Intrinsically photosensitive retinal ganglion cells (ipRGCs) are inner retinal photoreceptors that mediate non-image-forming visual functions, e.g. pupillary constriction, regulation of pineal melatonin release, and circadian photoentrainment. Five types of ipRGCs were recently discovered in mouse, but whether they exist in other mammals remained unknown. We report that the rat also has five types of ipRGCs, whose morphologies match those of mouse ipRGCs; this is the first demonstration of all five cell types in a non-mouse species. Through immunostaining and λmax measurements, we showed that melanopsin is likely the photopigment of all rat ipRGCs. The various cell types exhibited diverse spontaneous spike rates, with the M1 type spiking the least and M4 spiking the most, just like we had observed for their mouse counterparts. Also similar to mouse, all ipRGCs in rat generated not only sluggish intrinsic photoresponses but also fast, synaptically driven ones. However, we noticed two significant differences between these species. First, whereas we learned previously that all mouse ipRGCs had equally sustained synaptic light responses, rat M1 cells’ synaptic photoresponses were far more transient than those of M2–M5. Since M1 cells provide all input to the circadian clock, this rat-versus-mouse discrepancy could explain the difference in photoentrainment threshold between mouse and other species. Second, rat ipRGCs’ melanopsin-based spiking photoresponses could be classified into three varieties, but only two were discerned for mouse ipRGCs. This correlation of spiking photoresponses with cell types will help researchers classify ipRGCs in multielectrode-array (MEA) spike recordings.

Gonadotropin-releasing hormone (GnRH) neurons regulate reproduction though pulsatile hormone release. Disruption of GnRH release as measured via luteinizing hormone (LH) pulses occurs in polycystic ovary syndrome (PCOS), and in young hyperandrogenemic girls. In adult prenatally androgenized (PNA) mice, which exhibit many aspects of PCOS, increased LH is associated with increased GnRH neuron action potential firing. How GnRH neuron activity develops over the prepubertal period and whether this is altered by sex or prenatal androgen treatment are unknown. We hypothesized GnRH neurons are active before puberty and that this activity is sexually differentiated and altered by PNA. Dams were injected with dihydrotestosterone (DHT) on days 16 to 18 post copulation to generate PNA mice. Action potential firing of GFP-identified GnRH neurons in brain slices from 1-, 2-, 3-, and 4-week-old and adult mice was monitored. GnRH neurons were active at all ages tested. In control females, activity increased with age through 3 weeks, then decreased to adult levels. In contrast, activity did not change in PNA females and was reduced at 3 weeks. Activity was higher in control females than males from 2 to 3 weeks. PNA did not affect GnRH neuron firing rate in males at any age. Short-term action potential patterns were also affected by age and PNA treatment. GnRH neurons are thus typically more active during the prepubertal period than adulthood, and PNA reduces prepubertal activity in females. Prepubertal activity may play a role in establishing sexually differentiated neuronal networks upstream of GnRH neurons; androgen-induced changes during this time may contribute to the adult PNA, and possibly PCOS, phenotype.

Changes in gonadotropin-releasing hormone (GnRH) release frequency from the brain help drive reproductive cycles. In polycystic ovary syndrome (PCOS), persistent high GnRH/luteinizing hormone (LH) frequency disrupts cycles and exacerbates hyperandrogenemia. Adult prenatally-androgenized (PNA) mice exhibit increased GnRH neuron firing rate, elevated ovarian androgens, and disrupted cycles, but before puberty, GnRH neuron activity is reduced in PNA mice compared with controls. We hypothesized that ovarian feedback mediates the age-dependent change in GnRH neuron firing rate in PNA vs control mice. Extracellular recordings of green fluorescent protein (GFP)-identified GnRH neurons were made 5 to 7 days after sham-surgery, ovariectomy (OVX), or, in adults, after OVX plus replacement of sub-male androgen levels with dihydrotestosterone implants (OVX + DHT). In 3-week-old mice, OVX did not affect GnRH neuron firing rate in either group. In adult controls, OVX increased GnRH neuron firing rate, which was further enhanced by DHT. In adult PNA mice, however, OVX decreased GnRH neuron firing rate, and DHT restored firing rate to sham-operated levels. In contrast to the differential effects of ovarian feedback on GnRH neuron firing rate, serum LH increased after OVX in both control and PNA mice and was not altered by DHT. Pituitary gene expression largely reflected changes expected with OVX, although in PNA but not control mice, DHT treatment increased Lhb expression. These results suggest prenatal androgen exposure programs marked changes in GnRH neuron regulation by homeostatic steroid feedback. PNA lowers GnRH neuron activity in low-steroid states (before puberty, OVX), and renders activity in adulthood dependent upon ongoing exposure to elevated ovarian androgens.

Gonadotropin-releasing hormone (GnRH) neurons control anterior pituitary, and thereby gonadal, function. GnRH neurons are active before outward indicators of puberty appear. Prenatal androgen (PNA) exposure mimics reproductive dysfunction of the common fertility disorder polycystic ovary syndrome (PCOS) and reduces prepubertal GnRH neuron activity. Early neuron activity can play a critical role in establishing circuitry and adult function. We tested the hypothesis that changing prepubertal GnRH neuron activity programs adult GnRH neuron activity and reproduction independent of androgen exposure in female mice. Activating (3Dq) or inhibitory (4Di) designer receptors exclusively activated by designer drugs (DREADDs) were targeted to GnRH neurons using Cre-lox technology. In control studies, the DREADD ligand clozapine n-oxide (CNO) produced the expected changes in GnRH neuron activity in vitro and luteinizing hormone (LH) release in vivo . CNO was administered to control or PNA mice between two and three weeks of age, when GnRH neuron firing rate is reduced in PNA mice. In controls, reducing prepubertal GnRH neuron activity with 4Di increased adult GnRH neuron firing rate and days in diestrus but did not change puberty onset or GABA transmission to these cells. In contrast, activating GnRH neurons had no effect on reproductive parameters or firing rate and did not rescue reproductive phenotypes in PNA mice. These studies support the hypothesis that prepubertal neuronal activity sculpts elements of the adult reproductive neuroendocrine axis and cyclicity but indicate that other PNA-induced programming actions are required for full reproductive phenotypes and/or that compensatory mechanisms overcome activity-mediated changes to mitigate reproductive changes in adults.