Neurotoxicity studies with a tropomyosin-related kinase A inhibitor, ASP7962, on the sympathetic and sensory nervous systems in rats.

ASP7962 is a small molecule inhibitor for the nerve growth factor (NGF) receptor, tropomyosin-related kinase A (TrkA). NGF contributes to the survival of sensory and sympathetic neurons through TrkA receptor activation. Gross, microscopic, and quantitative effects to the nervous system were evaluated following oral ASP7962 administration to Sprague Dawley rats for 4 weeks and 13 weeks and after a recovery period. Histopathological findings included reversible neuronal atrophy but no neuronal death in the sympathetic ganglia (cervicothoracic ganglion, cranial mesenteric ganglion or superior [cranial] cervical ganglion). Stereological analysis showed reversible decreased ganglion volume and/or decreased neuron size in the superior (cranial) cervical ganglion in both the 4-week and the 13-week repeated dose studies. There were no test article related changes in the brain, dorsal root ganglia with spinal nerve roots or trigeminal ganglia and no functional deficits. ASP7962 did not cause any detectable dysfunction of the sympathetic and sensory nervous system in either study.

Efficacy of B-Type Natriuretic Peptide Is Coupled to Phosphodiesterase 2A in Cardiac Sympathetic Neurons.

Elevated B-type natriuretic peptide (BNP) regulates cGMP-phosphodiesterase activity. Its elevation is regarded as an early compensatory response to cardiac failure where it can facilitate sympathovagal balance and cardiorenal homeostasis. However, recent reports suggest a paradoxical proadrenergic action of BNP. Because phosphodiesterase activity is altered in cardiovascular disease, we tested the hypothesis that BNP might lose its efficacy by minimizing the action of cGMP on downstream pathways coupled to neurotransmission. BNP decreased norepinephrine release from atrial preparations in response to field stimulation and also significantly reduced the heart rate responses to sympathetic nerve stimulation in vitro. Using electrophysiological recording and fluorescence imaging, BNP also reduced the depolarization evoked calcium current and intracellular calcium transient in isolated cardiac sympathetic neurons. Pharmacological manipulations suggested that the reduction in the calcium transient was regulated by a cGMP/protein kinase G pathway. Fluorescence resonance energy transfer measurements for cAMP, and an immunoassay for cGMP, showed that BNP increased cGMP, but not cAMP. In addition, overexpression of phosphodiesterase 2A after adenoviral gene transfer markedly decreased BNP stimulation of cGMP and abrogated the BNP responses to the calcium current, intracellular calcium transient, and neurotransmitter release. These effects were reversed on inhibition of phosphodiesterase 2A. Moreover, phosphodiesterase 2A activity was significantly elevated in stellate neurons from the prohypertensive rat compared with the normotensive control. Our data suggest that abnormally high levels of phosphodiesterase 2A may provide a brake against the inhibitory action of BNP on sympathetic transmission.

Tlx3 controls cholinergic transmitter and Peptide phenotypes in a subset of prenatal sympathetic neurons.

The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.

Proliferation and cell cycle dynamics in the developing stellate ganglion.

Cell proliferation during nervous system development is poorly understood outside the mouse neocortex. We measured cell cycle dynamics in the embryonic mouse sympathetic stellate ganglion, where neuroblasts continue to proliferate following neuronal differentiation. At embryonic day (E) 9.5, when neural crest-derived cells were migrating and coalescing into the ganglion primordium, all cells were cycling, cell cycle length was only 10.6 h, and S-phase comprised over 65% of the cell cycle; these values are similar to those previously reported for embryonic stem cells. At E10.5, Sox10(+) cells lengthened their cell cycle to 38 h and reduced the length of S-phase. As cells started to express the neuronal markers Tuj1 and tyrosine hydroxylase (TH) at E10.5, they exited the cell cycle. At E11.5, when >80% of cells in the ganglion were Tuj1(+)/TH(+) neuroblasts, all cells were again cycling. Neuroblast cell cycle length did not change significantly after E11.5, and 98% of Sox10(-)/TH(+) cells had exited the cell cycle by E18.5. The cell cycle length of Sox10(+)/TH(-) cells increased during late embryonic development, and ∼25% were still cycling at E18.5. Loss of Ret increased neuroblast cell cycle length at E16.5 and decreased the number of neuroblasts at E18.5. A mathematical model generated from our data successfully predicted the relative change in proportions of neuroblasts and non-neuroblasts in wild-type mice. Our results show that, like other neurons, sympathetic neuron differentiation is associated with exit from the cell cycle; sympathetic neurons are unusual in that they then re-enter the cell cycle before later permanently exiting.

Somatic ATP release from guinea pig sympathetic neurons does not require calcium-induced calcium release from internal stores.

Prior studies indicated that a Ca(2+)-dependent release of ATP can be initiated from the soma of sympathetic neurons dissociated from guinea pig stellate ganglia. Previous studies also indicated that Ca(2+)-induced Ca(2+) release (CICR) can modulate membrane excitability in these same neurons. As Ca(2+) release from internal stores is thought to support somatodendritic transmitter release in other neurons, the present study investigated whether CICR is essential for somatic ATP release from dissociated sympathetic neurons. Caffeine increased intracellular Ca(2+) and activated two inward currents: a slow inward current (SIC) in 85% of cells, and multiple faster inward currents [asynchronous transient inward currents (ASTICs)] in 40% of cells voltage-clamped to negative potentials. Caffeine evoked both currents when cells were bathed in a Ca(2+)-deficient solution, indicating that both were initiated by Ca(2+) release from ryanodine-sensitive stores in the endoplasmic reticulum. Sodium influx contributed to generation of both SICs and ASTICs, but only ASTICs were inhibited by the presence of the P2X receptor blocker PPADs. Thus ASTICs, but not SICs, resulted from an ATP activation of P2X receptors. Ionomycin induced ASTICs in a Ca(2+)-containing solution, but not when it was applied in a Ca(2+)-deficient solution, demonstrating the key requirement for external Ca(2+) in initiating ASTICs by ionomycin. Pretreatment with drugs to deplete the internal stores of Ca(2+) did not block the ability of ionomycin or long depolarizing voltage steps to initiate ASTICs. Although a caffeine-induced release of Ca(2+) from internal stores can elicit both SICs and ASTICs in dissociated sympathetic neurons, CICR is not required for the somatic release of ATP.

Cholinergic neurons of mouse intrinsic cardiac ganglia contain noradrenergic enzymes, norepinephrine transporters, and the neurotrophin receptors tropomyosin-related kinase A and p75.

Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine beta-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.

Exocytotic release of ATP and activation of P2X receptors in dissociated guinea pig stellate neurons.

Activation of P2X receptors by a Ca(2+)- and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent release of ATP was measured using patch-clamp recordings from dissociated guinea pig stellate neurons. Asynchronous transient inward currents (ASTICs) were activated by depolarization or treatment with the Ca(2+) ionophore ionomycin (1.5 and 3 microM). During superfusion with a HEPES-buffered salt solution containing 2.5 mM Ca(2+), depolarizing voltage steps (-60 to 0 mV, 500 ms) evoked ASTICs on the decaying phase of a larger, transient inward current. Equimolar substitution of Ba(2+) for Ca(2+) augmented the postdepolarization frequency of ASTICs, while eliminating the larger transient current. Perfusion with an ionomycin-containing solution elicited a sustained activation of ASTICs, allowing quantitative analysis over a range of holding potentials. Under these conditions, increasing extracellular [Ca(2+)] to 5 mM increased ASTIC frequency, whereas no events were observed following replacement of Ca(2+) with Mg(2+), demonstrating a Ca(2+) requirement. ASTICs were Na(+) dependent, inwardly rectifying, and reversed near 0 mV. Treatment with the nonselective purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) blocked all events under both conditions, whereas the ganglionic nicotinic antagonist hexamethonium (100 microM and 1 mM) had no effect. PPADS also blocked the macroscopic inward current evoked by exogenously applied ATP (300 microM). The presence of botulinum neurotoxin E (BoNT/E) in the whole-cell recording electrode significantly attenuated the ionomycin-induced ASTIC activity, whereas phorbol ester treatment potentiated this activity. These results suggest that ASTICs are mediated by vesicular release of ATP and activation of P2X receptors.

How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion?

Sympathetic cholinergic postganglionic neurons are present in many sympathetic ganglia. Three classes of sympathetic cholinergic neuron have been reported in mammals; sudomotor neurons, vasodilator neurons and neurons innervating the periosteum. We have examined thoracic sympathetic ganglia in rats to determine if any other classes of cholinergic neurons exist. We could identify cholinergic sudomotor neurons and neurons innervating the rib periosteum, but confirmed that cholinergic sympathetic vasodilator neurons are absent in this species. Sudomotor neurons contained vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) and always lacked calbindin. Cholinergic neurons innervating the periosteum contained VIP and sometimes calbindin, but always lacked CGRP. Cholinergic neurons innervating the periosteum were usually surrounded by terminals immunoreactive for CGRP. We conclude that if any undiscovered populations of cholinergic neurons exist in the rat thoracic sympathetic chain, then they are indistinguishable in size, neurochemistry and inputs from sudomotor or cholinergic neurons innervating the periosteum. It may be that the latter two populations account for all cholinergic neurons in the rat thoracic sympathetic chain ganglia.

[Immunocytochemical characteristic of neurons of the mouse truncus sympaticus stellate ganglion in postnatal ontogenesis].

Neurotransmitter content in stellate ganglion neurons was studied immunocytochemically in mice of different ages (newborns, 10-, 20-, 30- and 60-days-old). Majority of the stellate ganglion neurons in mice of all the groups studied were tyrosine hydroxylase (TH)-positive. Most of neurons with the immunore-activity to choline acetyltransferase (ChAT) were shown to be also TH-positive in the newborn and 10-day-old animals. The percentage of neurons containing TH and neuropeptide Y increased from birth throughout all the age periods studied. The proportion of vasoactive intestinal peptide (VIP)- and ChAT-positive neurons was maximal in 10-day-old animals and then decreased up to 60 days of age. The somatostatin- and galanin-reactive cells were absent in all the mice. Thus, the maturation of neurotransmitter composition is complete in the mouse stellate ganglion by the end of the second month of life.

Nephroblastoma overexpressed gene (NOV) expression in rat hepatic stellate cells.

Using the expression-profiling method, we identified nephroblastoma overexpressed gene (NOV) mRNA as one member of the mRNA population that was upregulated in cultured activated hepatic stellate cell (HSC). Northern analysis showed that NOV mRNA was increasingly expressed during progressive activation of cultured rat HSCs, and a significant increase was observed in both the carbon tetrachloride-induced and bile duct ligation/scission rat models of liver fibrosis. RT-PCR showed human NOV mRNA was increased in most fibrotic livers compared with normal livers. The expression of NOV protein in fibrotic rat and human livers was predominantly located in areas of ductular proliferation and HSC of the fibrous septa. HSCs stimulated with transforming growth factor beta1 showed increased expression of NOV protein without changing its mRNA levels. Dexamethasone stimulated the expression of NOV mRNA and protein. Furthermore, we demonstrated that bile acids have a modulating effect on the induction of NOV mRNA expression. In conclusion, this study suggests that NOV is expressed during liver fibrogenesis and HSCs may be an important source of hepatic NOV.

Immunocytochemical properties of stellate ganglion neurons during early postnatal development.

Neurotransmitter features in sympathetic neurons are subject to change during development. To better understand the neuroplasticity of sympathetic neurons during early postnatal ontogenesis, this study was set up to immunocytochemically investigate the development of the catecholaminergic, cholinergic, and peptidergic phenotypes in the stellate ganglion of mice and rats. The present study was performed on Wistar rats and Swiss mice of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, and 60-day-old). To this end, double labeling for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), vasoactive intestinal (poly)peptide (VIP), neuropeptide Y (NPY), galanin (GAL), and somatostatin (SOM) was applied. The results obtained indicate that the majority of the neurons in the stellate ganglion of both species were TH-positive from birth onward and that a large part of these neurons also contained NPY. The percentage of neurons containing TH and NPY invariably increased with age up to 60 days postnatally. A smaller portion of the stellate ganglion neurons contained other types of neuropeptides and showed a distinct chronological pattern. The proportion of VIP- and ChAT-positive neurons was maximal in 10-day-old animals and then decreased up to 60 days of age, whereas the number of SOM-positive cells in rats significantly decreased from birth onward. In newborn rats, VIP-, ChAT- and SOM-positive neurons were largely TH-positive, while their proportions decreased in 10-day-old and older rats. Accordingly, the largest part of VIP-positive neurons also expressed SOM immunoreactivity at birth, after which the number of neurons containing both peptides diminished. The VIP- and SOM-positive cells did not contain NPY in any of the age groups studied. In rats up to 10 days of life, GAL-immunoreactive (-IR) neurons were scarce, after which their number increased to reach a maximal value in 30-day-old animals and then declined again. The SOM-reactive cells had the smallest size in all rats, while the largest neurons were those containing ChAT. In the mouse stellate ganglion, VIP- and ChAT-IR neurons were larger in comparison to NPY- and TH-IR cells. Our study further revealed some species differences: compared to mice the proportion of neurons containing TH and NPY was higher in rats at all ages under study. Furthermore, no GAL-immunostained neurons were found in mice and the number of SOM-positive cells in mice was limited compared to that observed in rats. In conclusion, the development of neurotransmitter composition is complete in rats and mice by their second month of life. At this age, the percentages of immunopositive cells have become similar to those reported in adult animals.

A novel approach employing ultrasound guidance for percutaneous cardiac muscle injection to retrograde label rat stellate ganglion neurons.

Stellate ganglion (SG) neurons provide the main sympathetic innervation to the heart and help to regulate cardiac function. The purpose of this study was to determine if ultrasound imaging could be employed to retrograde label rat SG neurons innervating the heart without employing thoracotomy. In addition, electrophysiological experiments were performed to characterize the modulation of Ca(2+) channels by neurotransmitters in unlabeled and dye-labeled SG neurons. Fluorescence imaging of actutely isolated cells revealed that dye uptake was successful within five days following injection of dye in the cardiac muscle. Whole-cell voltage-clamp recordings revealed that the majority of the Ca(2+) current was carried by N-type Ca(2+) channels. Finally, fluorescence dye uptake did not appear to affect the modulation of Ca(2+) currents following exposure of SG neurons to norepinephrine, adenosine and neurokinin A. These results demonstrate that ultrasound imaging-guided percutaneous injection can be effectively employed to retrograde label neurons innervating the heart.

Role of reactive oxygen species, glutathione and NF-kappaB in apoptosis induced by 3,4-methylenedioxymethamphetamine ("Ecstasy") on hepatic stellate cells.

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA), is a derivative of amphetamine with hepatotoxic effects that has been shown to induce apoptosis of cultured liver cells. In the present work, we studied the role played by oxidative stress in the apoptotic response caused by MDMA on a cell line of hepatic stellate cells (HSC). MDMA-treatment provoked oxidative stress determined as reactive oxygen species (ROS) accumulation and decrease of intracellular reduced glutathione levels. Pre-treatment with the antioxidant pyrrolidine dithiocarbamate blocked ROS production but did not prevent MDMA-induced apoptosis of HSC. The pro-oxidant menadione induced in HSC ROS production and apoptosis that were prevented by pyrrolidine dithiocarbamate, showing HSC to be susceptible to oxidative stress-induced apoptosis. Addition of exogenous GSH or its precursor NAC potentiated the apoptotic action of MDMA but blocked apoptosis induced by menadione. Pre-treatment of HSC with the cytochrome P450 inhibitor quinine diminished the extent of apoptosis caused by MDMA, suggesting the involvement of a metabolic derivative of MDMA on its apoptotic effect. Nuclear factor NF-kappaB was activated by MDMA in a oxidative stress independent fashion and played a protective role in the apoptotic response, since inhibition of NF-kappaB by treatment with parthenolide or by viral infection with a dominant-negative form of NIK (Ad5dnNIK) resulted in an increase of MDMA-induced cell death. In summary, MDMA-induced apoptosis of HSC is accompanied, but not caused by oxidative stress; a metabolic derivative of the drug is responsible for the apoptotic effect of MDMA, which is partially blocked by NF-kappaB activation.

Expression of vasoactive intestinal polypeptide and calcitonin gene-related peptide in human stellate ganglia after acute myocardial infarction.

Using the method of indirect immunofluorescence the distribution of vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) was investigated in autopsy specimens of human stellate ganglia following acute myocardial infarction (AMI). The dramatic increase of both VIP- and CGRP-immunoreactivities in principal ganglionic neurons as well as of calcitonin gene-related peptide in perineuronal nets was revealed. It was concluded that hypoxia and myocardial ischaemia following AMI are the main inducing factors for activation of both vasoactive regulatory neuropeptide synthesis. The upregulation of VIP and CGRP expression in sympathetic ganglionic neurons may provide regulatory and trophic support to the ischaemic heart.

Upregulation of vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) expression in stellate ganglia of children with congenital cardiovascular lesions.

The distribution patterns of vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) immunoreactivities (IR) in stellate ganglia of human neonates and infants with congenital heart and vascular lesions were investigated by the method of indirect immunohistochemistry. The results demonstrated upregulation of VIP and CGRP expression in principal ganglionic neurons independently of the type of lesion. It is suggested that the activation of neuropeptide synthesis in stellate ganglia is a compensatory reaction of ganglionic neurons in response to congenital cardiovascular lesions, in regulation of heart contractility, and as a trophic influence on the ischemic myocardium. Hypoxia is the main inducing factor for the upregulation of VIP and CGRP expression in sympathetic neurons.

Distribution of vasoactive intestinal polypeptide-, calcitonin gene-related peptide-, somatostatin- and neurofilament-immunoreactivities in sympathetic ganglia of human fetuses and premature neonates.

The distribution patterns of vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), somatostatin (SOM) and neurofilament (NF) immunoreactivities (IR) were studied in the stellate ganglia of human fetuses and neonates at 24-26 weeks gestation. Sizeable populations with some quantitative variations of VIP-, CGRP- and SOM immunoreactive nerve cells were detected in all ganglia studied. In marked contrast, neurofilament expression was down-regulated. The upregulation of VIP, CGRP and SOM expression suggested their inductor effect on growth and differentiation neurons as well as on the development of their neurotransmitter properties. The main neuropeptides-inducing factor of sympathetic ganglia in human prenatal ontogenesis may be considered as a relative hypoxia.

Tonically active protein kinase A regulates neurotransmitter release at the squid giant synapse.

1. Electrophysiological and microinjection methods were used to examine the role of cyclic AMP-dependent protein kinase A (PKA) in regulating transmitter release at the squid giant synapse. 2. Excitatory postsynaptic potentials (EPSPs) evoked by presynaptic action potentials were not affected by presynaptic injection of an exogenous active catalytic subunit of mammalian PKA. 3. In contrast, presynaptic injection of PKI-amide, a peptide that inhibits PKA with high potency and specificity, led to a reversible inhibition of EPSPs. 4. Injection of several other peptides that serve as substrates for PKA also reversibly inhibited neurotransmitter release. The ability of these peptides to inhibit release was correlated with their ability to serve as PKA substrates, suggesting that these peptides act by competing with endogenous substrates for phosphorylation by active endogenous PKA. 5. We suggest that the phosphorylation of PKA substrates is maintained at a relatively high state under basal conditions and that this tonic activity of PKA is to a large degree required for evoked neurotransmitter release at the squid giant presynaptic terminal.

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.

Hyperpolarization-activated cation currents in stellate and pyramidal neurons of rat entorhinal cortex.

Properties of hyperpolarization-activated cation currents (I(h)) were investigated in neurons of juvenile rat entorhinal cortex using the patch-clamp technique. A rat brain slice preparation containing the entorhinal cortex was used for whole-cell recordings of I(h) in pyramidal cells from layer IV and in stellate cells from layer II of the entorhinal cortex. In both stellate and pyramidal cells, I(h) activated at potentials more negative than -60 mV and did not show any time-dependent inactivation. Half-maximal activation of I(h) was achieved at -95.3 mV in pyramidal cells and at -95.0 mV in stellate cells. The channels were permeable for sodium and potassium ions. I(h) of pyramidal and stellate neurons was reduced by about 50% in the presence of 100 microM ZD7288. Extracellularly applied 1 mM Cs(+) decreased I(h) of pyramidal cells by 92%, whereas I(h) of stellate cells was only reduced by 70%. In both pyramidal and stellate neurons, I(h) was not significantly changed during the application of 1 mM Ba(2+). 8-Bromo-c-AMP increased amplitudes of I(h) in stellate cells, while I(h) of pyramidal cells remained unchanged. It is suggested that different types of hyperpolarization-activated cation channels are expressed in pyramidal and stellate cells of the entorhinal cortex.