Irving Zucker

Sex bias in neuroscience and biomedical research

Female mammals have long been neglected in biomedical research. The NIH mandated enrollment of women in human clinical trials in 1993, but no similar initiatives exist to foster research on female animals. We reviewed sex bias in research on mammals in 10 biological fields for 2009 and their historical precedents. Male bias was evident in 8 disciplines and most prominent in neuroscience, with single-sex studies of male animals outnumbering those of females 5.5 to 1. In the past half-century, male bias in non-human studies has increased while declining in human studies. Studies of both sexes frequently fail to analyze results by sex. Underrepresentation of females in animal models of disease is also commonplace, and our understanding of female biology is compromised by these deficiencies. The majority of articles in several journals are conducted on rats and mice to the exclusion of other useful animal models. The belief that non-human female mammals are intrinsically more variable than males and too troublesome for routine inclusion in research protocols is without foundation. We recommend that when only one sex is studied, this should be indicated in article titles, and that funding agencies favor proposals that investigate both sexes and analyze data by sex.

Light-dark rhythms in rat eating and drinking behavior

Abstract Following blinding there is a rapid and sustained increase in eating and drinking during the light portion of a light-dark (L-D) cycle. Group nocturnal rhythms in eating and drinking are retained in attenuated form for at least 10 days; sixty days after blinding these rhythms are absent. For sighted rats the food/water ratio is three times greater during the light than during the dark; this difference is greatly reduced almost immediately after blinding. Continuous illumination almost immediately suppresses water intake and abolishes group nocturnal rhythms in eating and drinking within 9–11 days. Nocturnal eating and drinking rhythms re-entrain within 7–9 days following an inversion of the L-D cycle. Animals blinded between 12 and 36 hr after such an inversion remain residually entrained to the original L-D cycle for at least 1 month, but animals blinded at 48–120 hr re-entrain to the new cycle. These differences persist for surprisingly long times and suggest that the initial period following a change in environmental illumination is most critical for re-entrainment of eating and drinking which may then proceed relatively normally in the absence of further visual stimulation. Nocturnal patterns of drinking are present in essentially adult form in rats 23 days old; blinding on Day 14 but not on Day 18 or later prevents the appearance of these rhythms. The early perinatal period is not critical for entrainment of eating and drinking rhythms since animals first exposed to alternating L-D patterns of illumination at 59 days of age display nocturnal behavior patterns shortly thereafter. The influence of daytime feeding schedules on nocturnal drinking patterns is described and the neural basis for rhythmic light-dependent behavior considered.

Female mice liberated for inclusion in neuroscience and biomedical research

The underrepresentation of female mice in neuroscience and biomedical research is based on the assumption that females are intrinsically more variable than males and must be tested at each of four stages of the estrous cycle to generate reliable data. Neither belief is empirically based. In a meta-analysis of 293 articles, behavioral, morphological, physiological, and molecular traits were monitored in male mice and females tested without regard to estrous cycle stage; variability was not significantly greater in females than males for any endpoint and was substantially greater in males for several traits. Group housing of mice increased variability in both males and females by 37%. Utilization of female mice in neuroscience research does not require monitoring of the estrous cycle. The prevalence of sex differences at all levels of biological organization, and limitations in generalizing findings obtained with males to females, argue for the routine inclusion of female rodents in most research protocols.

Hormonal determinants of sex differences in saccharin preference, food intake and body weight

Abstract Saccharin preferences (S.P.) are greater among female than male adult rats. This sex difference is mainly attributable to stimulatory effects of ovarian hormones in the female; ovariectomized rats have much lower S.P. than intact females, but S.P. of castrated males are only slightly greater than those of intact males. Thus testicular secretions exert at most a weak suppressive effect on S.P. Neither estradiol benzoate (EB) nor progesterone (P) acting alone is sufficient to elevate S.P. of spayed females; however, daily injection of a combination of both hormones increases preference for the sweet solution. The same EB-P treatments are ineffective in altering S.P. of castrated males. There is a sex difference in responsiveness of the substrate mediating S.P. to the activational actions of gonadal hormones. Hormonal influences on and sex differences in food and fluid intake and body weight are described and discussed.

Tracking the seasons: the internal calendars of vertebrates

Animals have evolved many season-specific behavioural and physiological adaptations that allow them to both cope with and exploit the cyclic annual environment. Two classes of endogenous annual timekeeping mechanisms enable animals to track, anticipate and prepare for the seasons: a timer that measures an interval of several months and a clock that oscillates with a period of approximately a year. Here, we discuss the basic properties and biological substrates of these timekeeping mechanisms, as well as their reliance on, and encoding of environmental cues to accurately time seasonal events. While the separate classification of interval timers and circannual clocks has elucidated important differences in their underlying properties, comparative physiological investigations, especially those regarding seasonal prolactin secretions, hint at the possibility of common substrates.

Sex differences in body weight and eating: Organization and activation by gonadal hormones in the rat ☆

Abstract Female rats injected on the fifth day of life with testosterone propionate (TP) or estradiol benzoate (EB) weigh more in adulthood than control animals injected with oil. If rats are ovariectomized prior to steroid treatment on Day 5, body weight differences in adulthood are reduced and their initial appearance during development is delayed. Neonatal TP or EB treatment increases responsiveness to the weight-promoting effects of androgens in adulthood and decreases adult responsiveness to the weight depressing actions of ovarian hormones. We propose that hormonal stimulation in infancy affects body weight by altering hormonal sensitivity of neural weight regulating mechanisms. Non-neural mechanisms and behavioral effects which might contribute to changes in body weight are also considered. The adult sex difference in body weight is multiply determined by postnatal organizational and activational effects of gonadal hormones as well as by non-hormonal genetic factors and/or prenatal testicular secretions. The sex difference in eating is less subject to early organizing actions of hormones and is primarily attributable to the different hormones secreted by male and female gonads beginning sometime after the end of the neonatal organizational period.

Light-dark rhythms in hamster eating, drinking and locomotor behaviors

Abstract Adult hamsters were exposed to alternating 12-hr periods of light (L) and darkness; 99% of daily wheel running activity occurred in the 12-hr dark (D) period. Water intake and food consumption also occurred more commonly in the dark but the extent to which these behaviors were displayed nocturnally, 85% and 58%, respectively, was significantly less than for wheel running. Activity cycles modulate drinking rhythms; nocturnal water intake was substantially reduced in hamsters with access to wheels while drinking during the light period was unaffected. Nocturnal drinking rhythms of hamsters phase-shifted very slowly after the L-D cycle was inverted; females required more than 40 days, whereas males re-established their normal nocturnal drinking rhythms in approximately 25 days. This sex difference disappeared after males and females were gonadectomized. Wheel running behavior of hamsters phase-shifted by 12 hr within 6–9 days of inversion of the L-D cycle. The great disparity in the rate of phase-shifting of hamster activity and drinking rhythms suggests different central pacemakers for these behaviors or different sensitivities to changes in the light-dark cycle in their respective phase-shifting and coupling mechanisms. Eating and drinking rhythms of blind hamsters were described and compared to those of blind rats. Constant illumination (L-L) decreased water intake of male and female hamsters; loss of entrainment to the previous L-D cycle occurred more slowly than for rats maintained in L-L. These findings are consistent with the differential effects of constant light on rhythmic reproductive phenomena in the two species. Hamster drinking and wheel-running behaviors are particularly appropriate endpoints for the analysis of neural and endocrine bases of circadian rhythmicity.

Absence of extraocular photoreception in diurnal and nocturnal rodents exposed to direct sunlight

1. 1. Entrainment of circadian wheel running activity was monitored over a period of several months for northern grasshopper mice (Onychomys leucogaster) and golden mantled ground squirrels (Spermophilus lateralis) housed out-of-doors and exposed to direct sunlight (average light intensity = 55,000 lx). 2. 2. Sighted animals entrained their activity rhythms to the natural photoperiod; the mice were nocturnal and the squirrels diurnal in their locomotor activity. 3. 3. The activity rhythms of all blind animals from each species failed to entrain to the light-dark cycle and free-ran with endogenous periods > 24 hr for squirrels and < 24 hr for grasshopper mice. 4. 4. Failure of entrainment in blind ground squirrels and grasshopper mice indicates that these species lack functional extraocular photoreceptors; they thus differ from non-mammalian vertebrates in which extraocular photoreception is well documented. 5. 5. The significance of exclusive reliance by mammals on ocular photoreceptors for entrainment of circadian rhythms is discussed.

Rat drinking rhythms: Central visual pathways and endocrine factors mediating responsiveness to environmental illumination

Abstract Continuous environmental illumination (L-L) suppressed water consumption in Sprague-Dawley rats. Bilateral transection of the inferior accessory optic tracts (IAOT) completely abolished this effect of light on drinking. On the other hand, bilateral ablation of the primary optic tracts (POT) increased the suppressive effect of light on water intake. POT rats were impaired in the acquisition of a light-dark discrimination in a Y-maze, whereas IAOT animals performed normally. These results suggest that the inferior accessory optic tracts mediate the inhibitory effects of excessive light on drinking and that the visual pathways which underlie pituitary responsiveness to illumination are distinguishable from those which mediate visually guided behavior. Pinealectomy did not attenuate the effect of L-L on drinking, but unilateral blinding diminished and hypophysectomy completely eliminated the suppressive effects of light. Rats maintained in a 12:12 light-dark (L-D) cycle displayed nocturnal rhythms in water intake; when the illumination cycle was inverted (D-L), they required 6–9 days to completely re-entrain drinking. Bilateral interruption of the inferior accessory and primary optic tracts and pinealectomy did not interfere with nocturnal drinking rhythms in L-D nor with their re-entrainment in D-L. Hypophysectomy attenuated nocturnal eating and drinking rhythms, and unilateral enucleation as well as hypophysectomy retarded re-entrainment in D-L. The effects of combined bilateral transection of the IAOT and POT were described; a drinkometer analysis of water intake confirmed results obtained with less refined measures of rhythmicity. Our findings indicate that excessive illumination influences the neuroendocrine drinking system of the albino rat via a completely decussated visual pathway. We propose that neither the primary nor the accessory optic tracts mediate the entrainment of drinking to the day-night cycle; this function may be fulfilled by a direct retino-hypothalamic pathway.

Photoperiodic Control of Reproduction in Olfactory-Bulbectomized Rats

30-day-old male rats were (1) sham-operated or subjected to (2) removal of the olfactory bulbs, (3) olfactory bulbectomy and blinding (4) olfactory bulbectomy and pinealectomy or (5) olfactory bulbectomy, blinding and pinealectomy. Animals were exposed from 30 to 110 days of age to long-day (14 h of light per day) or short-day (8 h of light per day) photoperiods. The reproductive system of neurologically-intact rats was not affected by exposure to short days. Nor did bulbectomy affect the reproductive system of rats exposed to long days. However, bulbectomized, short-day rats had significantly lower body weights, reduced testicular and seminal vesicle weights and lower plasma testosterone levels than did bulbectomized, long-day rats. The effects of short-day exposure on bulbectomized rats were prevented by pinealectomy. Short-day exposure and blinding exerted similar effects in bulbectomized rats. The testes of rats from all groups contained elongated spermatids; blinding and short-day treatment had no effect on spermatogenesis. Neither mating behavior nor the number of young sired was influenced by photoperiod in bulbectomized or intact rats. Removal of the olfactory bulbs unmasks photoperiodic responsiveness in rats; the antigonadal effects of short-day exposure are mediated by the pineal gland in bulbectomized rats as in species traditionally designated photoperiodic. The mechanisms by which bulbectomy renders rats responsive to short days are considered.