Vasoactive intestinal peptide and secretin produce long-term increases in tyrosine hydroxylase activity in the rat superior cervical ganglion.

Electrical stimulation of the preganglionic fibers innervating the rat superior cervical ganglion (SCG) produces both short-term and long-term increases in tyrosine hydroxylase (TH) activity that are not completely blocked by nicotinic antagonists. Vasoactive intestinal peptide (VIP) and secretin, two neuropeptides known to produce short-term increases in TH activity, were examined for their ability to produce long-term increases in this enzyme activity. Culturing the SCG in the presence of either peptide produced a 30-50% increase in TH activity measured 2 days later. The results raise the possibility that one of these peptides or a related molecule participates in the transsynaptic induction of ganglionic TH.

Phenotypic plasticity in adult sympathetic neurons: changes in neuropeptide expression in organ culture.

Vasoactive intestinal peptide (VIP)-like immunoreactivity is present at low levels in the superior cervical ganglion of the adult rat, where immunostained neural processes, but only an occasional immunostained cell body, are found. However, when ganglia are maintained for 24 or 48 hr in organ culture, their content of VIP-like immunoreactivity increases 6- or 31-fold, respectively. When examined at 24 hr, the increase in VIP-like immunoreactivity is totally blocked by an inhibitor of RNA or protein synthesis. Many neuronal cell bodies and processes with immunoreactivity for VIP and the related peptide histidine isoleucine amide (PHI) are seen in cultured ganglia. In addition, VIP/PHI mRNA is abundant in cultured ganglia but only barely detectable in ganglia prior to culture. Under the same culture conditions, neuropeptide Y-like immunoreactivity increases to a small extent, and tyrosine hydroxylase activity and total ganglion protein remain unchanged. These results support the idea that adult sympathetic neurons exhibit plasticity in neuropeptide expression and that this plasticity, in the case of VIP, depends on changes in gene expression.

Two new Wistar-Kyoto rat strains in which hypertension and hyperactivity are expressed separately.

Two inbred strains have been developed from a cross between SHR and WKY. WK-HTs are hypertensive but not hyperactive, and WK-HAs are hyperactive but normotensive. Together with SHR (that express both traits) and WKY (expressing neither trait) we used four strains to follow correlations of biological changes with the expression of hyperactivity or hypertension. We show that the well known sympathetic hyperreactivity of SHRs to acute stress is associated with the hyperactivity trait and not the hypertension among the four strains. Similarly, the well known ventricular hypertrophy in SHRs is more prominent among the hyperactive strains than the hypertensives. Examination of regional brain amine levels revealed an imbalance in forebrain serotonin transmission in the hyperactive strains, and no significant correlations with hypertension. On the other hand, neuropeptides in brainstem and spinal cord revealed a decrease, in hypertension, in neuropeptide Y and PNMT content of terminals of C1 fibers that innervate the spinal cord sympathetic outflow. Also, the two hypertensive strains showed increased TRH-and proctolin-like immunoreactivity in fibers that innervate the C1 cells in the rostral ventrolateral medulla. These findings illustrate the unique advantage provided by WK-HA and WK-HT strains as additional controls for SHRs in studying hypertension and hyperactivity.

Microtubule-associated protein-2 and neurofilament immunoreactivity in neurons and small, intensely fluorescent cells of an amphibian cardiac ganglion.

The localization of two cytoskeletal proteins was analysed in the cell bodies and processes of ganglionic neurons and small, intensely fluorescent cells of the parasympathetic cardiac ganglion of Necturus maculosus (mudpuppy). Antibodies against microtubule-associated protein-2 and against the highly phosphorylated isoforms of high and middle molecular weight neurofilament subunits were used as somatodendritic and axonal markers, respectively. The ganglionic neurons, which usually have only one major process, and small, intensely fluorescent cells, which have several processes, showed distinctly different staining patterns with the two antibodies. In control and denervated ganglia, the ganglionic cell bodies and several hundred micrometers of the proximal processes were labeled with the antibody against microtubule-associated protein-2, whereas small, intensely fluorescent cells and processes showed a paucity of immunoreactivity. The neurofilament antibody labeled numerous axons in the ganglion but did not label the proximal part of the postganglionic process or small, intensely fluorescent cell processes. Denervation resulted in the presence of phosphorylated neurofilament subunit immunoreactivity in the soma and proximal process of the ganglionic neuron. These data suggest that (i) small, intensely fluorescent cells and ganglionic neurons in the mudpuppy cardiac ganglion contain distinctly different cytoskeletal proteins, (ii) the proximal part of postganglionic "axons" contains dendrite-like and not axon-like cytoskeletal proteins, and (iii) deafferentation promotes the localization of phosphorylated forms of neurofilament subunits in the soma and proximal process of parasympathetic ganglionic neurons.

Galanin immunoreactivity in the mudpuppy cardiac ganglion.

The source of galanin-immunoreactive fibers in the cardiac ganglion and on cardiac muscle in mudpuppy (Necturus maculosus) has been determined utilizing immunohistochemical techniques. The galanin-immunoreactive fibers are not processes of afferent fibers originating in either the rostral four dorsal root ganglia or vagal sensory ganglia. Following colchicine treatment, all of the postganglionic parasympathetic neurons and a subpopulation of the small intrinsic neurons in the cardiac ganglion exhibit galanin immunoreactivity. The majority of the galanin-immunoreactive fibers that form complexes on the parasympathetic postganglionic neurons are derived from galanin-immunoreactive small intrinsic neurons, although some of these connections may represent collateral processes from other parasympathetic postganglionic neurons. All of the galanin-immunoreactive processes that innervate cardiac muscle are derived from postganglionic parasympathetic neurons in the cardiac ganglion.

Galanin-induced hyperpolarization and decreased membrane excitability of neurones in mudpuppy cardiac ganglia.

1. Membrane hyperpolarization and decreased excitability produced by galanin were investigated in vitro on parasympathetic postganglionic neurones in the cardiac ganglion of the mudpuppy, Necturus maculosus. 2. Galanin produced a slowly developing hyperpolarization which, in 2.5 mM-KCl, reversed at -105.4 +/- 2.7 mV. The reversal potential was shifted by 38.7 +/- 4.9 mV following a fourfold elevation of the extracellular potassium concentration. 3. Galanin inhibited action potential firing in spontaneously active neurones and decreased the number of spikes in a train produced by long (500-680 ms) depolarizing current pulses. Both effects were independent of the galanin-induced hyperpolarization. 4. Galanin increased the threshold for spike generation, prolonged the spike hyperpolarizing after-potential and decreased the maximum rate of rise, amplitude and maximum rate of fall of the sodium spike. These effects occurred independently of the galanin-induced hyperpolarization. 5. Galanin decreased the amplitude and duration of TTX-insensitive spikes initiated in cells maintained in a solution containing 9 mM-calcium, 20 mM-TEA and 1.5 microM-TTX. 6. These results suggest that a galanin-like peptide may act as an inhibitory transmitter in the mudpuppy cardiac ganglion.

The presence and possible role of a galanin-like peptide in the mudpuppy heart.

A correlated histochemical and pharmacological study was undertaken to establish the presence, origin, and possible function of nerve fibers containing a galanin-like peptide in the mudpuppy (Necturus maculosus) heart. Whole mount preparations of septum-sinus venosus or atria and sections of ventricular muscle were prepared for immunocytochemistry. Galanin-immunoreactive fibers were found coursing diffusely across the septum-sinus venosus to form complex networks over cardiac muscle strands. Individual atrial muscle strands were densely innervated by galanin-immunoreactive fibers and galanin-immunoreactive fibers were also observed in the epicardial and myocardial layers of the ventricle. Most of the parasympathetic postganglionic neurons in the cardiac ganglion and many of the small intensely fluorescent-like cells exhibited galanin immunoreactivity. Galanin-immunoreactive fibers were present in the nerve trunks connecting clusters of parasympathetic postganglionic neurons. Close associations between galanin-positive fibers and individual parasympathetic postganglionic neurons were also observed. The presence of the galanin-immunoreactive fibers was similar in preparations taken from animals pretreated with 6-hydroxydopamine to that seen in preparations taken from control animals, indicating that the galanin-positive fibers were not sympathetic postganglionic axons. Moreover, the galanin-immunoreactive nerve fibers were separate from fibers containing substance P and/or calcitonin gene-related peptide that have previously been shown to be processes of afferent fibers. In twitch-tension experiments, galanin in the range 1 x 10(-7) to 1 x 10(-6) M caused cardioinhibition of spontaneously beating isolated septal-sinus venosus preparations. Galanin also produced a concentration-dependent (1 x 10(-7) to 1 x 10(-6) M) decrease in the twitch-tension development of electrically stimulated atrial or ventricular preparations. Local application of galanin produced hyperpolarization of cardiac muscle fibers in both isolated septal-sinus venosus preparations and atrial preparations. The response of individual parasympathetic ganglion cells to local application of galanin varied between neurons; some neurons were depolarized whereas others were hyperpolarized. We conclude that a galanin-like peptide is contained in both the parasympathetic postganglionic neurons and small intensely fluorescent-like cells and their processes. Further, we hypothesize that in the case of the parasympathetic postganglionic neurons, the galanin-like peptide may work in conjunction with acetylcholine to regulate cardiac activity.

Monoamines in the caudal neurosecretory complex: biochemistry and immunohistochemistry.

Monoaminergic inputs to the caudal neurosecretory complex (CNc) of Poecilia latipinna have been identified using histofluorescence and immunohistochemical techniques. The present study was undertaken to identify specific monoamines and determine the relative contribution of indolamines and catecholamines in supraspinal and intrinsic innervation of the nucleus. The CNc was deafferented by transecting the spinal cord rostral to the CNc. Ten days subsequently, CNc of spinal-transected and control fish were processed for either biochemical or immunohistochemical analysis. Norepinephrine and serotonin were detected in pooled samples of control CNc. Following deafferentation, the content of both monoamines was diminished. Using immunohistochemical labeling for serotonin or for the catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) or dopamine-beta-hydroxylase (DBH), the number of monoamine fibers was decreased in deafferented CNc compared to control. A substantial serotonergic innervation remains after deafferentation, as evidenced by serotonin-positive neurons and heavy, varicose fibers. Occasional TH/DBH-positive cells and fibers remain after deafferentation. These data suggest that both norepinephrine and serotonin are associated with descending supraspinal projections, while serotonin predominates as the intrinsic monoamine.

Brain monoamines and metabolites in hypertensive and hyperactive rat strains.

Monoamines and metabolites were measured by HPLC-EC in brain regions of four Wistar-Kyoto derived rat strains, in whom the traits of genetic hypertension or hyperactive behavior were expressed together (SHR), separately (WK-HT and WK-HA strains, respectively), or not at all (WKY). These genetically related inbred strains were used to allow more discrete correlations between neurochemical changes and the hypertensive and/or hyperactive state, than was hitherto possible using SHR and WKY metabolite levels were present in the six brain regions examined, however, no correlations with hypertension were observed. Limited correlations were seen between hyperactivity and forebrain serotonergic systems. These findings demonstrate that neurochemical differences between SHR and WKY may be erroneously attributed to the hypertension and/or hyperactivity of the SHR, unless additional genetic control strains, such as WK-HT and WK-HA rats are utilized.

Organization of a vertebrate cardiac ganglion: a correlated biochemical and histochemical study.

A correlated biochemical and histochemical study was undertaken to identify and quantify the presence of different biogenic amines and a substance P-like peptide within the parasympathetic cardiac ganglion of the mudpuppy (Necturus maculosus). Tissue extracts of the cardiac septum containing the parasympathetic cardiac ganglia from control animals were found, by high-pressure liquid chromatography, to contain significant amounts of norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-HT. To allow neural elements of extraganglionic origin to degenerate, ganglia were explanted and maintained in organ culture for 8 d. Extracts from these explanted preparations had no detectable level of E, and NE was reduced, whereas DA and 5-HT levels were similar to those of control preparations. The results indicated that some of the neurons intrinsic to the cardiac septum contain DA and 5-HT and that most (greater than 70%) of the E and NE found in this tissue is of extrinsic origin. Histochemistry of control and explanted preparations showed 5-HT-immunoreactive and catecholamine-containing intrinsic neurons. A substance P-like peptide was identified by radioimmune assay in septal extracts. The peptide content diminished by one-third to one-fifth in preparations maintained in organ culture for 8-14 d, suggesting that a significant amount of the substance P-like peptide is derived from extraganglionic sources. Immunocytochemical studies demonstrated the presence of numerous long substance P-immunoreactive fibers coursing across the septum, branching over cardiac muscle fibers, and forming pericellular networks around individual parasympathetic ganglion cells and clusters of ganglion cells. In addition, numerous small intrinsic neurons exhibited immunoreactivity for substance P. Comparison of the substance P-staining patterns in control and explanted ganglia suggests that the majority of the long substance P-immunoreactive fibers innervating the mudpuppy cardiac ganglion cells are not parasympathetic preganglionic fibers. Rather, it is hypothesized that these fibers are processes of primary sensory fibers. The present observations indicate that the mudpuppy cardiac ganglion exhibits a complex organization similar to that of mammalian sympathetic and enteric ganglia.

Alterations in the noradrenergic projection to the cerebellum of the dystonic (dt) rat.

The genetically dystonic rat (dt) has elevated resting levels of cerebellar norepinephrine (NE) in comparison with phenotypically normal littermates. This difference is not secondary to cerebellar hypoplasia. Increased NE is observed as early as postnatal day 12, when clinical symptoms have become evident. The elevation in cerebellar NE levels in the dt rat involves all cerebellar areas, but is not generalized to all terminal fields of the locus coeruleus. Elevations in cerebellar NE are followed developmentally by a reduction in sensitivity to the NE-depleting effects of reserpine, a change which is also confined to the cerebellum. The effects of amphetamine and the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine were similar in normal and dt rats. Levels of the major cerebellar metabolite of NE, 3-methoxy-4-hydroxyphenylglycol, did not differ between mutant and normal animals. Nor were any changes noted in the number or affinity of beta-adrenergic receptors. These data indicate that there is a regional alteration in NE storage. Cerebellar morphology appears normal in the dt rat, except for a decrease in Purkinje cell size. This change and other evidence of biochemical abnormalities in the Purkinje cells suggest that the alterations in cerebellar NE in the dt mutant may be a secondary response to a functional change in the target neuron for this system, the Purkinje cell.

Decreased cerebellar 3',5'-cyclic guanosine monophosphate levels and insensitivity to harmaline in the genetically dystonic rat (dt).

The dystonic rat (dt) is an autosomal recessive mutant displaying a complex motor syndrome that includes sustained axial twisting movements. The syndrome is correlated with increased glutamic acid decarboxylase activity in the deep cerebellar nuclei and increased cerebellar norepinephrine levels in comparison with phenotypically normal littermates. Biochemical, behavioral, and anatomical techniques were used to investigate the possibility that the abnormalities noted in the cerebellum of the dt rat were indicative of altered function of the major projection neurons of the cerebellar cortex, the Purkinje cells. Phenotypically normal rats showed tremor in response to harmaline, a drug that acts on the inferior olive to produce bursting in the climbing fiber pathway. Dystonic rats were insensitive to the effects of harmaline but did respond to oxotremorine. Levels of the cyclic nucleotide 3',5'-cyclic guanosine monophosphate, a biochemical marker for Purkinje cells, increased in response to harmaline in normal rats but were significantly lower in dystonic rats under both basal and harmaline-stimulated conditions. Purkinje cell soma size was reduced in the dystonic rats but no other morphological correlates of the behavioral or biochemical deficits were noted. Taken together with other observations on this mutant, the results suggest an impairment in the cerebellum or in its connections with lower brainstem and spinal cord sites.

Decreased catalepsy response to haloperidol in the genetically dystonic (dt) rat.

The rat mutant dystonic displays an autosomal recessive neurological disease characterized by slow, twisting movements of the limbs and trunk. Rats displaying clinical signs also show a decreased behavioral response to the dopaminergic blocker, haloperidol. Investigation of the development of the cataleptic response to haloperidol in the dystonic (dt) rat indicated that the response of the dt rat in the bar test is similar to that of normal littermates until after the appearance of clinical symptoms in the mutants on postnatal day 10. Mutant rats did not differ from their normal littermates in response to another cataleptic agent, morphine. Assessment of the integrity of the nigrostriatal dopamine (DA) system did not indicate the presence of any degenerative process or of any alterations in DA metabolism. No reliable differences were found between normal and dt rats in striatal DA levels or turnover rates; in DA levels in response to gamma-hydroxybutyrolactone; or in the number and affinity of striatal DA muscarinic acetylcholine receptors. Nor did qualitative light microscopic examination of Golgi-impregnated tissue from dt rats indicate the presence of any morphological abnormalities in the striatum. These findings suggest that dystonic symptoms can occur in the absence of an alteration in striatal DA metabolism and that the dt rat may have a defect in a pathway efferent to the striatum.

Characterization of the rat mutant dystonic (dt): a new animal model of dystonia musculorum deformans.

An inherited neurological disorder characterized by sustained twisting movements during waking has been discovered in Sprague-Dawley rats. The mutation follows an autosomal recessive pattern of inheritance and has been named dystonic (dt). The rat mutants are indistinguishable from normal littermates in open field behavior and climbing activity prior to postnatal days 9 to 10. Clinical signs begin to appear on day 10 and include twisting of the axial musculature, hyperflexion of the trunk, self-clasping of forelimbs and hindlimbs, and poor placement of the limbs during locomotion. No morphological lesions of neural or non-neural tissues have been observed with routine light microscopy. Dystonic rats demonstrate significantly elevated cerebellar norepinephrine levels, although levels in other terminal fields of the locus ceruleus are similar to those of normal littermates. No differences in the pattern or density of noradrenergic innervation were apparent in cerebellar tissue from dt rats examined with histochemical fluorescence techniques. These mutants were less sensitive than unaffected littermates to the akinesic effects of the dopamine blocker haloperidol. However, striatal dopamine levels were not reliably different from normal in dt rats, and their response to the movement-stimulating effects of apomorphine appeared normal. These findings suggest the presence of biochemical disturbances in the extrapyramidal system of dt rats. The dt rat may provide a useful model for human dystonia musculorum deformans.