-adult fischer 344 rats following chronic intermittent hypoxia.

Previously, we have shown that chronic intermittent hypoxia (CIH) impairs baroreflex control of heart rate and augments aortic baroreceptor afferent function. In the present study, we examined whether CIH induces structural changes of aortic afferent axons and terminals. Young-adult Fischer 344 (F344, 4 months old) rats were exposed to room air (RA) or CIH for 35-45 days. After 14-24 days of exposure, they received tracer DiI injection into the left nodose ganglion to anterogradely label vagal afferent nerves. After surgery, animals were returned to their cages to continue RA or CIH exposure. Twenty-one days after DiI injection, the animals were sacrificed and the aortic arch was examined using confocal microscopy. In both RA and CIH rats, we found that DiI-labeled vagal afferent axons entered the wall of the aortic arch, then fanned out and branched into large receptive fields with numerous terminals (flower-sprays, end-nets and free endings). Vagal afferent axons projected much more to the anterior wall than to the posterior wall. In general, the flower-sprays, end-nets and free endings were widely and similarly distributed in the aortic arch of both groups. However, several salient differences between RA and CIH rats were found. Compared to RA control, CIH rats appeared to have larger vagal afferent receptive fields. The CIH rats had many abnormal flower-sprays, end-nets, and free endings which were intermingled and diffused into "bush-like" structures. However, the total number of flower-sprays was comparable (P>0.05). Since there was a large variance of the size of flower-sprays, we only sampled the 10 largest flower-sprays from each animal. CIH substantially increased the size of large flower-sprays (P<0.01). Numerous free endings with enlarged varicosities were identified, resembling axonal sprouting structures. Taken together, our data indicate that CIH induces significant remodeling of afferent terminal structures in the aortic arch of F344 rats. We suggest that such an enlargement of vagal afferent terminals may contribute to altered aortic baroreceptor function following CIH.

Impaired baroreflex control of renal sympathetic nerve activity in type 1 diabetic mice (OVE26).

To investigate the effects of chronic diabetes on baroreflex control of renal sympathetic nerve activity (RSNA), OVE26 diabetic (transgenic mouse line which develops hyperglycemia within the first 3 weeks after birth) and FVB control mice 5-6 months old were studied. Under anesthesia, RSNA in response to sodium nitroprusside (SNP)- and phenylephrine (PE)-induced mean arterial pressure changes (DeltaMAP) were measured. Baroreflex-induced inhibition of RSNA during PE infusion was characterized using the sigmoid logistic function curve. Baroreflex-induced excitation of RSNA during SNP infusion was characterized by the RSNA vs. DeltaMAP relationship. Mean arterial pressure (MAP) responses to the left aortic depressor nerve (ADN) stimulation were evaluated. Compared to FVB control, we found in OVE26 mice that (1) RSNA in response to MAP increase during PE infusion was dramatically reduced, as characterized by the maximal gain of the RSNA sigmoid logistic function curve (FVB: -20.0+/-5.1; OVE26: -7.6+/-0.8%/mm Hg, P<0.05); (2) RSNA in response to MAP decrease during SNP infusion was also attenuated (P<0.05); (3) MAP responses to ADN stimulation were reduced (P<0.05). We concluded that chronic diabetes impairs baroreflex control of RSNA in OVE26 diabetic mice. The use of the transgenic OVE26 diabetic mouse model may underlie a foundation for the further understanding of diabetes-induced autonomic neuropathy.

Attenuation of heart rate control and neural degeneration in nucleus ambiguus following chronic intermittent hypoxia in young adult Fischer 344 rats.

Chronic intermittent hypoxia (CIH) attenuates baroreflex control of heart rate (HR). In this study, we assessed whether CIH exposure reduced nucleus ambiguus (NA) control of HR and induced neural degeneration in the NA. Fischer 344 (age: 3-4 months) rats were exposed to either room air (RA: normoxia) or intermittent hypoxia for 35-50 days. At the end of these exposures, animals were anesthetized with pentobarbital. HR responses to arterial blood pressure (AP) changes induced by phenylephrine (PE) and sodium nitroprusside (SNP) were measured. In another set of rats, HR and AP responses to L-glutamate (L-Glu) microinjections (10 mM, 20 nl) into the left NA and electrical stimulation of the left cervical vagus nerve at 1-30 Hz (0.5 mA, 1 ms) for 20 s were measured. Brainstem slices at the level of -800, -400, 0, +400, +800 microm relative to the obex were processed in additional rats using Nissl staining. The NA was identified by retrogradely labeling vagal motoneurons using the tracer tetramethylrhodamine dextran (TMR-D) which was injected into the ipsilateral nodose ganglion. We found that CIH significantly 1) reduced the baroreflex control of HR (slope RA: -1.2+/-0.2 bpm/mmHg; CIH -0.5+/-0.1 bpm/mmHg; P<0.05); 2) attenuated the HR responses to l-Glu injections into the NA [HR: -280+/-15 (RA) vs. -235+/-16 (CIH) beats/min; P<0.05]; 3) augmented the HR responses to electrical stimulation of the vagus (P<0.05); 4) induced a significant cellular loss in the NA region (P<0.05). Thus, CIH induces a cell loss in the NA region which may contribute to attenuation of baroreflex sensitivity and NA control of HR following CIH.

Functional changes in baroreceptor afferent, central and efferent components of the baroreflex circuitry in type 1 diabetic mice (OVE26).

Baroreflex control of heart rate (HR) is impaired in diabetes mellitus. We hypothesized that diabetes mellitus induced functional changes of neural components at multiple sites within the baroreflex arc. Type 1 diabetic mice (OVE26) and FVB control mice were anesthetized. Baroreflex-mediated HR responses to sodium nitroprusside- (SNP) and phenylephrine- (PE) induced mean arterial blood pressure (MAP) changes were measured. Baroreceptor function was characterized by measuring the percent (%) change of baseline integrated aortic depressor nerve activity (Int ADNA) in response to SNP- and PE-induced MAP changes. The HR responses to electrical stimulation of the left aortic depressor nerve (ADN) and the right vagus nerve were assessed. Compared with FVB control mice, we found in OVE26 mice that (1) baroreflex-mediated bradycardia and tachycardia were significantly reduced. (2) The baroreceptor afferent function in response to MAP increase did not differ, as assessed by the parameters of the logistic function curve. But, the inhibition of Int ADNA in response to MAP decrease was significantly attenuated. (3) The maximum amplitude of bradycardic responses to right vagal efferent stimulation was augmented. (4) In contrast, the maximum amplitude of bradycardic responses to left ADN stimulation was decreased. Since Int ADNA was preserved in response to MAP increase and HR responses to vagal efferent stimulation were augmented, we conclude that a deficit of the central mediation of baroreflex HR contributes to the overall attenuation of baroreflex sensitivity in OVE26 mice. The successful conduction of physiological experiments on the ADN in OVE26 mice may provide a foundation for the understanding of cellular and molecular mechanisms of diabetes-induced cardiac neuropathy.

Morphology and topography of nucleus ambiguus projections to cardiac ganglia in rats and mice.

Vagal efferent axons from the nucleus ambiguus (NA) innervate ganglionated plexuses in the dorsal surface of cardiac atria, which in turn, may have different functional roles in cardiac regulation. However, the morphology and topography of vagal efferent projections to these ganglionated plexuses in rats and mice have not been well delineated. In the present study, we injected the tracer 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine methanesulfonate (DiI) into the left NA to label vagal efferent axons and terminals in cardiac ganglia and administered Fluoro-Gold (FG) i.p. to stain cardiac ganglia. Then, we used confocal microscopy and a Neurolucida 3-D Digitization System to qualitatively and quantitatively examine the distribution and structure of cardiac ganglia, and NA efferent projections to cardiac ganglia in the whole-mounts of Sprague-Dawley (SD) rats and FVB mice. Our observations were: 1) Cardiac ganglia of different shapes and sizes were distributed in the sinoatrial (SA) node, atrioventricular (AV) node, and lower pulmonary vein (LPV) regions on the dorsal surface of the atria. In each region, several ganglia formed a ganglionated plexus. The plexuses at different locations were interconnected by nerves. 2) Vagal efferent fibers ramified within cardiac ganglia, formed a complex network of axons, and innervated cardiac ganglia with very dense basket endings around individual cardiac principal neurons (PNs). 3) The percent of the PNs in cardiac ganglia which were innervated by DiI-labeled axons was 54.3+/-3.2% in mice vs. 53.2+/-3.2% in rats (P>0.10). 4) The density of axonal putative-synaptic varicosities on the surface of PNs was 0.15+/-0.02/microm(2) in mice vs. 0.16+/-0.02/microm(2) in rats (P>0.10). Thus, the distributions of cardiac ganglia and vagal efferent projections to cardiac ganglia in mice and rats were quite similar both qualitatively and quantitatively. Our study provides the structural foundation for future investigation of functional differentiation of ganglionated plexuses and the brain-heart circuitry in rodent models of human disease.

Calcium channel antagonists inhibit growth of subcutaneous xenograft meningiomas in nude mice.

BACKGROUND: We have previously shown that calcium channel antagonists inhibit in vitro meningioma growth. This study examines the effect of calcium channel antagonists on in vivo xenograft meningioma growth. METHODS: Meningioma cells taken from human patients were mixed with Matrigel and injected into the subcutaneous space in the flank of nude mice. These animals were treated with calcium channel antagonists in their drinking water. Tumor volumes were measured over time; comparison was made between control and treatment groups. Daily weights, average daily water consumption, and serum calcium channel antagonist levels were determined. Comparison of histology and proliferation index was made between control and treatment groups. RESULTS: Diltiazem treatment decreased tumor growth over time compared to control groups. Increased tumor growth inhibition was seen with increasing doses (p > 0.05). Treatment with verapamil had similar effects; however, there are no statistically significant dose dependent decreases in growth with increasing verapamil doses. There were no tumor "cures" or spontaneous regression of tumor in any group including the control groups. Animal daily weight and average daily water consumption was unaffected by increasing calcium channel antagonist doses compared to control groups. Mouse serum drug levels increased with increasing doses of drug in the drinking water of treatment groups (p > 0.05). Histology and proliferative index of treatment groups were similar to control groups. CONCLUSION: Calcium channel antagonists decrease but do not completely inhibit the growth of meningiomas in nude mice. Clinical correlations and potential applications are discussed.

Duration of neuronal stretch correlates with functional loss.

Postoperative cranial nerve weakness or paralysis is not uncommon in many otolaryngologic surgical procedures. Our study used a rat model to test the hypothesis that the length of time that a nerve is under tension may be an important variable in the amount of postoperative paresis. Forty Sprague-Dawley rats were divided into 4 groups that underwent either a sham operation or a traction injury for 1, 2, or 5 minutes. The traction injury was performed with a vessel loop placed around the sciatic nerve with 50 g of tension. Traction injury for 1 or 2 minutes did not result in any statistical differences in the motor capabilities of the lower limb. However, those animals with a stretch injury for 5 minutes had a significant loss of function (P < 0.01) when compared with all other groups. Histologic examination of nerves harvested on postoperative day 7 showed no evidence of mechanical injury. This study demonstrates that even minimal tension, if maintained for a significant amount of time, may result in postoperative weakness.

Calcium channel antagonist effect on in vitro meningioma signal transduction pathways after growth factor stimulation.

OBJECTIVE: We have previously demonstrated that calcium channel antagonists inhibit the growth of human meningiomas in culture after stimulation with growth factors. This study examined the effects of these drugs on signaling transduction pathways in an attempt to elucidate potential mechanisms by which this growth inhibition is mediated. METHODS: Primary cell cultures from patients with intracranial meningiomas were established. Cell growth studies were performed with inhibitors and stimulators of tyrosine kinase signal transduction. Intracellular calcium changes and inositol phosphate production were measured after growth factor exposure, with or without pretreatment by calcium channel antagonists. RESULTS: The growth of meningiomas in culture can be inhibited by tyrosine kinase receptor inhibitors. Inhibitors and stimulators of phospholipase C can stimulate or inhibit the growth of in vitro meningiomas, respectively. Calcium channel antagonists inhibit intracellular calcium changes induced by serum and epidermal growth factor. Inositol phosphate production is increased after growth factor stimulation, and calcium channel antagonists potentiate this effect. CONCLUSION: Calcium channel antagonists interfere with intracellular signaling pathways of cultured meningioma cells. This inhibition is unrelated to voltage-sensitive calcium channels. The findings of this project may aid in the understanding of the signal transduction mechanisms involved in growth factor-mediated meningioma proliferation and may lead to clinically relevant strategies for growth inhibition.

Neuroprotection due to irrigation during bipolar cautery.

OBJECTIVE: To test whether irrigation during bipolar cautery confers thermoprotection from neuronal injury. DESIGN: A rat animal model (15 rats for each treatment group) was used to test the thermoprotective effects of irrigation during bipolar cautery. In this model, the sciatic nerve was exposed, and a 1-second stimulus was applied using bipolar cautery forceps at 40 or 20 W placed directly on the nerve in the presence or absence of simultaneous irrigation. The effects of cautery were determined on the basis of clinical gait analysis by means of the Sciatic Functional Index, temperature response, and neuropathological findings. RESULTS: The degree of paresis was reduced with irrigation. Neuropathological examination of the sciatic nerve after cautery showed significant axonal loss (more small than large fibers) with concomitant demyelination, which was partially inhibited by irrigation (chi2; P = .04). The mechanism of thermoprotection by irrigation was not the result of a reduction in the temperature spike that followed cautery, but resulted from a reduced temperature response during the 15 seconds that followed 40- or 20-W stimulation with bipolar cautery. CONCLUSIONS: Simultaneous irrigation and bipolar cautery enhance temperature recovery to basal levels and protect the peripheral nerve from the effects of cautery.

Evaluation of a suture electrode for direct bladder stimulation in a lower motor neuron lesioned animal model.

The purpose of this study was to evaluate a "suture" type electrode for direct bladder stimulation in an animal model of a lower motor neuron lesion. During an initial surgery, five male cats were instrumented under anesthesia using multistranded, 316 LVM, stainless-steel, wire electrodes implanted on the bladder wall serosa above the trigone area. Electrodes were constructed with a needle attached to the end that was removed after suturing the electrode in place. Additional instrumentation included urinary bladder catheters (tubes) for pressure recording and filling, and hook type electrodes for leg and pelvic floor electromyography recording. Chronic bladder filling and stimulation studies were conducted in tethered animals three to four weeks following surgery. To test these electrodes in a spinal cord injury model, a lower motor neuron lesion was performed including the sacral cord and complete nerve roots at L6 and below. These animals were evaluated during weeks 3 and 10 after injury. Direct bladder stimulation induced active contractions and voiding both before and after spinal cord injury. Effective stimulation parameters consisted of 40 pulses per s, 300 micros to 1 ms pulse duration, a stimulation period from 3 to 4 s, and a stimulation current from 10 to 40 mA. Fluoroscopy revealed an open membranous urethra during stimulation and following stimulation. A small diameter penile urethra was observed to limit flow. Postmortem evaluation of the suture electrode revealed no abnormalities such as corrosion, migration into the bladder lumen or displacement. These findings indicate that suture electrodes are suitable and effective for short-term implantation in the lower motor neuron animal model.

Activation of various G-protein coupled receptors modulates Ca2+ channel currents via PTX-sensitive and voltage-dependent pathways in rat intracardiac neurons.

In the present study, we examined the ability of several putative neurotransmitters and neuromodulators to modulate voltage-dependent Ca2+ channel currents in adult rat intracardiac neurons. Of 17 compounds tested, acetylcholine (Ach), neuropeptide Y (NPY), norepinephrine (NE), and met-enkephalin (met-Enk) were effective modulators of the Ca2+ currents. The neurotransmitter-induced current inhibition was associated with slow activation kinetics and relief by a strong depolarizing prepulse. Overnight pretreatment of neurons with pertussis toxin (PTX, 500 ng/ml) significantly attenuated the neurotransmitter-induced current inhibition. Heterologous expression of transducin, a known chelator of G-protein betagamma subunits, almost completely abolished the neurotransmitter-induced current inhibition. Taken together, our data suggest that four different neurotransmitters inhibit the Ca2+ channel currents in adult rat intracardiac neurons via a pathway that is voltage-dependent, membrane-delimited, and utilizes betagamma subunits released from PTX-sensitive G-proteins. The Ca2+ channel inhibition by non-cholinergic neurotransmitters may play important roles in regulation of neuronal excitability and Ach release at synapses in intracardiac ganglia, thereby contributing to cholinergic control of cardiac functions.

Monoamine- and histamine-synthesizing enzymes and neurotransmitters within neurons of adult human cardiac ganglia.

BACKGROUND: Cardiac ganglia were originally thought to contain only cholinergic neurons relaying parasympathetic information from preganglionic brain stem neurons to the heart. Accumulating evidence, however, suggests that cardiac ganglia contain a heterogeneous population of neurons that synthesize or respond to several different neurotransmitters and neuropeptides. Reports regarding monoamine and histamine synthesis and neurotransmission within cardiac ganglia, however, present conflicting information or are limited in number. Furthermore, very few studies have examined the neurochemistry of adult human cardiac ganglia. The purpose of this study was, therefore, to determine whether monoamine- and histamine-synthesizing enzymes and neurotransmitters exist within neurons of adult human cardiac ganglia. METHODS AND RESULTS: Human heart tissue containing cardiac ganglia was obtained during autopsies of patients without cardiovascular pathology. Avidin-biotin complex immunohistochemistry was used to demonstrate tyrosine hydroxylase, L-dopa decarboxylase, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, tryptophan hydroxylase, and histidine decarboxylase immunoreactivity within neurons of cardiac ganglia. Dopamine, norepinephrine, serotonin, and histamine immunoreactivity was also found in ganglionic neurons. Omission or preadsorption of primary antibodies from the antisera and subsequent incubation with cardiac ganglia abolished specific staining in all cases examined. CONCLUSIONS: Our results suggest that neurons within cardiac ganglia contain enzymes involved in the synthesis of monoamines and histamine and that they contain dopamine, norepinephrine, serotonin, and histamine immunoreactivity. Our findings suggest a putative role for monoamine and histamine neurotransmission within adult human cardiac ganglia. Additional, functional evidence will be necessary to evaluate what the physiological role of monoamines and histamine may be in neural control of the adult human heart.

Proliferation of cultured human astrocytoma cells in response to an oxidant and antioxidant.

The role of reactive oxygen species (ROS) in initiation, promotion and progression of several (lung, skin, colon, bladder, breast) tumors is well-documented. Indirect evidence for ROS involvement in tumor proliferation is provided by numerous in vivo and in vitro studies that show antioxidants inhibit tumor proliferation. However, despite strong epidemiological and experimental support for ROS involvement in brain tumor proliferation, to date little is known about the role of ROS in brain tumor promotion at a cellular level. In the present study ROS involvement in proliferation of a cultured, human astrocytoma cell line (U373-MG) was tested by studying effects of an oxidant (hydrogen peroxide, H2O2), and an antioxidant (N-acetylcysteine, NAC) on astrocytoma on proliferation of these cultured cells. Proliferation was assessed by evaluating changes in cell counts and DNA synthesis. Results from these experiments clearly indicate that NAC inhibits tumor cell proliferation and DNA synthesis induced by both serum and H2O2 (10(-5) M). NAC alone did not have any significant effects on the proliferation of serum-starved cells. Thus, ROS are capable of inducing proliferation in cultured astrocytoma cells and antioxidants block ROS- and serum-induced proliferation. Further investigation using primary cultures and animal models will be needed to substantiate the therapeutic potential of antioxidants in future brain tumor therapy.

PLA2 activity regulates Ca2+ storage-dependent cellular proliferation.

The objective of this study is to determine the role of arachidonic acid (AA) in cell proliferation by inhibiting AA synthetic enzyme phospholipase A2 (PLA2) and to determine its involvement in the role of the second messenger intracellular calcium (Ca2+). Methods used to determine the effects on proliferation of cell cultures of primary meningioma and astrocytoma U373-MG included treatment with micromolar concentrations of PLA2 inhibitors 4-bromophenacylbromide and quinacrine. Effects of these drugs on proliferation were further investigated by the application of concentrations that inhibit growth by 50% while antagonizing these agents with AA replacement. Free cytosolic Ca2+ was measured with the use of fluorescent dye Fura-2 during PLA2 agonist/antagonist studies. These Ca2+ measurements were performed in the absence of extracellular Ca2+ to identify the contribution of intracellular Ca2+ sources. PLA2 inhibition resulted in decreased growth of cultured astrocytoma and meningioma cells in a dose-dependent manner in the micromolar range. This inhibitory effect was antagonized by the addition of AA. PLA2 inhibition caused an elevation of basal-cytosolic-free [Ca2+] while depleting internal Ca2+ stores. These Ca2+ changes were also antagonized by the addition of AA. In conclusion, these results demonstrate that AA, a PLA2 enzyme product, is involved in regulating the growth rate of these cell types. The PLA2 pathway also regulates the maintenance of the internal Ca2+ stores. Ca2+ is known to be a growth-related intracellular second messenger. These results suggest that the growth regulatory functions of AA are mediated by Ca2+-dependent mechanisms.

Thermoprotective mechanisms of irrigation during bipolar cautery.

Bipolar cautery is routinely used in operations of the head and neck, as well as in other specialties, both for dissection and for achieving hemostasis. Whereas simultaneous irrigation is frequently used to minimize neuronal injury, its effectiveness has not been tested under controlled conditions. Our objectives in this study were to test the hypothesis that including irrigation during bipolar cautery is thermoprotective and to identify the mechanisms underlying the thermoprotective effect. The thermoprotective role of irrigation with bipolar cautery was tested in a rat model in which the sciatic nerve was exposed and a 1-second stimulus at 40 or 20 watts was applied with bipolar cautery forceps placed directly on the nerve in the presence or absence of simultaneous irrigation. We used the Sciatic Functional Index as used to quantitate the degree of paresis induced. The results showed that simultaneous irrigation reduced the percentage of animals showing paresis. This effect was significant for animals exposed to 40- and 20-watt cautery. The mechanism for the reduction in the degree of paresis by irrigation could not be attributed to a lowering of the maximal temperature achieved after bipolar cautery. Instead, the thermoprotective mechanism of the irrigation involved an enhanced recovery to basal temperatures when measured at 15 seconds after nerve stimulation with 40 or 20 watts. Reducing the power from 40 watts to 20 watts did not significantly lessen the tissue temperature. The results of this study suggest that irrigation done simultaneously with bipolar cautery enhances temperature recovery to basal levels and plays a role in thermoprotection against the effects of cautery.

Matrigel augments xenograft transplantation of meningioma cells into athymic mice.

OBJECTIVE: We investigated the use of Matrigel to enhance the growth of human meningiomas in athymic (nude) mice. Tumor take and growth of xenograft meningioma cells in an in vivo model have previously been only partially successful. METHODS: The use of Matrigel has been reported to enhance tumorigenicity in a variety of solid tumors. This substance is derived from a mouse sarcoma and is a mixture of basement membrane proteins and growth factors. Meningioma cells obtained from human patients were placed in culture for 1 to 2 passages and then harvested and mixed with Matrigel and the mixture injected into the subcutaneous space in the flank of nude mice. Tumor volumes over time were measured at least three times a week and then harvested at 100 days postimplantation. Tumors were formalin-fixed, and histological examinations were performed. Immunohistochemistry was performed for human and mouse laminin, fibronectin, collagen Type IV, and epithelial membrane antigen. RESULTS: Tumors developed in all 40 mice studied. Growth of meningioma tumors was dependent on total number of cells injected and independent of the total volume of tumor cells and Matrigel matrix. Histologically and immunohistochemically, the xenograft tumors were very similar to the original human tumors. CONCLUSION: Matrigel is relatively easy to use and has a high rate of histologically confirmed meningioma tumor formation in an athymic mouse model. We plan to use this model for studying the growth of meningiomas in vivo.

Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway.

With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba2+ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 microM) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTPgammaS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). In conclusion, the M2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.

Calcium channel currents in acutely dissociated intracardiac neurons from adult rats.

With the use of the whole cell patch-clamp technique, multiple subtypes of voltage-activated calcium channels, as indicated by measuring Ba2+ currents, were pharmacologically identified in acutely dissociated intracardiac neurons from adult rats. All tested neurons that were held at -80 mV displayed only high-voltage-activated (HVA) Ca2+ channel currents that were completely blocked by 100 microM CdCl2. The current density of HVA Ca2+ currents was dependent on the external Ca2+ concentration. The Ba2+ (5 mM) currents were half-activated at -16.3 mV with a slope of 5.6 mV per e-fold change. The steady-state inactivation was also voltage dependent with half-inactivation at -33.7 mV and a slope of -12.1 mV per e-fold change. The most effective L-type channel activator, FPL 64176 (2 microM), enhanced the Ba2+ current in a voltage-dependent manner. When cells were held at -80 mV, the saturating concentration (10 microM) of nifedipine blocked approximately 11% of the control Ba2+ current. The major component of the Ca2+ channels was N type (63%), which was blocked by a saturating concentration (1 microM) of omega-conotoxin GVIA. Approximately 19% of the control Ba2+ current was sensitive to omega-conotoxin MVIIC (5 microM) but insensitive to low concentrations (30 and 100 nM) of omega-agatoxin IVA (omega-Aga IVA). In addition, a high concentration (1 microM) of omega-Aga IVA occluded the effect of omega-conotoxin MVIIC. Taken together, these results indicate that the omega-conotoxin MVIIC-sensitive current represents only the Q type of Ca2+ channels. The current that was insensitive to nifedipine and various toxins represents the R-type current (7%), which was sensitive to 100 microM NiCl2. In conclusion, the intracardiac neurons from adult rats express at least four different subtypes (L, N, Q, and R) of HVA Ca2+ channels. This information is essential for understanding the regulation of synaptic transmission and excitability of intracardiac neurons by different neurotransmitters and neural regulation of cardiac functions.

Determination of in situ dissociation constant for Fura-2 and quantitation of background fluorescence in astrocyte cell line U373-MG.

Accurate estimates of cytosolic free Ca2+ with fluorescence indicators are dependent on the determination of the in situ dissociation constants (kd) of the intracellular dyes and the correction for background fluorescence. The in situ dissociation constant for Ca2+ and indicator dye Fura-2/AM varies significantly from the in vitro published values due to differences in ionic strength, pH, viscosity and Ca2+ buffering by intracellular lipids and proteins. During the course of a measurement, background fluorescence changes may occur as the result of endogenous fluorescent compounds and compartmentalized Fura-2 indicator. The in situ dissociation constant value was determined for human astrocyte cell line U373-MG by creating several known intracellular Ca2+ concentrations while measuring total fluorescence and background fluorescence values for each. The background fluorescence was not constant, rather it demonstrated a linear relationship with the free cytosolic Ca2+ concentrations and total fluorescence intensities. The Ca2+ dependent and total fluorescence dependent background was expressed as a linear equation and subtracted appropriately from the total intensity measurements. The in situ dissociation constant was determined to be 3-fold greater than in vitro measurements after the background was corrected. The experimentally determined standard linear equations for background quantitation and the in situ dissociation constant for this line produce accurate cytosolic free Ca2+ estimates.

Testing peripheral somatosensory neuroprostheses by recording from raccoon cortex.

A topologically organized representation of the body surface exists within the mammalian somatosensory cortical areas such that stimulation of a part of the body surface will produce a response in a localized region of the contralateral somatosensory cortex. Because of this topography, we propose that the selectivity of a peripheral somatosensory neuroprosthetic electrode array can be tested by noting whether the locus of maximum activation in the cortex moves in a consistent manner when differing portions of the array are stimulated. We further propose that the raccoon might well be the ideal animal in which to test this hypothesis, since the raccoon has a rather unique cortical somatosensory area where each digit is represented in individual subgyri around the tri-radiate sulcus. To demonstrate the feasibility of this concept, a pilot study was carried out in one raccoon under barbiturate anesthesia. The median nerve was stimulated via selective quadrants of a nerve cuff array of four tripolar electrodes implanted around the nerve. Cuff stimulation produced short-latency evoked surface potentials in the digit areas of the raccoon first somatosensory cortex. Response selectivity could be demonstrated, as could a separation between thresholds for producing movement or producing cortical evoked potentials. The sensory and motor responses elicited were consistent with the orientation of the median nerve within the cuff as determined by a postmortem identification of the muscle innervation pattern of the nerve.