Susan Schwartz-Giblin

Intracerebral administration of antisense oligodeoxynucleotides to GAD65 and GAD67 mRNAs modulate reproductive behavior in the female rat

Increased GABA activity in the medial hypothalamus (HYP) and midbrain central gray (MCG), but not the preoptic area (POA), facilitates sexual receptivity in the female rat [40]. In the current experiments, ovariectomized females were chronically treated with estrogen (via silastic capsules) to maintain a continuously high level of lordosis response. Administration of crystalline antisense oligodeoxynucleotide to the GABA synthetic enzyme, GAD67, into the HYP and MCG significantly and reversibly reduced lordosis response for 1-2 days, but did not inhibit lordosis when administered into the POA. Administration of a control oligonucleotide, consisting of the same nucleotide bases but in a scrambled sequence, did not significantly modulate behavior when infused into any brain areas. When oligodeoxynucleotide antisense to GAD67 was suspended in oil and then infused into the HYP or MCG it was more effective and resulted in less inter-animal variability. Subsequent experiments involving infusions into the MCG compared the effectiveness of antisense oligonucleotides to the two different forms of GAD, known as GAD65 and GAD67. Oligodeoxynucleotides antisense to the mRNA for either gene were effective at reducing lordosis behavior but with a different time course. Oligonucleotide antisense to GAD67 significantly reduced behavior within 24 h of infusion and there was full recovery by 4 days post-infusion. GAD65 antisense oligonucleotide did not significantly reduce behavior until 48 h post infusion and animals did not fully recover to pretest levels of lordosis until 5 days post-infusion. When antisense oligonucleotide for the two genes was administered simultaneously, the inhibition of lordosis was maximal at 24 h and stayed depressed for 4 days. There did not appear to be an additive effect of the two different antisense oligonucleotides when administered together. Tissue GABA levels in HYP and MCG of individual rats assayed by HPLC were no longer correlated with lordosis score after antisense oligonucleotide infusion but were after infusions of scrambled control oligos. Immunoblotting for the two forms of GAD revealed that GAD67 antisense oligonucleotide infusion led to significant decreases in both GAD67 and GAD65 protein levels as compared to infusions of scrambled control oligo. In addition, the levels of a neuronal marker, neuron-specific enolase, also decreased (although nonsignificantly) suggesting either a temporary shutdown of protein synthesis or a degeneration of GABAergic neurons after GAD67 antisense oligonucleotide infusion.

Steroid hormone effects on picrotoxin-induced seizures in female and male rats

Picrotoxin (1 mg/kg, i.p.), evoked a single generalized seizure in 75% of ovariectomized rats. Pretreatment of matched pairs with silastic implants containing 100% estradiol had an anticonvulsant effect; it protected all rats against such seizures. Implants containing 10% estradiol in cholesterol were less effective in protecting against picrotoxin-induced seizures. With 2 mg/kg picrotoxin, 85% of the seizure-affected ovariectomized controls had multiple seizures. The incidence of seizures and the ratio of single to multiple seizures induced by the higher dose of picrotoxin were unaffected by estradiol silastic implants, intraperitoneal injections of progesterone (0.5 mg, 4-5 h before convulsant) or the combination of both hormones. At the 2 mg/kg dose, 8/8 intact males had no seizures while all paired ovariectomized females had seizures. By contrast, the incidence of seizures in pairs of gonadectomized males and females did not differ. Testosterone treatment improved the ratio of single to multiple seizures in males but not in females. Males had statistically fewer multiple seizures than did females after testosterone treatment. The distribution of latencies to a single seizure is statistically different from the distribution of latencies to the first of multiple seizures irrespective of dose, sex and hormone treatment. This suggests that the population of rats responding with a single seizure at the higher dose of picrotoxin have a higher threshold for acquiring multiple seizures and that testosterone predisposes males but not females to this population.

Steroid Regulation and Sex Differences in [3H]Muscimol Binding in Hippocampus, Hypothalamus and Midbrain in Rats

The gonadal steroids estradiol and progesterone have previously been shown to modulate the specific binding of the GABAA agonist, [3H]muscimol, in the CA1 region of the hippocampus, the ventromedial nucleus of the hypothalamus and the midbrain central gray of ovariectomized female rats. In this report we show a sex difference in the level of binding in the very caudal ventromedial nucleus of the hypothalamus. In contrast to females, there is no steroid modulation of [3H]muscimol binding in the ventromedial nucleus of the hypothalamus and midbrain central gray of males. These effects may be functionally related to GABAergic control of female sexual behavior. In contrast, steroid modulation of [3H]muscimol binding in the CA1 region of the hippocampus occurred to the same degree in males and females, and there was no difference in the level of binding in any region of the hippocampus between gonadectomized males and females.

Midbrain PAG Control of Female Reproductive Behavior: In Vitro Electrophysiological Characterization of Actions of Lordosis-Relevant Substances

Among the various innate behavioral repertoires of mammals, lordosis, as a major component of female reproductive behaviors is one of the best characterized. It is a stereotyped posture consisting of dorsiflexion of the vertebral column, which causes the elevation of the head and rump, accompanied by hind limb extension (Pfaff and Lewis, 1974). Somatosensory input given by a male partner is essential for every component of naturally occurring lordosis; it can be mimicked by an experimenter’s manual stimulation to the flanks, posterior rump, and perineum. The most important feature of lordosis is its steroid hormone dependency; an elevated level of estrogen is a prerequisite for the induction of lordosis both in naturally cycling and experimentally manipulated females. The nature and mechanisms of estrogen and progesterone control of lordosis have been extensively investigated and are well reviewed elsewhere (Pfaff and Schwartz-Giblin, 1988).

Ascending and descending projections to medullary reticular formation sites which activate deep lumbar hack muscles in the rat

SummaryThe purpose of this study was to determine ascending and descending afferents to a medullary reticular formation (MRF) site that, when electrically stimulated, evoked EMG activity in lumbar deep back muscles. In anesthetized female rats, the MRF was explored with electrical stimulation, using currents less than 50 μA, while EMG activity was recorded from the ipsilateral lateral longissimus (LL) and medial longissimus (ML). MRF sites that evoked muscle activity were located in the gigantocellular nucleus (Gi). At the effective stimulation site, the retrograde fluorescent tracer, Fluoro-Gold (FG), was deposited via a cannula attached to the stimulating electrode. In matched-pair control experiments, FG was deposited at MRF sites that were ineffective in producing EMG activity in LL and ML, for comparison of afferent projections to effective versus ineffective sites. Labeled cells rostral to FG deposition at effective MRF sites were located in the preoptic area, hypothalamus, limbic forebrain and midbrain, with particularly high numbers in the ipsilateral midbrain central gray, tegmentum, paraventricular nucleus and amygdala. At medullary levels, there was a heavy projection from the contralateral Gi. FG labeled cells were also located in the contralateral parvocellular reticular nucleus, and lateral, medial and spinal vestibular nuclei. Labeled cells with ascending projections were observed in greatest number in the rostral cervical spinal cord, with fewer cells at mid cervical levels and even fewer in the lumbar spinal cord. These labeled cells were located primarily in lamina V, VII, VIII and X. Locations of labeled cells following FG deposition at ineffective MRF sites were similar. However, there was a striking difference in the number of cells retrogradely labeled from the effective MRF sites compared to ineffective MRF sites. Significantly greater numbers of labeled cells were observed in the contralateral MRF, the midbrain, and the cervical spinal cord from the FG deposition at effective stimulation sites. These results suggest that one characteristic of MRF sites that activate epaxial muscles is a larger amount of afferent input, from the midbrain central gray and from contralateral Gi, compared to ineffective MRF sites. Ascending and descending inputs converge at the effective MRF sites, and the larger number of descending projections suggests a more powerful contribution of these afferents to deep lumbar back muscle activation.

Ovarian steroid modulation of [3H]muscimol binding in the spinal cord of the rat

[3H]Muscimol binding was measured in the lumbar spinal cord of female rats by in vitro quantitative autoradiography. Ovariectomized rats were treated subcutaneously with either oil, estradiol benzoate (EB) or EB plus progesterone (P) in a regime known to reliably induce sexual receptivity. The level of [3H]muscimol binding was highest in laminae I-III and in the region around the central canal. Binding was lower in laminae IV-VI and was frequently undetectable in the ventral horn. There was a significant increase in the level of binding in laminae I-III after EB treatment. There was also a significant increase after treatment with EB+P in comparison to both the ovariectomized and EB-treated groups in this same region of the spinal cord.

A histochemical study of lateral longissimus muscle in rat

Rat axial muscle previously has not been studied histochemically. We were interested in determining the fiber composition and fiber distribution in rat lateral longissimus (LL), the large epaxial dorsiflexor muscle active during sexual posturing in the female rat and to determine if estrogen replacement in ovariectomized rats would affect the histochemical profile. Staining for ATPase after acid preincubation at pH 4.5, pH 4.35, and after alkaline preincubation at pH 9.4 and staining for NADH-TR revealed that rat LL contains the three major types of fibers present in most mammalian hind limbs: fast-twitch glycolytic (FG); fast-twitch oxidative-glycolytic (FOG); and slow-twitch oxidative (SO). The muscle contains predominantly FG fibers; SO fibers are segregated superficially from L2--L6 where they comprise from 11 to 18% of the fiber population, and in an oxidative compartment in the medial deep region of L5 where they comprise 62% of all fibers. In the medial deep region of L5 most of the remaining fast fibers also contain oxidative enzyme. Spindles are most highly concentrated in this oxidative region of L5. Estrogen treatment did not affect the relative number, distribution, or diameter of the three muscle fiber types in rat LL. The concentration of SO and FOG fibers and spindles localized in the region of the lumbosacral joint is discussed by contrasting forceful movements (e.g., rump elevation during sexual behavior) with normal postural regulation.

Axial electromyogram and intervertebral length gauge responses during lordosis behavior in rats

Electromyogram activity from transversospinalis (TS) and lateral longissimus (LL) muscles was recorded concomitant with resistance changes of an intervertebral length gauge during manually elicited lordosis performance. Subjects were estrogen-treated ovariectomized rats. The EMG activity was evoked by manual stimulation of the cutaneous fields contacted by the male rat during mounting. During bilateral palpation of flanks at 2/s, time-locked bursts of EMG activity or more generalized EMG activation was recorded. The EMG response was accompanied by continuous and cumulative length gauge shortening, reflecting progressive rump elevation. The earliest recorded length gauge response had a latency of 10 ms from the first flank stimulus in a sequence of repetitive palpations. Perineal pressure recruited motor units in both the TS and LL which fired at high frequency. The latencies of EMG responses from the TS tended to be briefer than those from the LL; 24/35 TS responses were within 50 ms of the onset of perineal pressure. During some lordoses, the EMG response in both muscles was substantially briefer than the duration of the pressure stimulus and of the length gauge response. Activation of axial muscles during postural adjustments, standing, turning, and other behaviors is also reported. These data represent the first analysis of axial EMG and vertebral length gauge recordings during an endocrine-dependent mammalian behavior.

Reticulospinal and reticuloreticular pathways for activating the lumbar back muscles in the rat.

SummaryThese experiments tested hypotheses about the logic of reticulospinal and reticuloreticular controls over deep back muscles by examining descending efferent and contralateral projections of the sites within the medullary reticular formation (MRF) that evoke EMG responses in lumbar axial muscles upon electrical stimulation. In the first series of experiments, retrograde tracers were deposited at gigantocellular reticular nucleus (Gi) sites that excited the back muscles and in the contralateral lumbar spinal cord. The medullary reticular formation contralateral to the Gi stimulation/deposition site was examined for the presence of single- and double-labeled cells from these injections. Tracer depositions into Gi produced labeled cells in the contralateral Gi and Parvocellular reticular nucleus (PCRt) whereas the lumbar injections retrogradely labeled cells only in the ventral MRF, indicating that separate populations of medullary reticular cells project to the opposite MRF and the lumbar cord. In the second series of experiments the precise relationships between the location of neurons retrogradely labeled from lumbar spinal cord depositions of the retrograde tracer, Fluoro-Gold (FG) and effective stimulation tracks through the MRF were examined. The results indicate that the Gi sites that are most effective for activation of the back muscles are dorsal to the location of retrogradely labeled lumbar reticulospinal cells. To verify that cell bodies and not fibers of passage were stimulated, crystals of the excitatory amino acid agonist, N-methyl-d-asparate (NMDA) were deposited at effective stimulation sites in the Gi. NMDA decreased the ability of electrical stimulation to activate back muscles at 5 min postdeposition, indicating a local interaction of NMDA with cell bodies at the stimulation site. In the third series of experiments, electrical thresholds for EMG activation along a track through the MRF were compared to cells retrogradely labeled from FG deposited into the cervical spinal cord. In some experiments, Fast Blue was also deposited into the contralateral lumbar cord. Neurons at low threshold points on the electrode track were labeled following cervical depositions, indicating a direct projection to the cervical spinal cord. The lumbar depositions, again, labeled cells in MRF areas that were ventral to the locations of effective stimulation sites, primarily on the opposite side of the medulla. In addition, the lumbar depositions back-filled cells in the same cervical segments to which the Gi neurons project. These results suggest that one efferent projection from effective stimulation sites for back muscle activation is onto propriospinal neurons in the cervical cord, which in turn project to lumbar cord levels. In a final series of experiments, a stimulating electrode track through the MRF again identified low threshold and ineffective sites for activating lumbar epaxial EMG. Fluoro-Gold was deposited in the contralateral MRF (MRFc) at a low threshold stimulation site for activating back muscles on that side. Retrogradely labeled cells surrounded effective, but not ineffective, stimulation sites along the electrode track in the MRF. Thus, another projection from effective stimulation sites is to effective stimulation sites in the opposite MRF. These results suggest that neurons in Gi whose stimulation most effectively activates back muscle EMG do not project directly to the lumbar cord, but relay to cervical cord neurons, which in turn project onto lumbar neurons. The MRF commissural connections presumably amplify this descending MRF control of axial back muscles.

The pudendal nerve-evoked response in axial muscle.

SummaryIn 39 Urethane-anesthetized rats we have recorded the afferent volley in the dorsal roots and the electrical activity of the lateral longissimus muscle and its motor nerves during electrical stimulation of a cutaneous branch of the pudendal nerve. Male and female rats were used; the females were ovariectomized and either pretreated with estradiol or left without hormonal treatment. Conduction velocities in the pudendal nerve were 54 m/s for the largest Abeta fibers and averaged 10 m/s for A-delta fibers. Excitation of pudendal nerve afferents strongly potentiated the firing of axial motoneurons, at stimulus currents below threshold for A-delta fibers. Trains of three shocks to the pudendal nerve were considerably more effective than double or single shock trains. Repetition rates as low as 1/s had a long lasting excitatory effect on the lateral longissimus muscle and the magnitude of the responses increased gradually for several seconds with continued stimulation. Recordings from the axons of the epaxial motoneurons of female rats showed a strong activation of neuronal firing with an onset latency of 5.8 ms from the last shock of a three ms, three shock train; the onset in male rats, 8.4 ms, differed significantly. Peak spike activity occurred at mean latencies of 11, 22 and 102 ms in both sexes. A period of depressed firing was usually present from 34 to 50 ms. Males differed in having a larger peak in activity at 102 ms, but the overall profile of the responses was similar in males and females. No differences were seen in the overall response patterns of the estrogen-treated and untreated females. Responses of comparable magnitude were seen with ipsilateral or contralateral pudendal nerve stimulation; these were facilitated by bilateral stimulation. In electromyographic recordings, the onset of unit firings was seen at 6.4 ms latency in response to pudendal nerve stimulation. This unit activity was consistent with the firing pattern seen in the muscle nerves.