Microinjection Of Vasopressin Research Articles

Haemodynamic and renal sympathetic responses to V1b vasopressin receptor activation within the paraventricular nucleus.

What is the central question of this study? Does antagonism of V1b receptors prevent the haemodynamic and renal sympathetic nerve responses that occur with application of exogenous vasopressin into the paraventricular nucleus (PVN) of conscious, chronically instrumented rats? What is the main finding and its importance? Microinjection of vasopressin into the PVN increased mean arterial pressure, heart rate and renal sympathetic nerve activity, all of which were inhibited by pre-injection of the PVN with the V1b antagonist, nelivaptan. The administered vasopressin did not enter the peripheral circulation or increase plasma vasopressin. Ganglionic blockade prevented each of the responses, consistent with mediation by enhanced sympathetic output rather than an increase in circulating vasopressin. Vasopressin (VP) participates in regulation of haemodynamics and volume. Besides more classical actions as a circulating hormone, VP may act via release from axons and dendrites within the CNS. The paraventricular nucleus (PVN) possesses vasopressinergic neurons and a dense complement of VP receptors, including the V1b receptor, which has been implicated in several types of stress responses. We tested the hypothesis that antagonism of V1b receptors will prevent VP-induced increases in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA). Studies were performed in conscious male Sprague-Dawley rats chronically instrumented with vascular catheters, renal nerve electrodes and a cannula stereotaxically directed into the PVN. Unilateral microinjection of VP into the PVN significantly increased MAP, HR and RSNA, peaking at 10min. Pre-injection of the PVN with the selective V1b receptor antagonist, nelivaptan, did not alter baseline values but blocked the responses to VP. Ganglionic blockade with chlorisondamine decreased MAP and HR and abolished their increase in response to subsequent PVN application of VP. Injection of VP into the PVN did not alter plasma VP levels. Paraventricular nucleus injection with radiolabelled VP resulted in negligible radiolabelled VP in peripheral blood. These findings support the concept that, in basal conditions, PVN V1b receptor activation (rather than VP release into the periphery) may be implicated in the increases in MAP, HR and RSNA due to increased sympathetic outflow. While the role of V1a and oxytocin receptors cannot be excluded, these data suggest that further studies of the role of V1b receptor activation by endogenous VP during stress to effect neuroexcitation are warranted.

Paraventricular vasopressin-containing neurons project to brain stem and spinal cord respiratory-related sites.

We studied in the rat projections of vasopressin-containing neurons of the paraventricular nucleus (PVN) to phrenic nuclei and to the pre-Botzinger complex (pre-BotC). In addition, we determined vasopressin receptor expression within the pre-BotC and the physiological effects of vasopressin on respiratory drive and arterial blood pressure when injected into the pre-BotC. Retrograde tracing with cholera toxin B subunit (CT-b) showed that a subpopulation of vasopressin-containing PVN neurons project to phrenic nuclei and the pre-BotC. The latter region, identified by expression of neurokinin-1 receptors, contained a subpopulation of neurons that were immunoreactive for the vasopressin type 1 receptor (V(1)R). Microinjection of vasopressin in the pre-BotC (0.2 nmol/200 nl) significantly increased diaphragm electromyographic activity and frequency discharge (P<0.05). In addition, vasopressin increased blood pressure and heart rate (P<0.05). These data indicate that PVN vasopressin-containing neurons innervate respiratory-related regions of the medulla oblongata and spinal cord and when vasopressin is released at these sites, it may increase respiratory drive via activation of the distinct V(1)R.

Vasopressin-Mediated Modulation of Trigeminal Reflexes in Rats

In experiments on rats under urethane anesthesia, we studied how microinjections of vasopressin into the spinal trigeminal nucleus influence jaw-opening reflexes (JOR): nociceptive JOR, induced by stimulation of the tooth pulp, and non-nociceptive JOR, evoked by stimulation of Aα fibers of the infraorbital nerve. It was shown that injection of 10 μM vasopressin resulted in a pronounced decrease (by 45-50%) in the nociceptive JOR amplitude for 25-30 min, whereas the non-nociceptive JOR dropped only by 8-10% for 11-13 min. It is concluded that vasopressin predominantly affects the nociceptive JOR; this fact is obviously related to a central analgesic effect of this peptide.

Galanin antagonizes vasopressin-stimulated flank marking in male golden hamsters

Microinjection of vasopressin (VP) into the anterior hypothalamus (AH) of golden hamsters induces a rapid bout of flank marking, a stereotyped scent marking behavior used for olfactory communication. In rats, VP is colocalized with galanin (GAL) in several brain regions. GAL has been shown to antagonize the postsynaptic actions of other cosecreted neurotransmitters including acetylcholine and norepinephrine; however, the ability of GAL to modulate the postsynaptic actions of VP has not been assessed. Here, we report that coadministration of GAL can block VP-induced flank marking in golden hamsters in a dose dependent manner. These findings provide the first evidence in any species that GAL can antagonize the central actions of VP. Using slice binding and receptor autoradiography, we have identified GAL binding sites in the AH and two other regions implicated in flank marking behavior (the lateral septum and central grey). These findings raise the possibility that endogenous GAL may function as an inhibitory modulator of this stereotypic scent marking behavior.

Modified cardiovascular sensitivity of the area postrema to vasopressin in spontaneously hypertensive rats

Vasopressin has been shown to act at the area postrema to increase the sensitivity of the baroreceptor reflex. We have previously demonstrated that microinjection of vasopressin into the area postrema of Sprague-Dawley rats elicits pressor effects. We report here that vasopressin microinjection into the area postrema of spontaneously hypertensive rats is without effect on blood pressure, whereas microinjection into age-matched Wistar-Kyoto controls results in significant increases in blood pressure at 50 and 100 pg. These results suggest an altered sensitivity of the area postrema to vasopressin in this genetic model of hypertension.

Cardiovascular consequences of microinjection of vasopressin and angiotensin II in the area postrema

Microinjection of angiotensin II (ANG II) into the area postrema (AP) of urethan-anesthetized male Sprague-Dawley rats elicited statistically significant increases in mean arterial blood pressure at doses ranging from 10 pg to 500 ng (10 pg, mean +/- SE, 10.8 +/- 1.1 mmHg, P < 0.001; 250 ng, 15.2 +/- 2.6 mmHg, P < 0.001). Heart rate was also significantly increased at doses > 10 pg, although these increases were not dose dependent. Systemic administration of losartan (Dup-753), an AT1 antagonist, was able to significantly reduce the pressor response to 250 ng ANG (post-losartan: 81.9 +/- 9.5% reduction in blood pressure response, P < 0.0001), whereas PD123319, an AT2 antagonist, was without significant effect (P > 0.1). Microinjection of vasopressin (VP) (10 pg-500 ng) into the AP also resulted in statistically significant increases in blood pressure at doses ranging from 10 to 100 pg (10 pg, 7.0 +/- 1.5 mmHg, P < 0.05) and 100-500 ng (250 ng, 12.2 +/- 1.8 mmHg, P < 0.0001). Small but significant changes in heart rate were observed only at 100 pg and 100 ng. Systemic administration of a V1 antagonist significantly attenuated the increases in blood pressure in response to 50, 100, and 250 ng VP (250 ng, post-V1 antagonist: 66.4 +/- 8.6% reduction in blood pressure response, P < 0.001), whereas [desamino,D-Arg8]vasopressin (DDAVP), a V2 agonist, had a depressor effect when microinjected directly into the AP (250 ng, -9.9 +/- 1.6 mmHg, P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of microinjection of vasopressin in dorsal pontine reticular structures on the gain of vestibulospinal reflexes in decerebrate cats.

1. The possibility that vasopressin (VP) acts on the dorsal pontine reticular formation (pRF) and the related medullary inhibitory reticulospinal (RS) system to control posture as well as the vestibulospinal reflexes has been investigated by injecting small doses of VP in precollicular decerebrate cats. 2. Unilateral microinjection of VP (0.25 microliters at the concentration of 10(-11) micrograms/microliters saline) in the pRF decreased the extensor rigidity in the ipsilateral limbs, while that of the contralateral limbs either decreased or increased. The same injection also produced a moderate or a prominent increase in gain of the multiunit EMG responses of the ipsilateral triceps brachii to roll tilt of the animal (t-test, P less than 0.001 for either group of responses). In the first instance the response gain of the contralateral triceps brachii to animal tilt slightly increased, while the pattern of response remained always of the alpha-type, as shown for the ipsilateral responses (increased EMG activity during ipsilateral tilt and decreased activity during contralateral tilt). In the second instance, however, the response gain showed only slight changes, while the pattern of responses reversed from the alpha- to the beta-type. These findings occurred 5-20 min after the injection, fully developed within 30-60 min and disappeared in about 2-3 hours. 3. The structures responsible for the postural and reflex changes described above were located in the dorsal pontine tegmental region immediately ventral to the LC, and included the peri-LC alpha and the surrounding dorsal pRF. The induced effects depended upon the injected neuropeptide, since previous injection of an equal volume of saline stained by the pontamine sky blue dye into the same dorsal pontine area was ineffective. 4. We postulated that VP exerts an excitatory influence on ipsilateral dorsal pRF neurons. The increased discharge of these neurons and the related medullary inhibitory RS neurons would lead to a decreased postural activity in the ipsilateral limbs. However, since these inhibitory RS neurons fire out of phase with respect to the excitatory vestibulospinal neurons, it appears that the higher the firing rate of the RS neurons in the animal at rest, the greater the disinhibition that affects the limb extensor motoneurons during ipsilateral tilt. These motoneurons would then respond more efficiently to the same excitatory volleys elicited by given parameters of stimulation, thus leading to an increased gain of the EMG responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Microinjection of arginine-vasopressin into the periaqueductal gray stimulates flank marking in Syrian hamsters ( Mesocricetus auratus)

Syrian hamsters can communicate using a distinctive form of scent marking called flank marking. Vasopressin-sensitive neurons within the medial preoptic-anterior hypothalamic continuum (MPOA-AH) play a critical role in the control of this form of olfactory communication. Extrahypothalamic regions may also mediate hamster flank marking. Since the MPOA-AH and the periaqueductal gray (PAG) are reciprocally connected, the present study investigated whether PAG neurons are involved n the control of flank marking. The first study found that microinjection of vasopressin, but not oxytocin or saline, into the PAG induced high levels of flank marking in male ( n = 8) and female ( n = 5) hamsters ( P < 0.01). The second study demonstrated that microinjection of vasopressin into the PAG stimulated flank marking in a dose-dependent manner in both male ( n = 7) and female ( n = 11) hamsters ( P < 0.01). These data suggest that vasopressin-responsive neurons within the periaqueductal gray participate in the control of hamster flank marking.

Vasopressin in the septal area of the golden hamster controls scent marking and grooming

Microinjection of arginine vasopressin into the lateral septum and bed nucleus of the stria terminalis of male hamsters stimulates intense flank marking and flank gland grooming, while microinjections of vasopressin in sites immediately adjacent to these areas or in the lateral ventricle are ineffective. Microinjections of oxytocin, angiotensin II and the behaviorally active C-terminal fragment of vasopressin, metabolite neuropeptide, by comparison, do not stimulate flank marking. Effective sites for vasopressin injection are clearly superimposable upon autoradiographically defined sites of high V1-receptor density. Furthermore, vasopressin-sensitive neurons in the lateral septum and bed nucleus of the stria terminalis are necessary for the expression of naturally elicited flank marking since the microinjection of a V1-receptor antagonist into these sites was able to temporarily block flank marking triggered by odors from conspecifics.

In vivo conversion of vasopressin after microinjection into limbic brain areas of rats

The nonapeptide [Arg 8]vasopressin was rapidly degraded with a half-life of lower than 1 minute after local administration into the hippocampus. During the conversion of vasopressin C-terminal fragments were transiently generated. The profile of these metabolites indicated that they were formed by aminopeptidase activity. The aminopeptidase inhibitor amastatin partially inhibited the conversion of vasopressin. A minor pathway involved cleavages in the C-terminus. The results indicate a predominant involvement of aminopeptidase activity in the in vivo metabolism of exogenous vasopressin in the brain. Since products of this metabolic route have been shown to have potent behavioral activities, the behavioral effects seen after microinjection of vasopressin in the brain may be partially due to generation of vasopressin fragments.

Role of Central Vasopressin in Cardiovascular Regulation

Using techniques of intracerebroventricular administration of vasopressin, microinjection of vasopressin into specific brain nuclei, electrical stimulation, and ablation of specific nuclei, we found that vasopressin, through an effect on central neural structures, increases mean arterial pressure and heart rate via an increase in sympathetic outflow and that deoxycorticosterone acetate (DOCA)-salt hypertensive rats show increased sensitivity and responsiveness to the central effects of vasopressin. Furthermore, we found that an important central target area for the cardiovascular effects of vasopressin is the pontine nucleus locus coeruleus. Ablation of the locus coeruleus markedly attenuates the cardiovascular effects of vasopressin as well as the development of DOCA-salt hypertension. Taken together, our results suggest a link between vasopressin, the locus coeruleus, and the sympathetic nervous system in normal cardiovascular regulation as well as in the pathogenesis of DOCA-salt hypertension. Our studies support the hypothesis that vasopressin modifies sympathetic drive through an action on central neural target areas, such as the locus coeruleus controlling sympathetic outflow. Our data further suggests that vasopressin may participate in the pathogenesis of DOCA-salt hypertension by inducing stimulation of the sympathetic nervous system at the region of the locus coeruleus in this model.

Microinjection of vasopressin into the locus coeruleus of conscious rats.

To determine whether vasopressin plays a role in central neural control of cardiovascular function by acting on the locus coeruleus we monitored arterial pressure and heart rate responses to graded injections of vasopressin (1-30 ng, 0.1-0.3 microliter) in the locus coeruleus of conscious, restrained rats. Cannulas were stereotaxically implanted in the locus coeruleus 2-5 days prior to experiment. Injections of vasopressin into the locus coeruleus produced dose-related increases in mean arterial pressure (12 +/- 2 to 57 +/- 6 mmHg) and heart rate (27 +/- 6 to 123 +/- 16 beats/min), which lasted over 1 h at the highest dose. Injection of the antipressor vasopressin antagonist d(CH2)5Tyr(Me) arginine vasopressin (10 ng) into the locus coeruleus blocked the cardiovascular responses to vasopressin. Administration of vasopressin into an area lateral to the locus coeruleus had no effect on mean arterial pressure but produced an increase in heart rate. Equivalent doses of saline, angiotensin II, and norepinephrine (NE) had minimal or opposite (NE) effects on arterial pressure and heart rate. Peripheral alpha-adrenergic blockade with phentolamine and beta-adrenergic blockade with propranolol blocked the cardiovascular responses to injection of vasopressin in the locus coeruleus. These results suggest that vasopressin may act in the region of the locus coeruleus to exert a central action on the cardiovascular system that is mediated by a stimulation of sympathetic outflow.

Microinjection of vasopressin and two related peptides into the amygdala: enhancing effect on local dopamine neurotransmission

The effects of local microinjection of Arg 8-vasopression, cyclo[Lys-Gly] and l-Pro- l-Leu-GlyNH 2 (PLG) were studied on the α-MPT-induced disappearance of dopamine and noradrenaline in the amygdala and in a number of other brain regions. A dose-dependent increase in dopamine utilization was found in the amygdala after local microinjection of Arg 8-vasopressin, cyclo[Lys-Gly] and PLG at doses of 0.1, 2.7 and 18 pmol respectively. No effects were found on noradrenaline utilization in this brain region. Using a microdissection method, it was found that cyclo[Lys-Gly] enhanced dopamine utilization in the nucl. amygdaloideus centralis, whereas the effect of PLG was mainly located in the nucl. amgydaloideus corticalis. These effects of Arg 8-vasopressin, cyclo[Lys-Gly] and PLG on dopamine utilization in the amygdala are correlated with those on avoidance behavior and can be interpreted as in support of the role of dopamine as neurotransmitter involved in retrieval processes.