W. B. Quay

Circadian and ultradian changes in synaptic vesicle numbers in nerve endings on adrenomedullary noradrenaline cells, and their modifications by pinealectomy and sham operations.

Nerve endings (n = 1,298) on noradrenaline cells in adrenal medullas of male rats (n =117) were investigated by quantitative electron microscopy, with sampling at 8 times during a standardized light:d

Daily Rhythmic Changes in Synaptic Vesicle Contents of Nerve Endings on Adrenomedullary Adrenaline Cells, and Their Modification by Pinealectomy and Sham Operations

Nerve endings on adrenaline cells in male rat adrenal medullas were investigated by quantitative electron microscopy, with sampling at 8 times during a standard light: dark (12:12) photoperiod. Number (N) per unit area of 2 vesicle types (small clear vesicle, SCV; large granular vesicle, LGV) and % of LGV per total vesicle N were determined. Normal (non-operated) animals showed a circadian rhythm (p less than 0.005) in mean SCVN and one with nearly opposite phase relations in % LGVN. Doubly sham-operated animals had a similar rhythm in %LGVN but diminished circadian change in SCVN. Pinealectomized animals had a circadian pattern in SCVN, but this differed from that of normals in having a 2nd peak, a delay or phase shift of the primary peak, and an increased amplitude of the rhythmic changes.

Quantitative Cytological Analysis of Functional Changes in Adrenomedullary Chromaffin Cells in Normal, Sham-Operated, and Pinealectomized Rats in Relation to Time of Day: III. Nuclear Density

Adrenaline (A)‐ and noradrenaline (N)‐cells in juxtacortical and central areas of adrenal medullas of nonoperated, sham‐operated, and pinealectomized male rats (n = 125) were investigated by quantitative electron and light microscopy. Animals were killed at eight time points during a standardized 24‐h, light‐dark (12:12) cycle 14 d after surgery. Diameters of nucleoli (n = 9,600) and the nucleolar margination rate were studied chiefly by light microscopy.

Quantitative Ultrastructural Analysis of Differences in Exocytosis Number in Adrenomedullary Adrenaline Cells of Golden Hamsters Related to Time of Day, Pinealectomy, and Intracellular Region

This research analyzed differences mainly in the incidence of exocytotic figures in adrenaline cells (A ‐ cells) in pinealectomized (PX), sham‐operated (SPX), and non‐operated (NO) adult male golden hamsters, with the aim of determining whether these parameters change with the time of day and following pinealectomy, and whether intracellular regional differences exist in such changes. Animals acclimated to a standardized light: dark (LD) 12:12 photoperiod were sacrificed at 11 h after the onset of light (L‐11h) and 1 h after the onset of darkness (D‐1h) (8 animals/group/time) at 28 days postoperation. The adrenal medullas were examined and analyzed morphometrically by electron microscopy. The number of exocytoses per unit length (NEL) and the exocytosis index (a rough index of the number of exocytoses per cell) were measured in PF (perivascular‐space‐facing) and non‐PF plasma membranes. NEL increased from L‐11h (NO: 0.040 ± 0.010, X̄± SE) to D‐1h (0.078 ± 0.012) in all three experimental groups (ANOVA: P < 0.005), showing over fourfold higher levels in PF than in non‐PF membranes. NEL in PF membranes in PX animals showed higher levels than those in NO and SPX animals (P < 0.025), but in non‐PF membranes, no differences owing to time of day or surgery were seen. Exocytosis indices were (1) higher at D‐lh than at L‐llh in all three experimental groups (P < 0.005), (2) similar in PF and non‐PF membranes in control groups, and (3) higher in PF membranes in the PX group than in either non‐PF membranes or PF membranes in control groups. In conclusion, the exocytosis number in A cells changes in relation to time of day, rising in early dark phase, and its rise following pinealectomy can be seen only in PF membranes.

Hypothalamic dopamine-β-hydroxylase activity: Fluctuations with time of day and their modifications by intracranial surgery, adrenalectomy, and pinealectomy

Hypothalamic and plasma dopamine-β-hydroxylase (DBH, EC 1.14.2.1) activities were measured by a coupled radioenzymatic method. Animals representing five experimental groups (intact controls, adrenalectomized, pinealectomized, adrenalectomized + pinealectomized, doubly sham-operated) were killed and sampled at 8 times through the 24-hr daily cycle, 15 days postoperation, and at 50–52 days of age. Hypothalamic DBH in intact control animals had statistically significant fluctuations in relation to time of day. These changes were lost or dampened in groups that had had intracranial surgery and were characteristically shifted by adrenalectomy, either alone or with pinealectomy. Plasma DBH fluctuations in the same animals resembled those in hypothalamus in some features (e.g., peak near mid-dark; shift in daily maxima and minima after adrenalectomy) and differed in others (e.g., no effect of intracranial surgery or of sham operation; adrenalectomized + pinealectomized animals resembled the solely pinealectomized). Although temporal patterns in hypothalamic DBH activity thus differed in the experimental animal groups, the daily means of hypothalamic DBH activity were similar.

Effects of Melatonin on Adrenomedullary Dopamine-β-Hydroxylase Activity in Golden Hamsters: Evidence for Pineal and Dose Dependencies

Pineal influence in the control of adrenomedullary function in golden hamsters was investigated by examining changes in adrenal dopamine‐β‐hydroxylase (DBH) activity following pinealectomy, either alone or in combination with melatonin administration. Adult males acclimated to an LD 14:10 photoperiod were distributed in five experimental groups: intact controls (NO), sham‐pinealectomized (S), sham‐pinealectomized with black plastic shielding of the pineal region, pinealectomized (PX), and pinealectomized with the operated region shielded. Animals representing all of these groups were injected (between L11 and L11.75) with either vehicle, or a low dose (25 μg) or a high dose (2,500 μg) of melatonin daily for 28 days, after which they were killed, and the adrenals were collected for assay of DBH activity by means of a sensitive radioenzymatic method. We found that (1) PX + vehicle led to increased (P <.05) adrenal DBH activity in comparison with either NO or S groups; (2) daily 25 μg of melatonin resulted in lowered DBH activity in the NO group when compared with NO + vehicle (P <.001) or S + vehicle (P <.001) groups; (3) PX + 25 μ melatonin reversed the action of 25 μg melatonin in the NO + 25 μg group; (4) 2,500 μg melatonin was without effect on adrenal DBH in any of the injected surgical groups. These results show an inhibitory pineal influence on adrenal DBH activity, and that this was dose dependent. Furthermore, the inhibitory effect of exogenous melatonin on adrenomedullary DBH activity depended upon the presence of the pineal, suggesting a mediating role of the pineal in this particular action of melatonin. Since DBH catalyzes dopamine conversion to norepinephrine, a hormone and hormone precursor of the adrenal medulla, these results demonstrate, within at least one time frame, an inhibitory action of the pineal and melatonin on adrenomedullary catecholaminergic function.

The invagination complex in nerve endings on adrenomedullary adrenaline cells: quantitative ultrastructural description, and analysis of changes with time-of-day and their modification by sham-surgery and pinealectomy.

This work describes a special ultrastructural feature, the invagination complex (IC), involving the plasma membrane of nerve endings on adrenomedullary adrenaline cells. Quantitative characteristics of the IC and their changes were studied in 122 male albino (Holtzman strain) rats in 3 surgical groups: normal (non-operated, NO) sham-operated (SO) and pinealectomized (PX), all maintained in a standardized daily photoperiod (light : dark 12 : 12 h). Animals were decapitated 14 days postsurgery and at 8 specific times during the light : dark cycle. Left adrenal glands were removed, dn strain) rats in 3 surgical groups: normal (non-operated, NO) sham-operated (SO) and pinealectomized (PX), all maintained in a standardized daily photoperiod (light : dark 12 : 12 h). Animals were decapitated 14 days postsurgery and at 8 specific times during the light : dark cycle. Left adrenal glands were removed, dn strain) rats in 3 surgical groups: normal (non-operated, NO) sham-operated (SO) and pinealectomized (PX), all maintained in a standardized daily photoperiod (light : dark 12 : 12 h). Animals were decapitated 14 days postsurgery and at 8 specific times during the light : dark cycle. Left adrenal glands were removed, dissected and prepared for electron microscopy. In section profiles the diameter of each IC was usually 0.12-0.40 micrometers, and the depth 0.2-1.0 micrometers. They were frequently seen to be located near the synaptic complex (or the active zone). Coated pits, about 50 nm wide and 60 nm deep, often opened near the bottom of the invaginations of the IC. In NO animals, relative number and depth of the ICs showed daily rhythmic changes with minimal values about 1 h after onset and maximal (acrophase) values 3--5 h later (P less than 0.02 to less than 0.005, depending on index or measure). These changes occurred 3--5 h earlier, but less apparently, in SO animals, and appeared to be more greatly modified and dampened in PX animals. Two-way analysis of variance (ANOVA) and t-tests applied to 11 kinds of indices derived from counts and measurements of the ICs, support the differences between surgical groups, at least in many instances (P less than 0.05 to less than 0.001). It is concluded that the IC is a characteristic and dynamic feature of the nerve terminals and that it may possibly have a role in such phenomena as recycling of synaptic vesicles or related membrane constituents. It is also concluded that significant time-of-day and neuroendocrine effects are demonstrable in these structures, and that the time-of-day effects shown in these chronic studies have importance in the design of acute experiments designed to further test the functional relations and importance of ICs.

Effects of Pinealectomy on the Mitotic Activity of Adrenomedullary Chromaffin Cells in Relation to Time of Day

The frequency of mitoses of adrenaline (A) cells and noradrenaline (N) cells in the adrenal medulla of nonoperated (NO), sham‐operated (SPX), and pinealectomized (PX) male, 53‐day‐old Holtzman rats (n = 133) was investigated by means of light microscopy. Animals were killed at eight time points during a standardized 24‐h light‐dark (12:12) cycle 14 days after surgery. Mitotic indices (n/1,000) were determined in sections of adrenal medulla fixed with glutaraldehyde and OsO4. Overall frequency of mitoses was extremely low (mitotic index: 0.73 = 115/157,223). Daily mean mitotic index was maximum in A cells (0.83) and minimum in N cells (0.52) of PX group but did not show statistically significant differences between cell types or experimental groups. Neither cell type in NO animals showed 24‐h changes in mitotic index, but cells in SPX animals did, with highest value in the late dark phase and lowest in the late light phase, when values of two cell types were combined (P<0.01–0.001). In PX animals, mitotic index followed a similar but more distinct 24‐h change in A cells (P<0.009), but not in N cells, resulting in different time‐of‐day changes between two types of cells (P<0.01–0.05). The mitotic index was higher in PX than in control (NO and SPX) animals in the middark phase (P<0.05) and lower in operated (SPX and PX) than in nonoperated (NO) animals from late light to the early dark phase, suggesting that the latter was possibly due to a residual effect of the surgery. These results are consistent with the interpretation that the pineal has an inhibitory action on A cells and may coordinate the two types of cells in their mitotic activity, especially in the middark phase of the daily cycle.

Quantitative Cytological Analysis of Functional Changes in Adrenomedullary Chromaffin Cells in Normal, Sham‐Operated, and Pinealectomized Rats in Relation to Time‐of‐day: II. Nuclear‐Cytoplasmic Ratio, Nuclear Size, and Pars Granulosa of Nucleolus

Adrenaline(A)‐ and noradrenaline(N)‐cells in the adrenal medulla of nonoperated (NO), sham‐operated (SO), and pinealectomized (PX) male rats (n = 125) were investigated by quantitative electron and light microscopy. Animals were killed at eight time points during a standardized 24‐h, light‐dark (12:12) cycle 14 days after surgery. Nuclear‐cytoplasmic (N/P) ratios, diameters of nuclei, and the frequency of nucleoli showing a large amount of pars granulosa (granulated nucleoli), were the primary characteristics studied.

Elevation of γ-Aminobutyric Acid in Cultured Rat C6 Glioma Cells Following Methionine Supplementation

Methionine is recognized as the most toxic amino acid in various mammalian species investigated (Harper et al., 1970). Serum, liver, and brain levels of glycine and serine are antagonized by excessive methionine intake (Klavins, 1965; Daniel and Waisman, 1969). Both the behavioral and metabolic toxicities of methionine are ameliorated by coadministration of glycine or serine (Benevenga and Harper, 1967; 1970; Girard-Globa et al., 1972; Beaton, 1975). These effects and the metabolic interrelations of methionine are of special interest in the investigation of the neurochemistry of schizophrenia and several other brain disorders. Schizophrenics have been reported to be deficient in two enzymes of one-carbon metabolism: serine hydroxymethyltransferase (L-serine-tetrahydrofolate 5,10-hydroxymethyltransferase, EC 2.1.2.1), and methionine adenosyltransferase (ATP: L-methionine Sadenosyltransferase, EC 2.5.1.6.) (Carl et al., 1978). It appears possible that the unique behavioral reaction of schizophrenics to methionine loads may be related to the diminished levels of these enzymes in their blood cells (Cohen et al., 1974). In relation to our interest in the above problem and basic neurometabolic questions pertaining to it, we have studied changes in amino acid concentrations in cultured rat C6 glioma cells following incubation in media supplemented with methionine, glycine, and serine. We found that intracellular levels of glycine and serine were unaltered by incubation for 6 days in either 0.4 or 1.2 mwmethionine. Changes in most of the remaining amino acids paralleled more closely those changes previously reported for brain than for liver or serum. y-Aminobutyric acid (GABA) was a distinct exception, in that its intracellular concentration doubled whenever excess methionine was present in the medium. Neither glycine nor serine was effective either in elevating intracellular GABA or in diminishing the effect of methionine on levels of GABA.