Somatostatin Modulation of Excitatory Synaptic Transmission Between Periventricular and Arcuate Hypothalamic Nuclei In Vitro

Christophe Lanneau, Stéphane Peineau, Florence Petit, Jacques Epelbaum, and Robert Gardette

U.159 Institut National de la Santé et de la Recherche Medicale Centre Paul Broca, 75014 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lanneau, Christophe, Stéphane Peineau, Florence Petit, Jacques Epelbaum, and Robert Gardette. Somatostatin Modulation of Excitatory Synaptic Transmission Between Periventricular and Arcuate Hypothalamic Nuclei In Vitro. J. Neurophysiol. 84: 1464-1474, 2000. Hypophysiotropic somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) neurons are primarily involved in the neurohormonal control of growth hormone (GH) secretion. They are located in periventricular (PEV) and arcuate (ARC) hypothalamic nuclei, respectively, but their connectivity is not well defined. To better understand the neuronal network involved in the control of GH secretion, connections from PEV to ARC neurons were reconstructed in vitro and neuronal phenotypes assessed by single-cell multiplex RT-PCR. Of 814 stimulated PEV neurons, monosynaptic responses were detected in only 45 ARC neurons. Monosynaptic excitatory currents were detected in 29 ARC neurons and inhibitory currents in 16, indicating a 2/1 ratio for excitatory versus inhibitory connections. Galanin (GAL), NPY, pro-opiomelanocortin (POMC), and SRIF mRNAs were detected in neurons from both nuclei but GHRH mRNA almost exclusively in ARC. Among the five SRIF receptors, only sst1 and sst2 were expressed, in 94% of ARC and 59% of PEV neurons, respectively. Of 128 theoritical combinations between neuropeptides and sst receptors, only 22 were represented in PEV and 25 in ARC. For PEV neurons, neuropeptide phenotypes did not influence excitatory connections. However, the occurrence of presynaptic sst receptors on GAL and SRIF PEV neurons significantly increased their probability of connection to ARC neurons. GHRH ARC neurons expressing sst2, but not sst1, receptors were always connected with PEV neurons. Physiological responses to sst1 (CH-275) or sst2 (Octreotide) agonists were always correlated with the detection of respective sst mRNAs. In conclusion, 1) SRIF-modulated excitatory transmission develops in vitro from PEV to ARC neurons, 2) ARC GHRH neurons bearing sst2 receptors appears directly controlled by fast glutamatergic transmission from PEV neurons simultaneously expressing one to four neuropeptides, 3) GHRH neurons bearing sst1 receptors lack this control, and 4) these results suggest that fast excitatory neurotransmission and neuropeptide modulation can derive from a small subset of PEV hypothalamic neurons targeted at ARC neuronal subpopulations.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In mammalian CNS, the control of pituitary growth hormone (GH) secretion is achieved by an exquisite interplay between two hypothalamic neurohormones, a stimulatory GH-releasing hormone (GHRH) and an inhibitory one, somatostatin (SRIF) (for review see Tannenbaum and Epelbaum 1999). Their interactions result in the ultradian pulsatile pattern of GH release. In the rat, experimental findings led to the hypothesis that a 3.5-h rhythmic surge of each peptide (a fall of SRIF concentration associated with an increase in GHRH) is superimposed on their steady-state release, resulting in the ultradian rhythm of GH secretion (Wagner et al. 1998). The phasic release of SRIF appears to play a key role in determining GH pulsatility in humans too since it is preserved during constant infusion of GHRH (Vance et al. 1985). If there is good experimental evidence for the antiparallel rhythmicity of GHRH and SRIF neurons, the mechanisms of their cross-talk is less well understood. In rodents, GHRH hypophysiotropic neurons are located in the mediobasal hypothalamus, mostly in the ventrolateral portion of the arcuate nucleus (ARC) while hypophysiotropic SRIF neurons are located more anteriorly in the periventricular nucleus (PEV). In contrast to the physiological findings, neuroanatomical tract-tracing studies did not indicate a massive interrelation between GHRH and SRIF neurons. Indeed, if a few neurons from the periventricular zone of the paraventricular nucleus project to ARC (Toth and Palkovits 1998), most hypophysiotropic SRIF neurons (78%) project directly to the median eminence (Kawano and Daikoku 1988), without connecting with GHRH neurons (Willoughby et al. 1989) and only 3% of ARC GHRH neurons appear to contact PEV neurons (Fodor et al. 1994). However, ARC GHRH (Bertherat et al. 1992; Epelbaum 1992; McCarthy et al. 1992) neurons are endowed with SRIF binding sites and appear to be innervated by fibers originating from a subset of ARC somatostatinergic neurons (Daikoku et al. 1988; Liposits et al. 1988). Of the five SRIF receptor subtypes, ARC is one of the most enriched brain regions for both sst1 and sst2 mRNAs (Beaudet et al. 1995; Breder et al. 1992; Dournaud et al. 1996). Subsets of GHRH neurons express sst1 or sst2 mRNAs (Tannenbaum et al. 1998) while sst1 receptors have recently been located in close vicinity to PEV SRIF cell bodies and terminals in the external layer of the median eminence (Helboe et al. 1998). In mouse hypothalamic neuronal cultures, activation of the sst1 receptor subtype mediates SRIF-induced increase in sensitivity to glutamate (GLU) while decrease in the response to glutamate is linked to activation of the sst2 receptor subtype (Lanneau et al. 1998).

In the present work, we attempted to reconstruct the neuronal network involved in the control of GH secretion by co-incubating neurons from mouse embryos, dissected out from the anterior periventricular hypothalamic and arcuate regions, respectively. Since GLU appears as the major neurotransmitter involved in hypothalamic synaptic transmission (van den Pol and Trombley 1993), PEV neurons were electrophysiologically stimulated and excitatory postsynaptic currents recorded from ARC neurons. The peptidergic [galanin (GAL), GHRH, neuropeptide Y (NPY), pro-opiomelanocortin (POMC), SRIF] and SRIF receptors phenotype of interconnected neurons was concomitantly determined by single-cell RT-PCR.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cocultures of arcuate and periventricular neurons

Hypothalamic nuclear microdissection was performed according to the technique described previously (Loudes et al. 1997) and adapted to the mouse. A frontal slice of brain containing the hypothalamus was sampled from 10 to 18 17-day-old fetuses (albino Swiss, Janvier, Le Genest St Isle, France) and dissected into two fragments: a caudo-ventral one transected horizontally at the level of the median eminence and containing the arcuate nucleus and a thin, more rostro-dorsal segment adjacent to the third ventricle containing the periventricular area nucleus. Cells from the isolated regions were pooled, mechanically dissociated, briefly spun for 10 min at 450 g and the pellet resuspensed in 100-200 µl of defined medium (according to the number of fetuses), with 10% of fetal calf serum. Cells were plated along two lines in 35 mm Petri dishes at a density of one structure per 10 µl with a minimal distance (<1 mm) between the two structures. Cells were maintained for 24 h in a 37°C, 7% CO2 atmosphere saturated with water. After this period, 1.5 ml of serum-free medium was carefully added. Medium was changed twice a week. From 7 days in vitro (DIV) on, the cells were treated with 1 µM of cytosine arabinoside (Sigma, St. Quentin Fallavier, France) to block excessive glial cell proliferation.

Physiological recordings

A scheme of the experimental setup is summarized in Fig. 1. Extracellular stimulations of PEV neurons were performed using a Neurodata PG4000 digital stimulator (Neurodata, New York, NY) generating 4.5- to 10-V, 10- to 100-µs step stimulations. PEV neurons were stimulated under visual control by means of two patch pipettes (approximately 1 MOmega ) filled with extracellular solution and placed next to the cell bodies by the use of pneumatic micromanipulators (Narishige, Tokyo). The stimulating electrodes were separated by <50 µm, and one of them was placed in direct contact with the cell soma of the putative presynaptic neuron. This extracellular stimulation protocol was chosen to rapidly scan several PEV neurons to detect the presence or the absence of a synaptic connection with the recorded ARC neuron. Monosynaptic responses in ARC neurons (see RESULTS) were detected after a maximum of 28 stimulations in the PEV area. After detection, width and amplitude of the stimulation were adjusted just above the excitatory postsynaptic current (EPSC) threshold, and its frequency was set to 1 Hz. ARC neurons were defined as "non-responsive" in the absence of any synaptic response after stimulation of up to 35 neurons from PEV region, this number corresponding to all neurons that could be stimulated under visual control in one optical field under the microscope. Before harvesting cytoplasm (see Single-cell multiplex RT-PCR of neuropeptides and ssts mRNAs), displacement of the stimulating electrode followed by loss of the postsynaptic response was used to ascertain that the presynaptic neuron was indeed connected to the postsynaptic recorded ARC neuron.



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Fig. 1. Description of the experimental protocol. A: schematic drawing of the microdissection of periventricular (PEV) and arcuate (ARC) nuclei from 17-day-old mouse fetuses. The 2 nuclei are shown on the same anteriority level, although PEV is more rostral than ARC. B: phase contrast photomicrograph of a 15-day in vitro (DIV) coculture showing the pair of stimulating electrodes in the PEV and the recording electrode in the ARC. Numbers 1 to 35 represent the displacement of the stimulating electrode in PEV when exploring for monosynaptic responses in the recorded ARC neuron (as shown in inset). C: examples of phenotypic expression obtained, after cytoplasm harvesting and RT-PCR procedure, from a presynaptic PEV neuron [expressing somatostatin (SRIF) and sst2 mRNAs], and a postsynaptic coupled ARC neuron [expressing neuropeptide Y (NPY), pro-opiomelanocortin (POMC), SRIF, and sst1 mRNAs]. Control lane RT, processed without reverse transcriptase; MW, molecular weight marker.

Electrophysiological recordings from ARC neurons employed the whole cell tight seal recording configuration of the patch-clamp technique (Hamill et al. 1981). Patch-clamp electrodes (4-6 MOmega ) were obtained by a two-stage pull on a horizontal electrode puller (BB-CH Mecanex, Geneva, Switzerland) and filled with (in mM) sterile 120 KF, 3 KCl, 1 CaCl2, 2 MgCl2, 6 EGTA, 10 HEPES, with 1 mM of ultrapure ATP sodium salt and 0.5 mM of ultrapure GTP sodium salt (Clontech, Palo Alto, CA) at pH 7.4 (osmolarity 280-290 mOsm), with addition of QX314 2 mM (Alamone Labs, Jerusalem, Israel) to intracellularly block sodium channels. The external patch recording medium contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose at pH 7.35 (osmolarity 305-315 mOsm). TTX (0.5 µM; Latoxan, Rosans, France), octreotide (0.1 µM; SMS 201-995, gift from Novartis, Rueil-Malmaison, France) and CH-275 (0.1 µM; gift from C. Hoeger and J. Rivier, San Diego, CA), were dissolved in the external medium. Experiments were run on-line using an Axon TL-1 DMA interface (Axon Instruments, Foster City, CA) and a 386/20 Tandon computer. Delivery of command voltages and storage of current data into data files were driven by the Axon Clampex software.

ARC neurons were recorded after 11-16 DIV, at a holding potential of -80 mV and in the presence of 2 mM Mg2+. Glutamate or GABAA receptor-induced currents were distinguished from their reversal potential values (around 0 mV for Na+/K+-mediated glutamate current, and -55 mV for Cl--mediated GABAA current). Application of 0.5 µM TTX or decrease of extracellular Ca2+ level to 0.5 mM abolished these inward currents, thus showing that the recorded postsynaptic currents were indeed due to transmitter release from a presynaptic element (data not shown). The presence of 2 mM of the Na+ channel blocker QX314 in the recording pipette permits to reject a synaptic response linked to activation of autapses or of circular connectivity arising from the ARC postsynaptic recorded neuron, thus confirming that transmitter release does come from the stimulated presynaptic PEV neuron. Recorded signals were filtered at 10 kHz and series resistance compensated up to 80% with a lag of 8 µs. Monosynaptic connections were characterized by a latency lower than 5 ms (see RESULTS) and their ability to follow a frequency of stimulation up to 100 Hz. Rise times and latencies were continuously monitored during the recording period to reject cells showing changes in their recording configuration characteristics. Recordings lasted <20 min to avoid possible wash out of the cellular medium and/or rundown of the responses.

Only monosynaptic alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA)-mediated EPSCs, showing a reversal potential around 0 mV and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 5 µM, reflect a direct PEV-ARC glutamatergic projection. The effects of the drugs on polysynaptic responses were not tested due to the impossibility to precisely characterize the neuronal network involved in such events.

Electrophysiological data analysis

Analyses were performed off-line with Axon pClamp software package and winWCP Strathclyde whole cell program Beta test software (courtesy of J. Dempster, University of Strathclyde, Glasgow, UK). Mean values of evoked EPSCs peak current amplitude, rise time, latency, and T50% were calculated from a minimum of 100 unitary synaptic responses recorded under control conditions or in the presence of the drugs. All data are expressed as means ± SE (n), and statistical significance was evaluated by Student's t-test (for pooled values), ANOVA test (for paired values during control vs. drug applications), or chi 2 test (for comparison between cell populations).

Single-cell multiplex RT-PCR of neuropeptides and ssts mRNAs

After recording, cytoplasm was first harvested from the postsynaptic ARC neuron by gentle suction applied to the recording pipette. The presynaptic PEV neuron was then patched using a second recording pipette and its cytoplasm similarly retrieved.

For each sampled neuron, the tip of the pipette was broken in a PCR tube for reverse transcription. Retrotranscription of mRNA was performed in a final volume of 15 µl according to Lanneau et al. (1998), modified from Cauli et al. (1997). Coamplification of the cDNAs encoding the five sst1-5 somatostatinergic receptors, GAL, GHRH, NPY, POMC, and SRIF, was performed simultaneously. Primers are indicated in Table 1.


                              
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Table 1. Primer sequences for each molecular marker

RT reaction products were first amplified in the presence of every primer (25 pmol each) in a 100-µl volume in an automatic thermocycler (Genius Techne, Cambridge, UK) during 20 cycles (94°C for 30 s, 56°C for 30 s, 72°C for 30 s). An extension step was then performed at 72°C for 10 min. PCR products were purified from primers and salt using Nucleon QC microspin (Amersham, Rainham, UK).

A second round of PCR was then performed using 2 µl of the first purified PCR products as template. In this round, each marker was amplified individually using its specific primer pair for 28-35 cycles. All markers, but GHRH, were amplified as described for the first amplification step. GHRH cDNA was amplified in a 50-µl volume in 50 mM KCl, 10 mM Tris HCl, 0.15% TritonX100, 2.5 mM MgCl2, and 5 units of Taq polymerase (Promega Biotech Coger, Charbonières, France), with 0.25 mM of dNTPs, 50 pmol of the common sense primer, 50 pmol of reverse primer. Amplification was performed in an automatic thermocycler (Hybaid, Teddington, UK; 94°C for 30 s, 58-60°C for 30 s, 72°C for 30 s).

Fifteen microliters of each individual PCR reaction were then run on 2% agarose gel stained with Gel Star (FMC Bioproducts, Rockland, ME). In each experiment, cytoplasms with nucleus were submitted to the entire protocol without reverse transcriptase. The intronic sequence of SRIF amplicon was never detected, thus ruling out genomic DNA contamination.

All amplicons were sequenced by Eurogentec (Serraing, Belgium). The sequences obtained after purification on a low-melting agarose gel matched with those of the cloned sequences (BLASTN search Multibank).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of synaptic connections between presynaptic PEV and postsynaptic ARC neurons

Cocultures of PEV and ARC neurons were performed according to the scheme described on Fig. 1. After plating of the cells, glial cells, mostly derived from ARC, invaded the interval between the two cell populations and, after 9 days in vitro, a large number of neurites were observed extending between the two regions.

A total number of 814 PEV neurons were stimulated to determine their ratio of transmission to ARC. Monosynaptic responses were detected in 45 ARC neurons and polysynaptic responses (latency >5 ms) in 66 ARC neurons. Thus the ratio of monosynaptic transmission from PEV to ARC is 5.5% (45/814) and that of polysynaptic transmission 8.1% (66/814). Monosynaptic connections were either excitatory (29 cells, 3.5%) or inhibitory (16 cells, 2.0%).

Among the 29 cells receiving a monosynaptic excitatory input, only 9 displayed a pure monosynaptic response. For the 20 remaining neurons, the first monosynaptic event was followed by a polysynaptic response. The histogram of latencies of excitatory responses (91 individualized mean synaptic events, Fig. 2) showed a clear multigaussian distribution with 3 peaks indicative of mono- (extrapolated mean 3.2 ± 1.4 ms), di- (extrapolated mean 8.0 ± 1.4 ms), or tri-synaptic (extrapolated mean 12.8 ± 1.6 ms) events with a few responses at longer latencies.



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Fig. 2. Histogram of the distribution of excitatory postsynaptic current (EPSC) latencies in ARC neurons in response to PEV stimulation. Three peaks are evidenced corresponding to mono-, di-, or tri-synaptic events. Data were obtained from 29 ARC neurons and a total of 91 events (each event represents the mean of 10 unitary responses).

Phenotypic characterization of PEV and ARC neurons

The expression of mRNAs encoding for five neuropeptides (GAL, GHRH, NPY, POMC, and SRIF) and the five ssts was analyzed in three groups of neurons: 1) 27 pairs of PEV/ARC neurons displaying a characterized monosynaptic excitatory connection (8 pure monosynaptic and 19 mixed responses in which a polysynaptic component followed the monosynaptic one, Table 2), 2) 8 PEV for which an ARC neuronal target had not been found (Table 3A), and 3) 8 ARC neurons non-responsive to PEV stimulation (Table 3B).


                              
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Table 2. Distribution of the expression of neuropeptides and ssts mRNAs in PEV and ARC neurons linked by a monosynaptic excitatory connection


                              
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Table 3. Distribution of the expression of neuropeptides and ssts mRNAs in PEV neurons for which an ARC neuronal target could not be evidenced (A), and in ARC neurons non-responsive to PEV stimulation (B)

Neuropeptides

The total number of cells expressing each peptide ranked as follows (Fig. 3A):



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Fig. 3. Distribution of the expression of neuropeptides and ssts mRNAs in ARC and PEV neurons. A: percentages of cells expressing growth hormone-releasing hormone (GHRH), galanin (GAL), NPY, POMC, SRIF, sst1, sst2, sst1/sst2 mRNAs in ARC or PEV neurons. B: cell distribution as a function of the number of peptides coexpressed in ARC or PEV neurons. The number of cells is indicated above the bar graphs. *P < 0.05; **P < 0.01; ***P < 0.001.

in ARC: POMC(24) > NPY(21) > SRIF(20) > GHRH(16) GAL(8)

in PEV: SRIF(20) POMC(12) > NPY(10) > GAL(6) GHRH(1)

Three PEV neurons and one ARC neuron did not express any peptide mRNA.

GHRH mRNA was expressed in 16 ARC neurons and only 1 PEV cell (P < 0.001). Neurons expressing NPY and POMC mRNAs were significantly more numerous in ARC than PEV (NPY: P < 0.01 and POMC: P < 0.01, respectively). GAL and SRIF neurons were equally distributed in both ARC and PEV. The percentage of neurons expressing more than one neuropeptide mRNA was higher in ARC (33/35, 94%) than in PEV (14/35, 40%, P < 0.001; Fig. 3B). Of 32 theoritical neuropeptide phenotype combinations, only 12 were recorded in PEV and 19 in ARC.

sst receptors

Of the five sst mRNAs, only sst1 and sst2 were detected by single-cell RT-PCR. The rank of sst receptor expression for the total cell population in each region was as follows (Fig. 3A):

in ARC: sst2 (15) > sst1(13) sst1/sst2(5)

in PEV: sst1(10) > sst2 (9) sst1/sst2(2)

Fourteen PEV and two ARC cells did not express any sst receptors.

The occurrence of sst1 and sst2 alone or in combination was higher in ARC than in PEV neurons (33/35, 94% vs. 21/35, 60%, P < 0.001). Within each region, the number of neurons expressing sst1 or sst2 mRNAs was not different. Neurons co-expressing both receptors were significantly lower than those expressing either sst1 or sst2 in both regions.

Neuropeptide and receptors

The occurrence of coexpression of sst1 or sst2 with neuropeptide mRNAs is indicated on Table 2 for paired neurons, Table 3 for non-connected ARC or PEV neurons, and in Fig. 4 for the whole neuronal population. Of 128 theoritical combinations, only 22 were represented in PEV and 25 in ARC. The only paired mRNAs combination above 20% was SRIF/sst1 expresssing neurons in PEV (5/22, 23%). No combination reached 10% in ARC.



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Fig. 4. Distribution of the coexpression of the 5 neuropeptides with either sst1 or sst2 mRNAs in ARC and PEV neurons. A: percentages of ARC neurons coexpressing sst1 or sst2 mRNAs with neuropeptides mRNAs. Note the significantly lower coexpression of sst1 mRNA with GAL mRNA and the significantly higher coexpression of sst2 mRNA with POMC mRNA as compared with GAL mRNA. B: percentages of PEV neurons coexpressing sst1 or sst2 mRNAs with neuropeptides mRNAs. Note the significantly higher coexpression of sst1 mRNA with SRIF mRNA and that of sst2 mRNA with POMC and SRIF mRNAS as compared with GAL mRNA. Only 1 cell expressed GHRH mRNA. *P < 0.05; **P < 0.01; ***P < 0.001.

In ARC (Fig. 4A), GAL mRNA containing neurons represented a smaller proportion of sst1 expressing neurons (4/18, 22%) than NPY- (12/18, 67%, P < 0.01), or POMC- and SRIF- (11/18, 61%, P < 0.05) expressing neurons. GAL mRNA-containing neurons also represented a smaller proportion of sst2-containing neurons than POMC-expressing neurons (5/20, 25% vs. 15/20, 75%, respectively, P < 0.01).

In PEV (Fig. 4B), sst1 mRNA was significantly more often co-expressed with SRIF expressing (8/12, 66%) than GAL- (3/12, 25%, P < 0.05), NPY- (1/12, 8%, P < 0.01) or POMC- (2/12, 17%, P < 0.05) expressing neurons. Sst2 mRNA was significantly more often co-expressed with POMC- (8/11, 73%) or SRIF- (7/11, 64%) than GAL- (0/11, 0%, P < 0.01) or NPY- (3/11, 27%, P < 0.05) expressing neurons, and significantly less expressed with GHRH (1/11, 9%) than with POMC (P < 0.01) or SRIF (P < 0.05).

Relationships between excitatory synaptic transmission and phenotypic expression of PEV/ARC-coupled neurons

The comparison between experimental and theoritical probability of connection as a function of expressed PEV or ARC neuropeptides showed no preferential occurrence of synaptic connections between the two neuronal populations. In particular, the proportion of SRIF-expressing PEV neurons connected to GHRH-expressing (7/27, 26%) or non-GHRH-expressing (5/27, 19%) ARC neurons was not significantly different from that of non-SRIF-expressing PEV neurons connected to GHRH-expressing (3/27, 11%) or non-GHRH-expressing (9/27, 33%) ARC neurons.

However, the proportion of SRIF- and GAL-expressing PEV neurons monosynaptically connected to ARC neurons was significantly higher than random if they expressed sst receptor subtypes [(SRIF: 12 connected neurons/12 sst expressing neurons; GAL: 3/3, Table 2A) as compared with (SRIF: 2 connected neurons/7 neurons lacking sst expression, P < 0.001; GAL: 0/3, P < 0.05, Tables 2A and 3A)].

Conversely, the postsynaptic expression of sst receptors by GAL-, NPY-, POMC-, or SRIF-expressing ARC neurons did not increase the probability for an ARC neuron to receive a monosynaptic excitatory connection from a PEV neuron.

Finally, GHRH-expressing neurons could be divided in two subgroups, the presence of sst2 mRNAs being always associated with a connection with PEV neurons (10/10, Table 2B) and the presence of sst1 mRNA with its absence (5/5, Table 3B).

Effects of sst1- (CH-275) and sst2- (octreotide) selective agonists on excitatory synaptic transmission in phenotypically identified PEV/ARC coupled neurons

In 7 of 22 cells (32%), CH-275 elicited an increase in the amplitude of monosynaptic responses (30 ± 7%, P < 0.05, Fig. 5A). CH-275-induced changes developed rapidly (between 50 and 100 s), and recovery was complete within a few minutes wash. All seven CH-275-sensitive neurons expressed sst1 mRNA (Fig. 5C), but sst1 mRNA was also detected in four other cells that did not respond to this agonist.



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Fig. 5. Effect of the sst1 agonist CH-275 on the amplitude of monosynaptic EPSCs recorded in an ARC neuron in response to PEV stimulation. A: time plot of CH-275 effects. Each dot represents the mean amplitude ± SE of 5 consecutive evoked EPSCs. Insets show typical unitary monosynaptic evoked EPSCs before, during, and after CH-275 effects (scale bars: 50 pA, 1 ms). B: the presynaptic coupled PEV neuron expressed NPY, SRIF, and sst1 mRNAs. C: the postsynaptic coupled ARC neuron expressed NPY, POMC, and SRIF mRNAs, as well as sst1 (correlated to the CH-275 effect) and sst2 mRNAs.

In 17 of 26 cells (65%), octreotide induced a decrease in excitatory transmission (-24 ± 3%, P < 0.001, Fig. 6A). Octreotide induced changes developed slowly (2-5 min), and total recovery was rarely observed. All octreotide-sensitive neurons contained sst2 mRNA (Fig. 6C), and only one sst2-expressing neuron did not respond to this agonist.



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Fig. 6. Effect of the sst2 agonist Octreotide (OCT) on the amplitude of monosynaptic EPSCs recorded in an ARC neuron in response to PEV stimulation. A: time plot of OCT effects. Each dot represents the mean amplitude ± SE of 5 consecutive evoked EPSCs. Insets show typical unitary monosynaptic evoked EPSCs before, during, and after OCT effects (scale bars: 100 pA, 1 ms). B: the presynaptic coupled PEV neuron did not express any of the mRNAs studied. C: the postsynaptic coupled ARC neuron expressed GHRH and POMC mRNAs, as well as sst2 mRNA (correlated to the OCT effect).

Transmission failures were detected in 3 of the 22 cells under CH-275 (not illustrated) and 5 of the 26 cells under octreotide (Fig. 7A). No change in the probability of failures was found under CH-275 (from 0.048 ± 0.007 in control medium to 0.050 ± 0.006 in presence of CH-275), whereas this probability was significantly enhanced under octreotide (from 0.049 ± 0.023 in control medium to 0.320 ± 0.110 in presence of octreotide, P < 0.05). This increase in the number of failures was always correlated to the presence of sst2 mRNA in the presynaptic neuron (Fig. 7B).



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Fig. 7. Presynaptic effect of Octreotide on the excitatory monosynaptic transmission between a PEV and an ARC neuron. A: time plot of the ARC monosynaptic EPSC peak current during control and OCT perifusion. No change in the peak current was induced by OCT, but a marked increase in failure frequency was observed under OCT, indicative of a presynaptic effect. At the end of recording, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) perifusion totally abolished the response. Each dot on the time plot represents the amplitude of only 1 unitary response. Periods of perifusion of control medium (1), OCT (2 and 3), wash (4), and CNQX (5) are indicated by arabic numbers, and corresponding representative electrophysiological recordings of monosynaptic EPSCs are shown under the time plot (scale bars: 100 pA, 1 ms). B: the presynaptic coupled PEV neuron expressed sst2 mRNA (correlated to the presynaptic effect of OCT). C: the postsynaptic coupled ARC neuron expressed GAL, NPY, and sst1 mRNAs, but no sst2 mRNA (correlated to the lack of postsynaptic effect of OCT).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present work, we undertook to reconstruct an in vitro model of the intrahypothalamic network involved in the control of GH secretion, by allowing synaptic connections to develop between E17 mouse neurons microdissected from anterior periventricular nucleus region and mediobasal periventricular hypothalamus, the former containing somatostatin- and the latter GHRH-hypophysiotropic neurons. To test SRIF modulations in this network, monosynaptic excitatory connections were recorded since they represent a direct pathway between PEV and ARC neurons.

Synaptic connections within PEV/ARC network in vitro

Only a minority of PEV neurons were connected in vitro directly (5.5%) or indirectly (8.1%) to ARC neurons, in agreement with the fact that, in vivo, up to 78% of PEV neurons innervate directly the median eminence (Daikoku et al. 1988). A large proportion of hypohysiotropic SRIF cells in the PEV, which would normally project to the median eminence in vivo, might die from lack of connections rather than redirecting their connectivity toward ARC nucleus in vitro. At any rate, the PEV-ARC connections as observed in the in vitro model are in keeping with previous results reporting a physiological link between PEV and ARC, some ARC neurons showing a hyperactivation rebound following an initial inhibition at the end of an electrical stimulation of PEV (Dickson et al. 1994). Thus the pattern of connections as observed in vitro appears reminiscent of the hypothalamus in vivo.

In vitro, excitatory connections between PEV and ARC neurons outnumbered inhibitory ones, at variance with results obtained with cultures of uncharacterized hypothalamic neuronal cells (Muller et al. 1997). The high occurrence of polysynaptic events recorded in ARC neurons after PEV stimulation indicates that the connections between the two nuclei involve interneurons, reminiscent of local ARC GABAergic or glutamatergic circuitry (Belousov and van den Pol 1997).

Patterns of neuropeptide expression

As for the synaptic connections, the rank of neuropeptide neuronal phenotypes, in both ARC and PEV neurons, corresponds to previously reported data obtained in vivo in rats (see Meister 1989 for review). Indeed, all but one GHRH mRNA-expressing neurons were restricted to the ARC region. This neuron is likely to correspond to the smaller contingent of GHRH neurons located in the paraventricular nucleus (Bloch et al. 1984; Jacobowitz et al. 1983; Sawchenko et al. 1985). Interestingly, the rate of coexpression between neuropeptides in both nuclei is not a random phenomenon since <25% of all statistical combinations between neuropeptides and receptors were observed.

ARC nucleus contains over 15 identified neurotransmitters and neuropeptides (Chronwall 1985). The high rate of neuropeptide coexpression found in mouse ARC nucleus herein is therefore not surprising. Thus if GHRH can be colocalized with neurotransmitters such as GABA (Meister and Hökfelt 1988) or dopamine (Everitt et al. 1986) in rat arcuate nucleus, it is also coexpressed with other peptides such as neurotensin or GAL (Hökfelt et al. 1986). GHRH and POMC coexpression in vivo appears controversial in ARC, being detected (Chronwall 1985) or not (Sawchenko et al. 1985). Finally, it should be mentioned that Ciofi et al. (1987) have shown that almost all the NPY-immunoreactive neurons in the medial part of ARC are also stained with antisera against hpGHRH44, in keeping with the GHRH/NPY coexpression found in the present study.

In PEV, the occurrence of peptide coexpression was much lower than in ARC, in keeping with a previous study showing that neurons expressing simultaneously GAL with neurotensin or corticotropin-releasing factor were detected in much lower density in rat PEV than in other paraventricular nucleus subdivisions (Ceccatelli et al. 1989).

Patterns of ssts expression

The in vitro model also allows to define more precisely the localization of somatostatin receptor expressing neurons in mouse hypothalamus. sst1 and sst2 were the only receptors detected at the single-cell level, but there was no preferential expression of one or the other subtype either within or between each nucleus. According to the expression of sst1 receptors by hypophysiotropic SRIF neurons in the rat anterior periventricular hypothalamic nucleus (Helboe et al. 1998), it is noteworthy that, in the in vitro model, SRIF/sst1 expressing neurons in PEV are the only population above 20% of the combinations of sst1 or sst2 coexpression with neuropeptides. Sst receptors were much more often expressed in ARC (94% of the neurons) than PEV (59%), indicating that ARC displays a richer expression of ssts than both PEV or total hypothalamus (66% of the neurons) (Lanneau et al. 1998). This result is compatible with the extensive perikarial SRIF binding network that has been visualized in the ARC region in the rat (Bertherat et al. 1992). Moreover, SRIF-immunoreactive axons form synaptic junctions on GHRH neurons in ARC (Daikoku et al. 1988; Liposits et al. 1988; Willoughby et al. 1989), and this neuronal population expresses sst1 or sst2 receptor genes (Tannenbaum et al. 1998). POMC-expressing cells also bear SRIF1 (sst2/3/5) receptors (Fodor et al. 1998) as also observed herein. These results indicate that the distribution of ssts is not restricted to one single neuronal ARC population. In ARC, SRIF may directly modulate GHRH release by acting on GHRH expressing neurons or indirectly through the modulation of other peptidergic expressing neurons.

Relationships between neuropeptide and sst mRNA expression, and synaptic connections

According to neuropeptide expression, preferential PEV/ARC connections were not evidenced in the cocultures. This stochastic distribution is in favor of a rather diffuse control of ARC neurons by PEV neurons. However, sst receptor expression appeared not at random among neuropeptide ARC phenotypes. Indeed, only these GHRH neurons that expressed sst2 receptors received afferences from PEV, while those expressing sst1 receptors did not. This suggests that the sst2 receptor subtype may be directly involved in the regulation of GHRH neurons through PEV afferences, whereas the control of these neurons by the sst1 receptor subtype (Tannenbaum et al. 1998) might involve different neuronal connections.

In keeping with the present observations indicating projections from SRIF- and POMC-expressing PEV neurons to GHRH-expressing ARC neurons, SRIF- (Liposits et al. 1988) and POMC- (Daikoku et al. 1988) immunoreactive fibers have been described in close connection to ARC GHRH cell bodies. On the contrary, although we found that activation of NPY- or GAL-expressing PEV neurons could lead to an excitatory response in GHRH-expressing ARC neurons, NPY- or GAL-immunoreactive fibers close to GHRH perikaria have yet to be reported (Chan et al. 1996).

Glutamate and neuropeptide coexpressions

In our experimental model, stimulated PEV neurons are presynaptic to ARC neurons, being therefore glutamatergic and simultaneously coexpressing one to four neuropeptides. Up to now, coexpression of glutamate was mostly reported with one neuropeptide only [substance P: De Biasi and Rustioni 1988; Nicholas et al. 1990; SRIF: Sur et al. 1994; neurotensin: Todd et al. 1994; calcitonin gene-related peptide: Freund et al. 1997; NPY: Makiura et al. 1999; pituitary adenylate cyclase activating polypeptide (PACAP): Hannibal et al. 2000] with the notable exception of the coexpression of glutamate with both CGRP and SP in some terminals in the rat dorsal horn (Merighi et al. 1991).

In keeping with our previous results showing that SRIF, through sst1 and sst2 receptors, differentially modulated exogenous glutamate actions in mouse hypothalamic neurons in culture (Gardette et al. 1995; Lanneau et al. 1998), we now bring evidence that SRIF, which is found coexpressed with glutamate in PEV neurons, modulates the glutamatergic transmission induced by the activation of the very neuron coexpressing the two molecules. Furthermore, sst2-mediated inhibitions of hypothalamic neurons seem to involve not only postsynaptic mechanisms (Lanneau et al. 1998), but also presynaptic mechanisms as previously reported on rat hippocampal autapses (Boehm and Betz 1997). These results introduce a new level of complexity in the regulation of neuronal intrahypothalamic networks potentially involved in the control of the secretion of hypophysiotropic hormones.

In conclusion, we have developed an in vitro model that allows for the physiological characterization of the neuronal network involved in the intrahypothalamic control of GH secretion. It demonstrates a high degree of complexity in terms of peptidergic expression, SRIF receptor distribution, and connectivity within and between PEV and ARC nuclei. This model has been used to assess the somatostatin modulation of excitatory transmission and will be developed to define the importance of other components (such as inhibitory transmission) of the network. Most interestingly, the glutamatergic transmission that is modulated by SRIF agonists comes from PEV neurons simultaneously expressing various hypothalamic neuropeptide mRNAs. This strongly suggests that both excitatory neurotransmission and neuropeptidergic modulation can be elicited by the same hypothalamic PEV neurons targeted at specific ARC neuronal subpopulations such as GHRH neurons expressing the sst2 somatostatinergic receptor subtype.


    ACKNOWLEDGMENTS

We thank Dr. Jens Hannibal for kindly providing his in press manuscript, and Drs. Annie Faivre and Catherine Loudes for helpful advice in setting up the microdissection protocol.

This work was supported by INSERM and a fellowship grant from Novartis to C. Lanneau.


    FOOTNOTES

Address for reprint requests: J. Epelbaum, INSERM U159, 2ter rue d'Alésia, 75014 Paris, France (E-mail: epelbaum{at}broca.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 March 2000; accepted in final form 5 June 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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