Castration rapidly decreases hypothalamic gamma-aminobutyric acidergic neuronal activity in both male and female rats.

The postcastration LH response is greater and somewhat more rapid in male than female rats. We have previously demonstrated that hypothalamic gamma-aminobutyric acid (GABA)ergic neuronal activity decreases following gonadectomy in male rats. To investigate whether these same hypothalamic GABA neurons decrease their activity postcastration in female rats, and whether more rapid and or greater postcastration decreases occur in male rats, we determined the timing and magnitude of the postcastration decreases in GABA turnover which are associated with the sexually dimorphic postcastration LH response. Adult male and 4-day cycling female rats were castrated between 0800 and 1000 h (females ovariectomized on diestrus day 1). Serum LH levels increased significantly by 12 h postcastration in both males and females with the magnitude of the increases being 6.2-fold in males and 2.8-fold in females. GABA turnover was determined in 16 microdissected brain structures by the GABA transaminase inhibition method at 0 h (sham-operated controls), 6 h, 12 h and 1, 2, 4 and 6 days postcastration. In male rats, in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], the rate of GABA turnover decreased significantly already by 6 h postcastration compared with the 0 h controls, and remained suppressed through 6 days. This rapid down regulation of DBB(ovlt) GABAergic neurons also occurred in female rats, however, the duration of the decrease was not as prolonged as in male rats. Similar changes occurred in the tuberoinfundibular GABAergic (TIGA) neurons projecting to the median eminence in both males and females. Down regulation of these GABAergic neurons precedes or is coincident with increased postcastration LH secretion in both sexes, and the duration of the decreases is consistent with the less robust postcastration LH response in female rats. In addition, the rate of GABA turnover decreased after castration in the interstitial (bed) nucleus of the stria terminalis, ventral aspect (INSTv), the medial preoptic nucleus, dorsomedial aspect (MPNdm) and the ventromedial nucleus, ventrolateral aspect (VMNvl) in male rats, and in the INSTv and VMNvl of female rats, while there was no effect of castration in other hypothalamic regions or control structures. The result in the female VMNvl is consistent with reports that GABA facilitates lordosis behavior in this hypothalamic structure. These findings are consistent with the hypothesis that discrete hypothalamic populations of sex steroid-sensitive GABAergic neurons mediate the postcastration LH responses in both male and female rats, and may underlie other sexually dimorphic adult phenotypes such as sex behavior.

Sex differences in GABA turnover and glutamic acid decarboxylase (GAD(65) and GAD(67)) mRNA in the rat hypothalamus.

GABAergic neurons are estimated to make up more than half of the neuronal population of the hypothalamus and they likely account for some of the structural and functional sexual dimorphisms observed in the mammalian brain. We previously reported sex differences in the rate of GABA turnover in discrete hypothalamic structures of adult rats. In the present study, we extended our search for sex differences in GABA turnover to additional structures, and further determined whether these differences were associated with differences in GAD(65) and or GAD(67) mRNA levels. Utilizing the GABA transaminase inhibition method, we determined GABA turnover in 14 microdissected brain regions. The rate of GABA turnover was about 2-fold greater in male than in diestrous day one (D(1)) female rats in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], anteroventral periventricular nucleus (AVPv), median eminence (ME), and dorsomedial portion of the ventromedial nucleus (VMNdm). A sex difference also was noted in the DBB(ovlt) for GAD(65) mRNA determined by microlysate RNase protection assay. Here, GAD(65) levels were almost 2-fold greater in male rats, which suggests that differences in the activity of this GAD enzyme isoform contributes to the difference in turnover in this area. Additionally, in the dorsomedial nucleus (DMN), the GAD(65) mRNA level was significantly higher in female rats, and in the medial amygdaloid nucleus (Am), GAD(67) mRNA was higher in male rats. These data reveal striking sexual dimorphisms in the rate of GABA turnover and in GAD mRNA levels in specific populations of hypothalamic GABAergic neurons. The functional relationships between these GABAergic neurons and sexually dimorphic phenotypes associated with these structures, such as gonadotropin secretion, reproductive behaviors, seizure threshold and others, warrant further investigation.

Effects of ethacrynic acid on intraocular pressure of anesthetized rats.

Ethacrynic acid (ECA) lowers intraocular pressure (i.o.p.) by an effect usually ascribed to increased drainage of aqueous humor by the trabecular meshwork. Here, we describe the effects of a continuous 2-hr intracameral infusion of balanced salt solution (BSS), with or without 2 mM ECA (sodium salt), on IOP of pentobarbital anesthetized rats. The infusion was divided into a constant (0.05 microliter/min) and a periodic (0.25 microliter/min) component that cycled 4 min on then 4 min off. This permitted the calculation of dynamic changes in resistive (trabecular and uveoslceral drainage) and nonresistive (aqueous synthesis, episcleral venous pressure) components of IOP by fitting a second-order transfer function to the responses. ECA markedly blunted the BSS-induced rise in IOP (P < 0.01). The rise in resistive mechanisms (ocular impedance) was transiently blunted by ECA (P < 0.05) during the third and fourth 8-min cycles, and nonresistive mechanisms were reduced by ECA from cycles 3-10 (P < 0.05). Then, at the end of the infusion, the control and ECA dynamic values were similar (P < 0.05), although IOP of ECA-treated rats was still slightly reduced (P < 0.05). The most likely explanation is a summation of small changes in both resistive and nonresistive components of IOP dynamics. Systemic blood pressure was unchanged within either group. The well-known effects of ECA on the trabecular meshwork, alone, are insufficient to explain the dynamic changes in IOP observed in this model.

Aqueous humor dynamics in anesthetized rats infused with intracameral apraclonidine.

We studied the acute effects of the ocular hypotensive drug, apraclonidine (AP), on intraocular pressure (IOP) and aqueous humor dynamics of anesthetized rats during infusion-induced ocular hypertension. Two infusions were made into the anterior chamber of the eye: one was constant at a rate of 0.05 microl/min, the other was cyclic, at a rate of 0.25 microl/min, with the pump on for 4 min, then off for 4 min. Data were processed by complex demodulation and analysis of a second-order transfer function. This permitted separate calculations of resistive components (Ao), i.e., trabecular meshwork and uveoscleral outflows, and residual pressure (RP) estimating nonresistive components, i.e., aqueous synthesis and episcleral venous pressure. A balanced salt solution (BSS) and AP (0. 0005%) were tested. AP markedly delayed the within-group rise in IOP: 20 min for BSS vs. 60 min for AP (p < 0.001). IOP of AP rats was less than control for 100 min (p < 0.05). The infusions raised Ao in both groups (p < 0.05). AP initially had a transient inhibitory effect (p < 0.05). Infusions raised RP in both groups. AP had a strong inhibitory effect for the first 8 cycles (p < 0.05). These data document that the acute effects of AP in this in vivo rat model of ocular hypertension were to delay increases in IOP, mainly by reducing nonresistive components of aqueous humor dynamics. Transient inhibition of resistive mechanisms also occurred.

Timolol effects on aqueous humor dynamics in eyes of anesthetized rats.

Anterior chambers of the eyes of male rats were cannulated under pentobarbital anesthesia for intracameral infusions of balanced salt solution (BSS) and intraocular pressure (IOP) recording. Blood pressure was recorded from a femoral artery. IOP was recorded during a 2-h intracameral infusion composed of a constant component (0.05 microl/min) and a periodic component (0.25 microl/min), cycling at 4 min on and then 4 min off. After a 20-min baseline period, 1 drop of timolol (0.5%) or BSS was applied to the cornea and repeated 1 h later. Intracameral infusions of BSS and 0.05% timolol were also compared. Topical timolol slightly delayed the BSS-induced IOP rise (p <.05). Complex demodulation and the estimated gain parameter of a second-order transfer function fit to the periodic responses revealed that topical timolol also reduced (p <.05) passive outflow resistance. Intracameral timolol markedly delayed the BSS-induced rise in IOP. Initially, timolol decreased both outflow impedance and nonresistive components (p <.05) of IOP, but these effects dissipated by 2 h when IOPs were similar. In all experiments, within-group blood pressure was unchanged. Topical and intracameral timolol have different effects on IOP. The data support the opinion that, in vivo, timolol acts at beta-receptors that control both outflow impedance and nonresistive mechanisms, probably vascular, to lower IOP.

Mechanisms for vasopressin effects on intraocular pressure in anesthetized rats.

Continuous intracameral infusions of a balanced salt solution (0.175 microliter min-1) have been reported to raise intraocular pressure (IOP) in anesthetized rats. Palm et al. (1995) previously reported that this effect was attenuated significantly by inclusion of arginine-vasopressin (AVP, 10 ng 0.175 microliter-1) in the infusate. This study used experimental and computer simulation methods to investigate factors underlying these changes in IOP. First, constant intracameral infusions of artificial cerebrospinal fluid (aCSF) at different fixed rates (0.049-0.35 microliter min-1) were used to estimate the outflow resistance. Secondly, IOP responses were measured during an 2 hr intracameral infusion of either aCSF or AVP that was the sum of a small constant component (0.05 microliter min-1) and a larger periodic component (0.25 microliter min-1, cycling for 4 min on, then 4 min off); the mean infusion rate was 0.175 microliter min-1. As shown previously for 0.175 microliter min-1 constant infusions, the periodic aCSF infusion induced a significant rise in IOP that was attenuated by AVP administration. Complex demodulation analysis and the estimated gain parameter of a second order transfer function fit to the periodic responses indicated that outflow resistance increased significantly during the infusions in both aCSF and AVP groups, but that the indices of resistance did not differ significantly between aCSF and AVP infused eyes. This finding implies that changes in outflow resistance do not explain the difference in IOP responses to intracameral aCSF and AVP. The two responses differed significantly, though, in damping factors, such that the aCSF responses were considerably more underdamped than the AVP responses. It is hypothesized that aCSF-induced increase in IOP reflects both (1) a small component reflecting increased outflow resistance and (2) a larger non-resistive component. Since the non-resistive component is insensitive to pretreatment with acetazolamide, it is suggested that the aCSF-induced elevation in IOP reflects primarily vascular perfusion changes that are reduced by local vasoconstrictor actions of AVP. The latter mechanism likely maintains vascular perfusion of the globe when intraocular hypertension develops.

Comparison of anterior chamber infusates on the intraocular pressure of intact rat eyes.

1. Infusions of balanced salt solutions (BSS) into the eye often cause a delayed, gradual increase in intraocular pressure or outflow facility, known as the "washout" effect. The reason(s) is occult, especially at low input rates when drainage mechanisms are not overloaded by excessive volume input. However, direct, quantitative comparisons of BSSs used in ocular research have been reported infrequently. 2. We compared the effects of three BSSs on intraocular pressure and the estimated resistance to drainage after a 1-hr, low-volume infusion into the anterior chambers of the eyes of anesthetized rats. 3. The BSSs tested raised intraocular pressure (P < 0.05) after a 20-40-min delay, and the highest IOPs occurred at 1 hr. Recovery of intraocular pressure to baseline only occurred with one BSS (Dulbecco). 4. Fitting the ascending and descending portions of the mean pressure curves to an exponential revealed differences among the infusates. The Dulbecco solution resulted in minimal changes in time constant, gain, and offset during the ascending and descending periods. 5. The data obtained show that different BSSs yield pressure curves that appear grossly similar, even though there were large differences in composition and osmolality. However, the underlying changes in ocular dynamics were not identical. Thus, it may be prudent to test more than one solvent to study "washout," or to deliver drugs directly into the anterior chamber.

Interaction between head-down tilt and anterior chamber infusions on intraocular pressure of anesthetized rats.

Head-down tilt or infusions of a balanced salt solution into the anterior chamber of the eye raise intraocular pressure. We measured intraocular pressure directly in adult male Sprague-Dawley rats, anesthetized with pentobarbital, and subjected to 45 degrees head-down tilt alone, tilt with an anterior chamber infusion (0.087 microliter min-1), or tilt with an infusion containing arginine vasopressin. The intraocular pressure of the three groups differed during the 1 hr tilt and recovery periods. In the case of tilt alone, intraocular pressure quickly reached a peak after tilting, partially decreased during the tilt period, recovered to baseline immediately after tilt, then a secondary rise occurred. Combined infusion and tilt caused a slower rise to peak intraocular pressure, and only a partial recovery occurred during the 1 hr recovery period. Combined vasopressin infusion and tilt caused a gradual rise in intraocular pressure of a lesser magnitude than the other groups, followed by a rapid recovery to baseline pressure and no secondary rise. Systemic arterial pressure was stable within and between the groups. The underlying mechanism for these differing response patterns is unknown. However, some evidence indicates that infusions are independent of aqueous synthesis rate, and that vasopressin, acting on a V1 receptor subtype reached from the anterior chamber, exerts a vascular effect.

Pentobarbital alters the response of cerebrospinal fluid pressure evoked by two provocative tests in rats.

The intracerbroventricular (ICV) route of administration is a commonly used pharmacologic procedure in rats. However, little attention is given to changes in cerebrospinal fluid pressure (CSFp) when the ventricular system is perturbed, especially in conscious animals. This communication examined CSFp in pentobarbital-anesthetized rats subjected to two pertubations where CSFp had been previously measured in conscious rats. Pentobarbital treatment resulted in marked, qualitative differences after both protocols. Whereas conscious rats responded to a 10-min ICV infusion (8 microliters/min) by a delayed, secondary increase in CSFp, this effect was completely eliminated (p < 0.001) by pentobarbital. Also, whereas conscious rats could maintain CSFp at normal values during an hour of 45 degrees head-down tilt, pentobarbital-treated rats could not. Anesthetized rats displayed an immediate fall in CSFp to near zero values (p < 0.001), followed by a rapid recovery (p < 0.001) after tilting. Such differences in CSF dynamics raise important issues. For example, what mechanism(s) underlie the changes in CSF dynamics? Also, are these differences in conscious and anesthetized rats potential variables for drug distribution when the ICV route is used?