Ranbir K. Bhatnagar

Periventricular noradrenergic systems are critical for angiotensin-induced drinking and blood pressure responses

Deficits in experimentally induced drinking and pressor responses after administration of 6-hydroxydopamine (6-OHDA) into the lateral cerebral ventricle (LCV) have been reported. Questions have arisen, however, as to whether these effects are due to non-specific actions of the neurotoxin and, if specific, whether the depletion of dopamine (DA) or of norepinephrine (NE) is the critical factor for producing the impairments. In the present report methods to deplete brain catecholamine (CA) differentially were employed in order to test the hypothesis that central 6-OHDA injections act on brain CA substrates per se to produce behavioral and physiological response deficits to angiotensin II (ANG II) challenges. The findings support the interpretation that forebrain dopamine is essential for the mediation of sensorimotor integration involved in response to acute homeostatic stressors. In addition, the outcome identifies an important role for forebrain noradrenergic systems in the mediation of ANG II-elicited drinking and blood pressure responses.

Assessment of the effects of neonatal subcutaneous 6-hydroxydopamine on noradrenergic and dopaminergic innervation of the cerebral cortex.

Female rats, treated at birth with 6-hydroxydopamine (3 x 100 mg/kg s.c. at 24 h intervals) or vehicle, were subjected at 112 days of age to unilateral electrolytic lesions of the locus coeruleus. Two weeks later regions of the telencephalon, both ipsi- and contralateral to the lesion, were simultaneously assayed for norepinephrine (NE) and dopamine (DA) content, and for tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities. In the vehicle-treated rats the lesion resulted in at least an 80% reduction of NE and DBH on the ipsilateral side, relative to the contralateral side. TOH was reduced to a similar extent only in the parietal cortex and hippocampus. In the prefrontal cortex and cingulate gyrus TOH was decreased by only 31% and 64% respectively; the remainder was interpreted to be associated with projections of the mesocortical dopamine system. From this data it was possible to calculate that the ratio of TOH to DA in dopaminergic terminals is about 10-fold greater than the ratio of TOH to NE in noradrenergic terminals. Neonatal 6-hydroxydopamine treatment resulted in practically total elimination of noradrenergic terminals throughout the telencephalon, and the locus coeruleus lesion had no additional effect. The drug treatment produced no significant change in DA content or in the TOH to DA ratio in the prefrontal cortex and cingulate gyrus, indicating complete sparing of the mesocortical DA projections.

Regional development of norepinephrine, dopamine-β-hydroxylase and tyrosine hydroxylase in the rat brain subsequent to neonatal treatment with subcutaneous 6-hydroxydopamine

Neonatal rats were injected subcutaneously with 100 mg/kg 6-hydroxydopamine (6-OHDA), or vehicle, on postnatal days 1, 2 and 3. At several times thereafter, determinations of tyrosine hydroxylase (TOH) and dopamine-beta-hydroxylase (DBH) activities, and norepinephrine (NE) concentration were made in the parietal cortex, cerebellum and pons-medulla in order to assess the extent of initial noradrenergic degeneration induced, and the rate of any ensuing regeneration. By the day following completion of the treatment (postnatal day 4), degeneration of noradrenergic terminals in the parietal cortex and cerebellum was very extensive, with NE levels and DBH activities reduced by more than 80%, and TOH activities reduced by 50%. In the parietal cortex noradrenergic degeneration remained virtually complete; and 9 and 70 days postnatal NE concentration and DBH and TOH activities were all decreased by more than 90--95%. In the cerebellum a progressive regeneration and apparent sprouting of NE fibers was observed. By postnatal day 9, NE, DBH and TOH in this tissue had all recovered to near control levels, and by day 70 these measures exceeded control levels by 95%, 115% and 50% respectively. In the pons-medulla, the initial effect of 6-OHDA on any of the measured parameters was negligible. By postnatal day 9 an increase in NE concentration was apparent, which increased further by day 70 to surpass the control level by 70%. At this same time DBH activity was increased by only 15% and TOH activity was unchanged. Separate analysis of the rostral half of the pons, which contains the locus coeruleus, revealed that on day 70 NE and DBH levels were increased much more substantially than in the whole pons-medulla, and TOH activity was also significantly elevated. This data indicates that the initial amount of degeneration induced by the 6-OHDA treatment is similar in both the parietal cortex and cerebellum, but regeneration proceeds only in the cerebellum. This suggests that noradrenergic fiber growth and regeneration in each target tissue is under independent regulation, possibly by the individual target neurons themselves.

Characteristics of [3H]hemicholinium-3 binding to rat striatal membranes: evidence for negative cooperative site-site interactions.

Abstract: The characteristics of [3H]hemicholinium‐3 ([3H]HC‐3) interactions with rat striatal membranes were investigated. Under the described assay conditions, [3H]‐HC‐3 binds with a saturable population of membrane binding sites having the following regional distribution: striatum « hippocampus ≧ cerebral cortex > cerebellum. The specific binding of [3H]HC‐3 showed an obligatory requirement for NaCl; other halide salts of sodium or KCl failed to substitute for NaCl. The Scatchard transformation of saturation isotherm data generated a curvilinear plot with high‐and low‐affinity components of binding. The dissociation of [3H]HC‐3 at infinite dilution was also multiexponential. The dissociation could, however, be accelerated if unla‐beled HC‐3 was included in the diluting buffer, and this increase in dissociation appeared to be dependent on the concentrations of unlabeled HC‐3 used, with the maximal increase demonstrable at 100 nM The dissociation was also dependent on the fractional saturation of binding sites with labeled HC‐3, such that, at higher fractional saturation of binding sites, the overall dissociation was faster and the difference in the dissociation observed between „dilution only” and „dilution + unlabeled HC‐3” was reduced. This occupancy‐dependent change in dissociation could also be influenced by temperature and pH. Based on the results of these kinetic studies, the steady‐state [3H]HC‐3 binding data were analyzed for a homogeneous population of binding sites undergoing site‐site interactions of the negative cooperative type. Such an analysis yielded a KD of 9.3 nM for the high‐affinity state and a KD of 22.8 nM for the low‐affinity state of binding sites, with a Bmax of 434 fmol/ mg of protein. Competitive binding studies showed that unlabeled HC‐3 was most potent in displacing [3H]HC‐3, followed by choline. Other drugs known to have little influence on the synaptosomal sodium‐dependent high‐affinity choline uptake system (SDHACU) had no significant effect on [3H]HC‐3 binding sites. Similarities in ionic dependencies, regional distributions, and pharmacological selectivi‐ties of [3H]HC‐3 binding with synaptosomal SDHACU suggest that [3H]HC‐3 selectively labels SDHACU sites located on presynaptic cholinergic neurons in rat CNS. We suggest that the two affinity states of [3H]HC‐3 binding sjtes represent the different „functional” states of the SDHACU system. The binding of HC‐3 (or choline) with the high‐affinity state of the binding sites induces negative cooperative site‐site interactions among the binding sites, resulting in the formation of a low‐affinity binding state. Because the affinities of HC‐3 and choline for this low‐affinity state of the [3H]HC‐3 binding sites correspond to the affinities of these agents for the SDHACU system, we also suggest that the low‐affinity binding state represents the „functional” form for SDHACU.

Ca2+-Dependent, ATP-Induced Conversion of the [3H]Hemicholinium-3 Binding Sites from High- to Low-Affinity States in Rat Striatum: Effect of Protein Kinase Inhibitors on This Affinity Conversion and Synaptosomal Choline Transport

Abstract: Tritium‐labeled hemicholinium‐3 ([3H]HC‐3) was used to characterize the sodium‐dependent high‐affinity choline carrier sites in rat striatal preparations. In an earlier study, we had shown that [3H]HC‐3 labels choline carrier sites with high and low affinities and had suggested that the low‐affinity sites represent “functional” carrier sites. The objective of the present study was to examine the mechanisms involved in the regulation of the two affinity states of [3H]HC‐3 binding. Here, we demonstrate that these two affinity states are totally interconvertible; addition of 0.1 mM ATP in the binding assay medium quantitatively converted all the binding sites to the low‐affinity state, whereas addition of 1 mM β,γ‐methylene 5′‐ATP quantitatively converted all the binding sites to the high‐affinity state. Preincubation of the tissue (for 15 min at 37°C) before the binding assay also converted the binding sites to the high‐affinity state, whereas supplementation of the assay medium with ATP (0.5 mM) again induced expression of the low‐affinity state of the binding sites. This effect of ATP was found to be selective for this nucleotide. Neither ADP (1 mM) nor cyclic AMP could mimic such an effect. Other nucleotide triphosphates–CTP (0.5 mM) and GTP (0.5 mM)–also could not substitute for ATP. GTP, however, caused nearly a 35% reduction in the number of binding sites, accompanying a loss of the low‐affinity component of binding. This effect of GTP was also shared by 5′‐guanylyl‐imidodiphosphate but not by GDP or cyclic GMP. This ATPdependent low‐affinity conversion of [3H]HC‐3 binding sites requires divalent metal ions. Tissue treated with EDTA did not elicit the ATP effect; however, the sensitivity to ATP could be restored fully by supplementation with Ca2+ and Mg2+. Neither a Ca2+/phospholipid‐dependent protein kinase inhibitor (staurosporin) nor Ca2+/calmodulin‐dependent protein kinase inhibitors (trifluoperazine and W‐7) blocked the Ca2+‐dependent ATP‐induced low‐affinity conversion of [3H]HC‐3 binding sites. The low‐affinity conversion could, however, be blocked by gossypol, an agent shown previously to be an inhibitor of certain calcium‐dependent protein kinases. In a similar concentration range, gossypol also inhibited synaptosomal [14C]choline uptake. Staurosporin was inactive as a choline uptake inhibitor, whereas calmodulin antagonists potently inhibited choline uptake with the rank order of calmidazolium > pimozide > trifluoperazine > W‐7 > W‐13 > W‐5 > W‐12. We suggest that the Ca2+‐dependent ATP‐mediated modification of choline carrier sites is important in the activation of the choline carrier. The sensitivity of the synaptosomal choline uptake system to calmodulin inhibitors and the relative insensitivity of [3H]HC‐3 binding parameters to these agents suggest that calmodulin‐dependent regulation of choline uptake is mediated through a site other than [3H]HC‐3 recognition sites or through a certain modification of the carrier sites that is not reflected in the binding site parameters tested.

Regenerative critical periods for locus coeruleus in post- natal rat pups following intracisternal 6-hydroxydopamine: a model of noradrenergic development

Rat pups were injected intracisternally with 20, 40 or 80 microgram of 6-hydroxydopamine (6-OHDA) at various ages over the first 12 postnatal days in order to determine the critical period of the noradrenergic regenerative-sprouting response in the cerebellum. Twenty-four hours after the treatment NE fibers in the cerebellum had become extensively degenerated. NE levels were reduced by greater than 90-95% and histofluorescence microscopy revealed an absence of innervation except for lesioned axon stumps in the basal white matter and peduncles. The 80 microgram dose produced considerable cellular degeneration in the locus coeruleus and no regenerative growth was seen to follow this treatment. Following the two lower doses, however, regenerative growth did occur. This was maximal in those rats treated closest to birth and declined progressively to become insignificant in rats which were treated on postnatal days 5-12, depending upon the cerebellar subregion. This decline in regenerative potential paralleled the time course for development of NE levels in control cerebella. For this reason the mechanism(s) controlling noradrenergic developmental and regenerative growth in the cerebellum appear to be similar. Such regenerative growth may thus be a useful model for the study of developmental growth of locus coeruleus axons. Contrary to the cerebellar projection, regenerative growth of the forebrain noradrenergic projection was not detected until the rats were between 7 and 12 days old at the time of treatment. This regeneration in the cerebral cortex was preceded by incomplete initial destruction of NE fibers there, in apparent similarity to regenerative growth described to occur in the adult rat forebrain.

Critical periods for noradrenergic regeneration in rat brain regions following neonatal subcutaneous 6-hydroxydopamine

Abstract The critical period for 60HDA-induced noradrenergic hypertrophy was assessed in several subregions of the cerebellum by treating rat pups at various ages over the first postnatal week. Noradrenergic recovery was greatest closest to birth and declined gradually over this period. Within the cerebellum recovery appeared to decline most rapidly in the middle region. In contrast, in the cerebral cortex noradrenergic fibers apparently increased their resistance and survivability over the first postnatal week. The results are discussed in terms of a hypothesized target-elicited retrograde control of locus coeruleus growth.

Methylpiperidine analog of hemicholinium-3: A selective, high affinity non-competitive inhibitor of sodium dependent choline uptake system

The potency of hemicholinium-3 (HC-3) and its analogs to inhibit sodium dependent high affinity choline uptake were evaluated in rat striatal synaptosomal preparation. Hemicholinium-3 inhibited sodium dependent high affinity choline uptake (IC50 = 18 nM) while the half molecule of HC-3, HC-15, was inactive. The order of potency for choline uptake inhibition of piperidine substituted HC-3 molecule was as follows: 4-methylpiperidine (A-5 and CA-5) much greater than HC-3 much greater than unsubstituted piperidines (CA-1 and A-1) much greater than 2- or 3-methylpiperidine (A-2 and A-3) and 4-hydroxypiperidine (A-7). The tertiary amine derivative of 4-methylpiperidine substituted HC-3 (A-4) was nearly 10-fold less potent than its corresponding quaternary derivative (A-5). Choline uptake was inhibited competitively by HC-3 and non-competitively by A-5. The inhibition of choline uptake by A-5 was readily reversible by washing. A-5 did not inhibit the uptake of dopamine and gamma-aminobutyric acid. These findings suggest that the N-methyl,4-methylpiperidine analog of HC-3 (A-5) is the most potent of all known inhibitors of sodium dependent high affinity choline uptake and that the inhibition of choline uptake by this compound is mediated through a mechanism distinct from a simple competitive one.

Distribution of hypertrophied locus coeruleus projection to adult cerebellum after neonatal 6-hydroxydopamine

Following treatment as neonates with a high subcutaneous dose of 6-hydroxydopamine (6-OHDA), the projections of the locus coeruleus were mapped in the brains of adult rats. This was done using the technique of unilateral lesions in the nucleus followed by simultaneous determinations of norepinephrine (NE) levels, dopamine-beta-hydroxylase (DBH) activity and synaptosomal [3H]NE uptake in various terminal areas. In particular the cerebellum was subdivided into 3 areas in order to assess any changes from normal in the distribution of the hypertrophied noradrenergic projections here. In vehicle-control rats the lesions resulted in an 80--85% loss of NE in the parietal cortex ipsilateral to the lesion and a 15--20% loss contralaterally. In the control cerebellum the locus coeruleus projection, based upon changes in all 3 markers, is distributed 2/3 ipsilaterally and 1/3 contralaterally with the same pattern present in all 3 subregions. The neonatal 6-OHDA treatment resulted in virtually complete loss of noradrenergic terminals in the cerebral cortex. Following neonatal 6-OHDA treatment cerebellar levels of NE, DBH and [3H]NE uptake increased by between 20--60%, with the smallest increases occurring in [3H]NE uptake. In these rats the locus coeruleus accounted for at least 75--80% of the cerebellar noradrenergic parameters. Unlike control rats however, the lesions in these rats produced only ipsilateral decreases in NE and DBH. On the other hand changes in [3H]NE uptake indicated a normal 2/3 ipsilateral, 1/3 contralateral pattern. It is suggested that two separable events occur in the noradrenergic projection to the cerebellum. The first is the regeneration of an increased number of nerve terminals, or sprouting, and the second is a build-up of synaptic vesicles, or collateral accumulation. The sprouting, judging from the [3H]NE uptake data, occurs with apparently normal distribution, but the accumulation of NE and DBH is confined predominantly to the ipsilaterally projecting axon terminals. This may be the consequence of collateral accumulation resulting from the degeneration of the largely ipsilateral coeruleocortical projection.

Dopaminergic nature of amphetamine-induced pecking in pigeons.

d-Amphetamine was found to induce a pecking response in pigeons. The pecking response induced by d-amphetamine was antagonized by chlorpromazine, haloperidol or bulbocapnine indicating that this pecking response was caused by dopaminergic receptor stimulation. Pretreatment of pigeons with alpha-methyltyrosine (alpha-MT) reduced d-amphetamine-induced pecking, while the combined treatment of pigeons with alpha-MT and L-dihydroxyphenylalanine (L-DOPA, 100 mg/kg) partially restored the pecking response. d-Amphetamine-induced pecking was not reduced by a dopamine-beta-hydroxylase inhibitor, 1-phenyl-3-(2-thiazolyl)-2-thiourea (U-14,624). Alpha-MT reduced brain dopamine but not norepinephrine level, whereas U-14,624 decreased brain norepinephrine but not dopamine. Thus there is a correlation between brain dopamine level and d-amphetamine-induced pecking response. It is concluded that d-amphetamine-induced pecking is mediated indirectly by the release of dopamine.