The effects of firocoxib on cautery disbudding pain and stress responses in preweaned dairy calves.

Perioperative analgesic effects of oral firocoxib following cautery disbudding were investigated in preweaned calves. Twenty Holstein calves approximately 4 to 6wk old received a single oral dose of firocoxib, a nonsteroidal antiinflammatory, at 0.5mg/kg (n=10) or placebo (n=10) in a randomized controlled clinical trial. Responses, including ocular temperature determined by infrared thermography, pressure algometry measuring mechanical nociception threshold, and heart rate, were evaluated at 2, 4, 7, 8, and 24h after cornual nerve block and cautery disbudding. Blood samples were collected over 96h and analyzed for plasma cortisol and substance P concentrations by RIA. Additionally, ex vivo prostaglandin E2 concentrations were determined over a 72-h study period using an enzyme immunoassay. Data were analyzed using a linear mixed effects model with repeated measures. An inhibition of ex vivo prostaglandin E2 synthesis was observed from 12 to 48h following disbudding in calves treated with firocoxib. Cautery disbudding was associated with an increased nociception for the duration of sampling (24h). During the initial 24-h period following disbudding, no difference in response between treatment groups was noted. Following 24h, mean cortisol concentrations diverged between the 2 study groups with placebo-treated calves having increased cortisol concentrations at approximately 48h after disbudding. Furthermore, the overall integrated cortisol response as calculated as area under the effect curve tended to be reduced in firocoxib-treated calves. The prolonged effects of cautery dehorning require further investigation. Moreover, the effect of firocoxib on cortisol reduction observed in this study requires additional exploration.

The economic value of organic dairy farms in Vermont and Minnesota.

This study quantifies the overall economic values of organic dairy farms in Vermont and Minnesota and estimates the economic impacts of organic dairy farm sales relative to an equivalent level of sales from conventional dairy farms in those states. This question is of interest because the development of the organic dairy sector has allowed some farms that likely would not have remained in the conventional dairy business to continue being economically viable as organic dairy farms. Thus, these sales provide an economic impact in regions when this milk is exported to nonproducing regions. Organic and conventional dairy farm financial data in Vermont and Minnesota were collected and assembled to develop dairy farm production functions by region and dairy type. These production functions were then used in state-level input-output models to calculate economic impacts. The opportunity costs of organic dairy farm production were also estimated by comparing the relative statewide economic impacts of organic and conventional dairy farms if both experience a hypothetical 5-million-dollar increase in sales. Between 2008 and 2010, Vermont's 180 organic dairy farms annually contributed $76.3 million in output (the value of an industry's production within the state), 808 jobs, $34.1 million in gross state product, and $26.3 million in labor income to Vermont's economy. Between 2009 and 2011, Minnesota's 114 organic dairy farms annually contributed $77.7 million in output, 552 jobs, $32.1 million in gross state product, and $21 million in labor income to Minnesota's economy. In Vermont, organic dairy farm sales revenue would result in greater state-wide impacts of 3% in output, 39% in labor income, 33% in gross state product, and 46% in employment relative to the impacts from an equivalent level of sales revenue to conventional dairy farms. In Minnesota, these economic impacts are 4, 9, 11, and 12% greater, respectively, for organic dairy farms relative to conventional dairy farms. This study concludes that organic dairy farms may contribute more to the local economy than average and similar-size conventional dairy farms in the Northeast and Upper Midwest and that organic dairy farm milk production supports economic development in rural communities.

Love thy neighbor--but does that include a six hundred eighty-four cow dairy operation? A survey of community perceptions.

The juxtaposition of nonfarming residences to operating dairy farms often precipitates conflict over appropriate land use. This was the situation facing the residents of the town of Charlotte, Vermont, in 2002 when a local dairy farmer proposed expanding from 225 to 684 cows with the construction of a new dairy facility and manure storage lagoon. The proposal raised considerable concern within the community and presented a unique opportunity for extension researchers to examine and analyze the attitudes and concerns of local residents toward the planned expansion, including their reasons for supporting or opposing the expansion, and to develop recommendations for farm operators considering future expansions. A survey instrument was developed and inserted in a local newspaper that was delivered to all households of Charlotte to identify important concerns of the community and explanatory factors differing between supporters and nonsupporters. Of those responding to the survey, 44.3% opposed the proposed dairy facility, 30.6% supported it, 17.9% needed more information to make a decision, and 7.2% had no opinion or were unaware of the proposal. There were no significant demographic (age, gender, educational attainment) differences between supporters and nonsupporters. Yet, the closer the proximity of the respondent's residence to the farm, the more likely he or she was to oppose it (beta = 1.018). The concerns of greatest importance were water quality (4.42/5), effect on property values (3.07/5), and animal welfare (3.58/5). Responses to the open-ended questions on the survey revealed strong views toward the farmer personally as well as concentrated animal feeding operations in general. The results indicate that farmers and extension need to take proactive steps to provide education and information relevant to the facts and issues surrounding new dairy facilities for 500 to 700 dairy cows.

Regulation of neuronal pituitary adenylate cyclase-activating polypeptide expression during culture of guinea-pig cardiac ganglia.

The trophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) increases in many different neuron types following injury; a response postulated to support cell survival and regeneration. In acutely isolated cardiac ganglia, approximately 1% of the cardiac neurons exhibited PACAP immunoreactivity whereas after 72 h in culture, approximately 25% of the neurons were PACAP immunoreactive. In contrast, there was no increase in vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) cells. Using a combination of immunocytochemical and molecular techniques, we have quantified PACAP expression, during explant culture of guinea-pig cardiac ganglia. Using real time polymerase chain reaction, PACAP transcript levels increased progressively up to 48 h in culture with no further increase after 72 h. PACAP transcript levels were reduced by neurturin at 48 h in culture but not after 24 or 72 h in culture. In addition, neurturin partially suppressed the percentage of PACAP-IR neurons after 72 h in culture, an effect mediated by activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways. The addition of different known regulatory molecules, including ciliary neurotrophic factor (CNTF), interleukin-1 beta (Il-1beta), tumor necrosis factor-alpha (TNFalpha), fibroblast growth factor basic (bFGF), transforming growth factor-beta (TGF-beta) and nerve growth factor (NGF) did not increase the percentage of PACAP-IR neurons after 24 h in culture; a result indicating that the generation and secretion of these factors did not stimulate PACAP expression. The presence of 20 nM PACAP or 10 muM forskolin increased the percentage of PACAP-IR cardiac neurons in 24 h cultures, but not in 72 h cultures. Neither treatment enhanced the number of VIP-IR neurons. The addition of the PACAP selective receptor (PAC(1)) receptor antagonist, M65 (100 nM) suppressed the 20 nM PACAP-induced increase in percentage of PACAP-IR cells in 24 h cultures indicating the effect of PACAP was mediated through the PAC(1) receptor. However, 100 nM M65 had no effect on the percentage of PACAP-IR cells in either 24 or 48 h cultures not treated with exogenous PACAP, suggesting that endogenous release of PACAP likely did not contribute to the enhanced peptide expression. We postulate that the enhanced PACAP expression, which occurs in response to injury is facilitated in the explant cultured cardiac ganglia by the loss of a target-derived inhibitory factor, very likely neurturin. In intact tissues the presence of neurturin would normally suppress PACAP expression. Lastly, our results indicate that many common trophic factors do not enhance PACAP expression in the cultured cardiac neurons. However, the stimulatory role of an, as yet, unidentified factor cannot be excluded.

Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells.

In maize endosperm, genes encoding the 22-kD zein class of storage proteins are regulated by the OPAQUE2 locus. The Opaque2 (O2) protein shares homology with the basic domain/leucine zipper class of transcriptional activators. Using microprojectile bombardment, we have shown that O2 is capable of transactivating a 22-kD zein promoter in maize endosperm suspension cultures and in longitudinal sections of intact endosperm. Two mutant forms of the O2 gene were constructed by deleting regions that encode either the basic domain or the first 175 N-terminal residues of the O2 protein. When either of these mutant O2 genes was coexpressed with wild-type O2 in a maize endosperm expression system, O2-mediated transactivation of the 22-kD zein promoter was inhibited specifically and in a dose-dependent manner. Electrophoretic mobility shift assays and immunoprecipitation studies indicated that the mutant O2 proteins form heterodimers with wild-type O2 in vitro. The mutant lacking the basic domain forms heterodimers with wild-type O2, which can no longer bind DNA. In contrast, the product of the N-terminal truncation allele forms homodimers and heterodimers with wild-type O2, both of which can still bind DNA. Because the N-terminal region contains an activation domain, it is likely that these latter complexes are deficient in transactivation. Dominant negative inhibitors of gene expression, such as those constructed here, provide an alternative to antisense RNA approaches for inactivation of gene function in plants.

Step-arrest mutants of FLP recombinase: implications for the catalytic mechanism of DNA recombination.

The site-specific recombinase (FLP) encoded by the yeast plasmid 2 micron circle belongs to the integrase (of phage lambda) family of recombinases. The sparse homology within the members of this family contrasts with the invariance of three residues, His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them. We report here results on substrate recognition and catalysis by FLP proteins altered at these residues. Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination. We provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate.

A cost and returns evaluation of alternative dairy products to determine capital investment and operational feasibility of a small-scale dairy processing facility.

This study examines the economic feasibility of 50- and 500-cow dairy processing facilities for fluid milk, yogurt, and cheese. Net present value and internal rate of return calculations for projected costs and returns over a 10-yr period indicate that larger yogurt and cheese processing plants offer the most profitable prospects, whereas a smaller yogurt plant would break even. A smaller cheese plant would have insufficient returns to cover the cost of capital, and fluid milk processing at either scale is economically infeasible. Economic success in processing is greatly contingent upon individual business, financial management, and marketing skills.

Trophic factor modulation of cocaine- and amphetamine-regulated transcript peptide expression in explant cultured guinea-pig cardiac neurons.

The present study investigated the influence of trophic factors on the expression of cocaine- and amphetamine-regulated transcript peptide (CARTp) in guinea-pig cardiac ganglia maintained in explant culture. In acutely isolated cardiac ganglia preparations, <1% of the cholinergic cardiac neurons exhibited CARTp immunoreactivity. In contrast, this number increased to >25% of the cardiac neurons after 72 h in explant culture. This increase in the number of CARTp neurons in cultured cardiac ganglia explants was accompanied by an increase in CARTp transcript levels as assessed by real time polymerase chain reaction. Treatment of cardiac ganglia cultures with neurturin or glial-derived trophic factor (both at 10 ng/ml) for 72 h prevented the increase in neurons that exhibited CARTp immunoreactivity. In contrast, treatment with ciliary neurotrophic factor (50 ng/ml) for 72 h produced a small significant increase in the percentage of CARTp-immunoreactive cardiac neurons and treatment with nerve growth factor (100 ng/ml) had no effect. Neurturin treatment also decreased cardiac neuron CARTp levels after 72 h in explant culture. Cardiac neurons exhibited immunoreactivity to the neurturin receptor GFRalpha2 whereas non-neural cells preferentially exhibited immunoreactivity to the glial-derived neurotrophic factor receptor GFRalpha1 and neurturin transcripts were detected in cardiac tissue extracts. We hypothesize that a target-derived inhibitory factor, very likely neurturin, is a critical factor suppressing the expression of CARTp in guinea-pig cardiac neurons. These observations contrast with those reported in sympathetic neurons that suggest up-regulation of trophic factors after axotomy or during explant culture is a key factor contributing to the up-regulation of many neuropeptides.

Mechanisms mediating pituitary adenylate cyclase-activating polypeptide depolarization of rat sympathetic neurons.

The direct effects of pituitary adenylate cyclase-activating polypeptides (PACAP) on sympathetic neurons were investigated using rat superior cervical ganglion neurons. Electrophysiological and pharmacological analyses were used to evaluate PACAP modulation of sympathetic neuron membrane potentials and to investigate potential ionic and intracellular signaling mechanisms mediating the responses. More than 90% of the sympathetic neurons were depolarized by the PACAP peptides even when stimulated release was blocked, indicating that the PACAP peptides elicited primary responses in the postganglionic neurons. The response profile was consistent for activation of PACAP-selective PAC(1) receptors: nanomolar concentrations of PACAP27 and PACAP38 were required to stimulate depolarization, whereas vasoactive intestinal peptide failed to evoke any response. Furthermore, depolarizations elicited by PACAP27 were reduced by the PAC(1) receptor antagonist PACAP(6-38). Both sodium influx and inhibition of a potassium current contributed to the peptide-induced depolarizations. Activation of neither pertussis toxin- nor cholera toxin-sensitive G-proteins was required for generation of the depolarizations. cAMP and diacylglycerol production and activation of protein kinase A or protein kinase C also were not requisite for the responses. By contrast, phospholipase C (PLC)-dependent inositol 1,4,5-triphosphate (IP(3)) synthesis was crucial to the PACAP-mediated depolarizations. Although calcium release from IP(3)-sensitive stores was not required for the PACAP-induced responses, inhibition of IP(3) receptors reduced the depolarizations. Thus, among the many signal transduction pathways coupled to the PAC(1) receptor, the PACAP-induced depolarization of sympathetic neurons appears to require activation of PLC and subsequent generation of IP(3).

Desensitization and recovery at the frog neuromuscular junction.

The time course of carbachol-induced desensitization onset and recovery of sensitivity after desenitization have been compared at the frog neuromuscular junction. The activation-desensitization sequence was determined from input conductance measurements using potassium-depolarized muscle preparations. Both desensitization onset and recovery from desensitization could be adequately described by single time constant expressions, with tauonset being considerably shorter than taurecovery. In nine experiments, tauonset was 13+/-1.3 s and taurecovery was 424+/-51 s with 1 mM carbachol. Elevating the external calcium or carbachol concentration accelerated desensitization onset without changing the recovery of sensitivity after equilibrium desensitization. Desensitization onset was accelerated by a prior activation-desensitization sequence to an extent determined by the recovery interval that followed the initial carbachol application. The time course of return of tauonset was closely parallel to, but slower than the time course of recovery of sensitivity. These results are consistent with a cyclic model in which intracellular calcium is a factor controlling the rate of development of desensitization.

Desensitization onset and recovery at the potassium-depolarized frog neuromuscular junction are voltage sensitive.

The influence of voltage on the time-course of desensitization onset and recovery has been studied at the frog neuromuscular junction. The activation-desensitization sequence was determined from carbachol-induced end-plate currents in potassium-depolarized fibers voltage-clamped either to -40 mV or +40 mV. The time-course of both desensitization onset and recovery developed exponentially, with onset occurring more rapidly than recovery. Desensitization onset was voltage dependent, the onset time constant being 8.3 +/- 1.3 s (11 fibers) at -40 mV and 19.3 +/- 3.4 s (15 fibers) at +40 mV. Recovery from desensitization was also influenced by voltage. The extent of recovery after 2 min was 80.4 +/- 6.3% in those fibers voltage-clamped to -40 mV and 57.4 +/- 3.6% in those fibers voltage-clamped to +40 mV. The voltage dependence of desenistization onset and recovery did not result from a difference in ability to control voltage at these two levels of membrane potential. These results demonstrate that in the potassium-depolarized preparation the processes controlling both desensitization onset and recovery of sensitivity from the desensitivity from the desensitized state are influenced by membrane voltage.

The interaction between caffeine and calcium in the desensitization of muscle postjunctional membrane receptors.

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.

Influence of polyvalent cations on the activation of muscle end plate receptors.

The influence of polyvalent cations on the activation of end plate receptors has been studied in vitro on the sartorius muscle of the frog. In the absence of extracellular calcium, the sensitivity of the receptors to depolarizing quaternary ammonium salts was markedly reduced. Maximum receptor activation occurred in those fibers equilibrated in 1.8 mM calcium Ringer solution, with the response being reduced as the calcium concentration was raised or lowered. Magnesium was less efficient than calcium in regulating the sensitivity of the end plate receptors, the maximum receptor response occurring in those fibers equilibrated in 8 mM magnesium Ringer solution. In the presence of lanthanum the end plate response to carbamylcholine or acetylcholine was enhanced. Lanthanum increased the conductance change produced by carbamylcholine both in polarized and in potassium-depolarized fibers. The application of 10(-2) mM lanthanum to the end plate increased MEPP's amplitude, rise time, and half-fall time by 19, 54, and 45%, respectively. The results suggest that polyvalent cations influence postjunctional membrane receptor processes in addition to their well-documented prejunctional action.

Factors in the inactivation of postjunctional membrane receptors of frog skeletal muscle.

Several factors which influence the rate of inactivation of muscle postjunctional membrane (PJM) receptors during the sustained application of carbamylcholine (CARB) have been studied by two methods. The rate of inactivation was increased by elevating the tonicity of the bathing medium, by increasing the CARB concentration, by raising the calcium ion concentration, and by substituting SO(4) (=) for Cl(-) ions in the extracellular fluid. The relative effectiveness of calcium and other divalent cations in receptor inactivation was compared. In the absence of calcium, other divalent cations such as magnesium, strontium, or manganese were not efficient substitutes for calcium. In the presence of calcium, the addition of strontium or manganese ions accelerated the rate of receptor inactivation, but the addition of magnesium (up to 12 mM) inhibited this process. The inactivation of the membrane receptors in denervated muscle fibers was found to be similar to that in innervated muscle fibers. Various factors in PJM receptor inactivation are discussed. It is suggested that PJM receptor inactivation is influenced by the binding of calcium ions to sites on the internal surface of the PJM.

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells.

Excitatory postsynaptic currents (EPSCs) have been studied in voltage-clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range --90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/-0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.

Voltage dependence of desensitization. Influence of calcium and activation kinetics.

The voltage dependence of carbachol-induced desensitization has been analyzed in potassium-depolarized frog sartorius muscle preparations with voltage clamp techniques over a wide voltage range (-120 to +40 mV). Desensitization developed exponentially at all voltages with tau, the time constant of desensitization onset, varying as a logarithmic function of membrane voltage. The voltage dependence of tau remained in calcium-deficient solutions and was not altered by elevating either the level of extracellular or intracellular calcium. We have analyzed our results according to a simple sequential kinetic scheme in which the rate-limiting step in the development of desensitization is a transition of the receptor channel complex from the activated conducting state to a desensitized, nonconducting state. We conclude (a) that the observed voltage sensitivity of desensitization primarily resides in the voltage dependence of this transition, and (b) the kinetics of activation appear to have a greater influence on the observed rate of desensitization than on its voltage dependence. The magnitude of the voltage dependence suggests that a greater change in free energy is required for the transition to the desensitized state than for the transition between the open and closed states of the receptor channel complex.

Sexually dimorphic distribution of a galanin-like peptide in the central nervous system of the teleost fish Poecilia latipinna.

Immunohistochemical techniques were used to visualize areas of the brain and spinal cord containing a galanin-like peptide in the teleost fish, the sailfin molly. Galanin-like immunoreactivity (GAL-LI) in both males and females was identified in neurons in the nucleus preopticus periventricularis, nucleus lateralis tuberis, and nucleus commissuralis. GAL-LI fibers had a comparable distribution in the forebrain, preoptic, hypothalamic, and visceral sensory areas of both sexes. In striking contrast to these areas, the optic tectum, torus semicircularis, brainstem tegmentum, and spinal cord of the male contained much higher levels of GAL-LI than the female. GAL-LI in these dimorphic areas in the female was limited to single fiber bundles in the ventromedial tegmentum and in the trigeminal system. Additionally, a population of neurons in the preoptic nucleus was found to contain GAL-LI in the male only. Sexual dimorphism was especially prominent in the spinal cord, where extensive GAL-LI fibers were found in the male only. These fibers were oriented in the longitudinal plane and confined largely to the gray matter. Comparative studies were performed on the goldfish spinal cord, in which GAL-LI was localized solely in the dorsal horn and exhibited no sexual dimorphism. Further, examination of spinal cord material from neonatal mollies revealed a lack of spinal GAL-LI at this developmental stage. As the extent of GAL-LI in the male molly spinal cord differs from both the goldfish and from that reported for the mammalian spinal cord, and a prominent sexual dimorphism in GAL-LI extends from the diencephalon to the caudal spinal cord, it is suggested that a galanin-like peptide may play a unique, sex-specific role in this species.

Source of sexually dimorphic galanin-like immunoreactive projections in the teleost fish Poecilia latipinna.

A galanin-like peptide has a sexually dimorphic distribution in the teleost fish, the sailfin molly. An extensive system of galanin-like immunoreactive (GAL-LI) fibers has been described in the brainstem and spinal cord of the male molly, which is absent in the female (Cornbrooks and Parsons, companion paper). As GAL-LI in the mammalian spinal cord has been localized to neurons of origin in the dorsal root ganglia and dorsal and ventral horns, the present study was undertaken to determine whether the sexually dimorphic GAL-LI in the male molly may originate in part from corresponding sources in this species. Colchicine treatments of the spinal cord and dorsal root ganglia did not result in GAL-LI staining in neuronal somata in these regions. Following complete transection of the spinal cord and at any level of the spinal cord, there was a complete absence of GAL-LI caudal to the lesion site. In fish that received unilateral spinal transection, there was a loss of GAL-LI ipsilateral and caudal to the lesion. Finally, in fish that received lesions in the rostral hypothalamus, there was a complete loss of GAL-LI in the sexually dimorphic fiber system in the brainstem and spinal cord, but not in non-dimorphic GAL-LI regions of the brainstem. Thus the sexually dimorphic fiber system in the male molly may originate in neurons of the preoptic nucleus that are sexually dimorphic for a GAL-LI peptide. This preoptico-spinal pathway may mediate sex-specific behaviors in this species.

Origin of neuronal nitric oxide synthase (NOS)-immunoreactive fibers in guinea pig parasympathetic cardiac ganglia.

This study was conducted to determine the origin(s) of neuronal nitric oxide synthase-immunoreactive (NOS-IR) fibers within guinea pig atrial whole-mount preparations containing the cardiac ganglia. Intrinsic NOS-IR cardiac neurons exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they were cholinergic as well as nitrergic. Comparison of control versus 72-hour explant culture preparations indicated that most of the nitrergic fibers within cardiac ganglia were extrinsic. The extrinsic NOS-IR fibers were not IR for ChAT (marker of preganglionic parasympathetic neurons), tyrosine hydroxylase (marker of catecholaminergic sympathetic postganglionic axons), or calcitonin gene-related peptide (CGRP) (marker of afferent fibers). Separate NOS-IR and ChAT-IR neurons were present within medullary regions containing the cardiovascular regulatory nuclei (nucleus ambiguus and dorsal motor nucleus of the vagus), but no cells were found that exhibited both NOS immunoreactivity and ChAT immunoreactivity. The small size and location of the medullary NOS-IR neurons suggested they were probably interneurons. Only an occasional sympathetic postganglionic cell in the stellate ganglion complex exhibited NOS immunoreactivity. NOS-IR cells were present in dorsal root ganglia (thoracic 1-5), but these typically also exhibited CGRP immunoreactivity. NOS-IR cells were also present in the nodose ganglia, but only some exhibited CGRP immunoreactivity. We concluded that virtually all the extrinsic NOS-IR nerve fibers represented an afferent fiber input that was separate from the substance P (SP)/CGRP-containing population of sensory fibers. Furthermore, much of this NOS innervation is probably derived from the nodose ganglia.

Origin of pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive fibers innervating guinea pig parasympathetic cardiac ganglia.

The present study investigated the origin of pituitary adenylate cyclase-activating polypeptide (PACAP) -immunoreactive (IR) fibers innervating guinea pig cardiac ganglia. Immunohistochemistry was performed on whole-mounts containing cardiac ganglia, and sections of stellate, nodose, and dorsal root ganglia (DRG, thoracic levels 1-4), and caudal medulla. In control preparations, only 4% of the cardiac neurons were PACAP-IR, although most cardiac ganglion cells were surrounded by a network of PACAP-IR fibers. After 3-7 days in explant culture, the number of PACAP-IR cardiac neurons increased approximately eightfold. However, virtually all PACAP-IR fibers surrounding the cardiac neurons had degenerated, demonstrating that the major source of the PACAP-IR fibers was extrinsic to the cardiac ganglia preparation. PACAP- and choline acetyltransferase (ChAT) immunoreactivity were colocalized in fibers within the stellate ganglia but not within neuropeptide Y (NPY) -IR cell bodies and fibers. PACAP-IR cells and fibers were present in the nodose ganglia. PACAP immunoreactivity also was present in fibers and primarily small neurons in thoracic DRGs. In situ hybridization demonstrated the presence of proPACAP mRNA within neurons in the region of the dorsal motor nucleus of the vagus and nucleus ambiguus. PACAP immunoreactivity was colocalized with ChAT immunoreactivity, but not with NPY immunoreactivity or SP immunoreactivity, in fibers surrounding neurons within cardiac ganglia. We conclude that PACAP-containing fibers innervating the postganglionic parasympathetic neurons in guinea pig cardiac ganglia are primarily preganglionic parasympathetic axons.