mNTS neurons produced by angiotensin II and arginine-vasopressin

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Letters, 151 (1993) 121-125 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-39401931% 06 00

Neuroscience

NSL 09313

Increases in cytosolic Ca2+ in rat area postrema/mNTS neurons produced by angiotensin II and arginine-vasopressin M. Hay, G.L. Edwards, K. Lindsley, S. Murphy, R.V. Sharma, R.C. Bhalla and AK. Johnson The Departments

of Internal Medrcme. Psychology,

Pharmacology,

and Anatomy, and The Cardiovascular

Center, Umverslly of Iowa College of

MedIcme, Iowa Czty, IA 52242 (USA)

(Received 6 July 1992; Revised version received 19 November 1992; Accepted 20 November 1992) Key words.

Area postrema, Medial nucleus tractus sohtanus, Angiotensm II; Argmme-vasopressm, AT, receptor subtype; Fura- loading, Mtcroscoptc dtgttal image analysis

Intracellular Ca” concentration; Angiotensm

It is well established that neurons m the dorsal medulla, including the area postrema and the medial nucleus tractus sohtanus (mNTS), are mvolved m the central actions of ctrculatmg peptides such as angtotensm II (ANG II) and argmme vasopressm (AVP) This report describes a preparatton that permits the tdenttfication and maintenance of area postrema/mNTS neurons m culture in which the cellular and potenttally subcellular responses to neurotransmttters and neuropeptides on area postrema/mNTS cells can be Investigated. Followmg 15-21 days m culture, the effects of ANG II and AVP on changes m mtracellular Ca” concentration ([Ca*‘],) were examined Both ANG II and AVP resulted m a rapid and transient increase m [Ca”], reachmg maximum m 15 s and returning towards basehne values withm 180 s. The ANG II-mediated increase m [Ca”], was almost completely abohshed by the selecttve angiotensm AT, receptor subtype antagomst, losartan (DuP 753) These results suggest that ANG II and AVP modulate area postrema/mNTS neuronal acttvtty by mcreasmg mtracellular Ca”.

The central effects of circulating vasoactive peptides such as ANG II and AVP on cardiovascular function have been demonstrated in a number of different animal models. For example, in rats and dogs, circulating ANG II has been shown to attenuate the arterial baroreflex [5, 9-l 1, 161. Circulating AVP, on the other hand, has been found to facilitate arterial baroreflex function in both dogs and rabbits [7, 14, 18, 211. Both of these effects are known to be dependent on an intact area postrema, a circumventricular organ located in the medulla oblongata. Like other circumventricular organs, the area postrema is devoid of a blood brain barrier and is susceptible to modulation by circulating factors. Because the area postrema has direct projections to neurons involved in cardiovascular control such as the mNTS and the parabrachial nucleus (PBN) [22], it is hypothesized that circulating peptides such as ANG II and AVP act on area postrema neurons to modulate cardiovascular function. ANG II and AVP have also been suggested to act as neurotransmitters within the central nervous system. ANG II and AVP receptors, and ANG II and AVP-containing processes and terminals have been localized Correspondence A K Johnson, Dept of Psychology, University of Iowa, 11 Seashore Hall E, Iowa City, IA 52242-1407, USA

within the mNTS and other cardiovascular related regions [6, 15, 201. Furthermore, direct microinjections of ANG II and AVP into these regions result in significant changes in efferent sympathetic activity and blood pressure [2,4, 8, 12, 131. Cytosolic free calcium concentration is a fundamental regulator of cellular functions such as metabolic activity, gene expression and cell division [19]. Microscopic digital image analysis of Fura- loaded single cells provides a non-invasive and specific technique to measure changes in [Ca*‘], in intact cells [23]. Using this method, the purpose of the present study was to determine the effects of ANG II and AVP on [Ca”], in area postrema/ mNTS neurons in culture. All area postrema/mNTS neurons were obtained from 14 to 16-day-old rat pups (Sprague-Dawley). Pups were anesthetized with halothane and rapidly decapitated. The hindbrain and cerebellum were then exposed, removed and placed in cold HEPES-buffered, Earl’s balanced salt solution (HEBSS) which contained (in mM) NaCl 138, KC1 5.0, NaHCO, 4.2, NaH,PO, 1.0, glucose 13.9, MgSO, 0.03, BSA 0.3%, and HEPES 25.0, pH 7.4. Under a dissecting microscope the area postrema was readily identified. The area postrema and the tissue immediately ventral and ventrolateral to the area postrema

122

(mNTS) were carefully removed. This tissue was transferred to a cold HEBSS solution and minced.

specific neurofilament monoclonal antibodies

Procedures used for isolation and culturing are similar to those described by Dutton [3]. Tissue was incubated m

These procedures from non-neuronal

a culture flask for 30-40 mm at 37°C in 10 ml HEBSS containing 0.25 mg/ml trypsin (Sigma). Following incubation, 10 ml HEBSS, 0.1 mg/ml DNase and 1 mg/ml

phological characterization of these neurons. Figure 1B shows 12-day-old area postrema/mNTS neurons with positive staining for neurofilament reaction product. No-

soybean trypsin inhibitor (Type Is, Sigma) was added to stop trypsinization. Tissue was gently triturated for 20-

tice the absence of staining m the flat cells also in culture. To identify the neural connectivity of these neurons to PBN neurons, some cells were retrogradely labeled wtth

30 s, underlayed

with 5 ml of 4% BSA and then centrr-

fuged at 100 x g for 5 min. Cells were re-suspended m Eagle’s minimum essential media with 10% FBS, 2.5% chick embryo extract, KC1 2 0 mM, glucose 6 gm/l, glutamme 2 mM and gentamicin 180 PM, pH 7.4, and transferred to two poly-I-lysine coated coverslips Neurons were maintained m culture for 12-21 days. In order to identify area postrema/mNTS neurons with projecttons to the parabrachial nuclei, 12-day-old rat pups anesthetized with methoxyflurane were placed m a stereotaxic head holder. A micropipette filled with fluorescent indodicarbocyanme perchlorate (DiI, Molecular Probes) was positioned 8.0 mm posterior and 1.7 mm lateral from bregma and then lowered 5.5 mm into the parabrachial nucleus. DiI was injected bilaterally using a custom pressure microinjection system. The volume of the injectate was measured directly by momtormg the movement of the meniscus m the mlcropipette barrel during the injection with a microscope equipped with a fine reticle. A total of 30 nl was injected bilaterally over approximately 15520 s. [Ca”], was measured using Furaratio fluorescence and a video microscopic digital image analysis system (Photon Technology International, PTI) as described previously [ 1, 171. The time course of increases m [Ca”], was determined by acquiring 340/380 image pairs at 5-10 s intervals for 5 min after the addition of agonists. The fluorescent images of cells at 340 nm excitation were used to define a mask. The masked images were used to compute intracellular calcium concentration from the ratio images (340/380 nm). [Ca”], values reported are the average of whole cell [Ca”], measurements Cells selected for study were identified by the followmg morphological criteria. As opposed to a ghal cell or fibroblast, neurons possessed phase bright cell bodies and processes. Fig. 1A is a photograph taken from a phase contrast microscope showing 3-day-old area postrema/mNTS neurons in culture. Neurons ranged m size between 10 and 25 pm and had developed bipolar neurites extending approximately 20 ,um or more from the cell body. After 12-15 days m culture, the neurite outgrowth was markedly greater. Further identification of neurons in culture was confirmed by immunocytochemical localization of neuron

DL

protein raised

reaction against

product using neurofilament.

allowed dtscrimination of neuronal cells m culture and provided a mor-

Fig. 1C shows a DiI retrogradely

trema/mNTS neuron. similar morphological

DiI-labeled characteristics

labeled

area pos-

neurons possessed as those described

above. Approximately 5% of the neurons m culture were retrogradely labeled from the PBN To determine if area postrema/mNTS neurons m culture were physiologically responsive to neuropeptides, we examined the effects of ANG II and AVP on [Ca”], m these cells. Because of limitations of our particular imaging system during the Furaexperiments, we were unable to simultaneously identify neurons which had been prelabeled with rhodamme fluorescent DiI from the parabrachial nucleus. Neurons were selected for Furaimaging by the above morphological criteria. In 38 out of the 46 cells tested. application of ANG II resulted m a marked, transient increase m [Ca”], Fig. 1D is a pseudocolor computer generated image showmg the temporal and spatial distribution of Increased [Ca”], m response to ANG II. ANG II resulted m a rapid increase in [Ca”], and reached maximum m this cell m 10 s and returned to resting level within 70 s. [Ca’+], appeared to be distributed non-homogeneously m the cytosol of the cells tested. The [Ca”], values given m Fig. 2A, B and C were averaged from the entire cellular region by summmg and averaging [Ca”], for all pixels representing the cellular area. The basal values of [Ca”‘], were acquired before addition of the agonist. Fig. 2A shows the averaged data for 38 neurons Peak responses appeared within 15 s and returned toward baseline withm 180 s. Baseline [Ca”], averaged 133 + 17 nM. ANG II significantly mcreased [Ca”], approximately 143% to 290 + 23

-3

Fig

1 A area postrema/mNTS

Neurons

were phase bnght

vertical

bar = 200 pm)

neurons

show posltlve stammg

= 50 pm)

neurons

B fluorescently

neuron

m d Fura-

followmg

of ANG II Cellular

were used to define the mask mtracellular

calcmm

(right side

area postremdmNTS reaction

product

(bdr

labeled

with

D pseudocolor

loaded area postrema/mNTS

The masked

concentration

3 days m culture neurltes

retrogradely

nucleus (bar = 50pm)

of [Ca”], dlstrlbutlon apphcatlon

labeled

for neurofilament

C area postrema/mNTS

DII from the pdrabrachlal

followmg

and had well developed

Image neuron

Images of 340 mm excltatlon Images were used to compute

from the 340 nm/380 nm ratlo Imdges

123

D

B

sewnds Fig 2 A time course of increase responses

m [Ca”],

m cultured

to ANG II m the absence and m the presence of increase

area postrema/mNTS of the angotensm

in [Ca*‘], m area postrema/mNTS

mM. ANG II-mediated increase in [Ca*‘], was repeatable for a given cell following a 30 min wash period. This response was also blocked by pretreatment of neurons with the selective angiotensin AT, receptor subtype antagonist, losartan (DuP 753). Fig. 2B shows the averaged [Ca”], responses to ANG II in the presence and absence of DuP 753 (n = 5). While DuP 753 had no effects on baseline [Ca’+],, this antagonist virtually eliminated the increase in [Ca”‘], caused by ANG II. Six neurons which were tested for the effects of ANG II were also tested with AVP. In all six of these cells, application of 100 nM AVP resulted m a marked mcrease in [Ca”], similar to that seen with ANG II (Fig. 2C). Results from this study are the first to describe a preparation for culturing and identifying prelabeled area postrema/mNTS neurons. By injecting the retrograde label DiI into the parabrachial nucleus we were able to identify specific projections of the neurons of interest for study. Furthermore, the isolation and culturing of area postrema/mNTS neurons yields a preparation in which the cellular and potentially subcellular effects of neurotransmitters and neuropeptides on these cells can be investigated In this study the effects of ANG II and AVP on [Ca”], m isolated area postrema/mNTS neurons in culture were characterized. Both ANG II and AVP acted to rapidly and transiently increase [Ca”], and both of these effects were identified to occur within the same neuron. These results suggest that the effects of circulating peptides such as ANG II and AVP on area postrema/mNTS neurons may involve increases in intracellular calcium. In addition, the blockade of the ANG II response by pretreatment of the neurons with DuP 753 indicates that the effects of ANG II on area postrema/mNTS neurons are mediated by AT, receptors. Future studies will determine

neurons

m response

AT, receptor neurons

subtype

to 100 nM ANG

antagonist,

m response

II (n = 38) B Averaged

losartan

[Ca”],

(DuP 753) (n = 5) C time course

to 100 nM AVP (n = 6)

whether these increases in Ca” are due to release from cytosolic stores or possibly influx through voltage gated Ca2’ channels.

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