Respiratory alkalosis provokes spike-wave discharges in seizure-prone rats.

Hyperventilation reliably provokes seizures in patients diagnosed with absence epilepsy. Despite this predictable patient response, the mechanisms that enable hyperventilation to powerfully activate absence seizure-generating circuits remain entirely unknown. By utilizing gas exchange manipulations and optogenetics in the WAG/Rij rat, an established rodent model of absence epilepsy, we demonstrate that absence seizures are highly sensitive to arterial carbon dioxide, suggesting that seizure-generating circuits are sensitive to pH. Moreover, hyperventilation consistently activated neurons within the intralaminar nuclei of the thalamus, a structure implicated in seizure generation. We show that intralaminar thalamus also contains pH-sensitive neurons. Collectively, these observations suggest that hyperventilation activates pH-sensitive neurons of the intralaminar nuclei to provoke absence seizures.

Anatomically segregated basal ganglia pathways allow parallel behavioral modulation.

In the basal ganglia (BG), anatomically segregated and topographically organized feedforward circuits are thought to modulate multiple behaviors in parallel. Although topographically arranged BG circuits have been described, the extent to which these relationships are maintained across the BG output nuclei and in downstream targets is unclear. Here, using focal trans-synaptic anterograde tracing, we show that the motor-action-related topographical organization of the striatum is preserved in all BG output nuclei. The topography is also maintained downstream of the BG and in multiple parallel closed loops that provide striatal input. Furthermore, focal activation of two distinct striatal regions induces either licking or turning, consistent with their respective anatomical targets of projection outside of the BG. Our results confirm the parallel model of BG function and suggest that the integration and competition of information relating to different behavior occur largely outside of the BG.

The Parabrachial Nucleus Directly Channels Spinal Nociceptive Signals to the Intralaminar Thalamic Nuclei, but Not the Amygdala.

The parabrachial nucleus (PBN) is one of the major targets of spinal projection neurons and plays important roles in pain. However, the architecture of the spinoparabrachial pathway underlying its functional role in nociceptive information processing remains elusive. Here, we report that the PBN directly relays nociceptive signals from the spinal cord to the intralaminar thalamic nuclei (ILN). We demonstrate that the spinal cord connects with the PBN in a bilateral manner and that the ipsilateral spinoparabrachial pathway is critical for nocifensive behavior. We identify Tacr1-expressing neurons as the major neuronal subtype in the PBN that receives direct spinal input and show that these neurons are critical for processing nociceptive information. Furthermore, PBN neurons receiving spinal input form functional monosynaptic excitatory connections with neurons in the ILN, but not the amygdala. Together, our results delineate the neural circuit underlying nocifensive behavior, providing crucial insight into the circuit mechanism underlying nociceptive information processing.

A disinhibitory nigra-parafascicular pathway amplifies seizure in temporal lobe epilepsy.

The precise circuit of the substantia nigra pars reticulata (SNr) involved in temporal lobe epilepsy (TLE) is still unclear. Here we found that optogenetic or chemogenetic activation of SNr parvalbumin+ (PV) GABAergic neurons amplifies seizure activities in kindling- and kainic acid-induced TLE models, whereas selective inhibition of these neurons alleviates seizure activities. The severity of seizures is bidirectionally regulated by optogenetic manipulation of SNr PV fibers projecting to the parafascicular nucleus (PF). Electrophysiology combined with rabies virus-assisted circuit mapping shows that SNr PV neurons directly project to and functionally inhibit posterior PF GABAergic neurons. Activity of these neurons also regulates seizure activity. Collectively, our results reveal that a long-range SNr-PF disinhibitory circuit participates in regulating seizure in TLE and inactivation of this circuit can alleviate severity of epileptic seizures. These findings provide a better understanding of pathological changes from a circuit perspective and suggest a possibility to precisely control epilepsy.

Modulation of thalamo-cortical activity by the NMDA receptor antagonists ketamine and phencyclidine in the awake freely-moving rat.

Non-competitive N-methyl-d-aspartate receptor antagonists mimic schizophrenia symptoms and produce immediate and persistent antidepressant effects. We investigated the effects of ketamine and phencyclidine (PCP) on thalamo-cortical network activity in awake, freely-moving male Wistar rats to gain new insight into the neuronal populations and brain circuits involved in the effects of NMDA-R antagonists. Single unit and local field potential (LFP) recordings were conducted in mediodorsal/centromedial thalamus and in medial prefrontal cortex (mPFC) using microelectrode arrays. Ketamine and PCP moderately increased the discharge rates of principal neurons in both areas while not attenuating the discharge of mPFC GABAergic interneurons. They also strongly affected LFP activity, reducing beta power and increasing that of gamma and high-frequency oscillation bands. These effects were short-lasting following the rapid pharmacokinetic profile of the drugs, and consequently were not present at 24 h after ketamine administration. The temporal profile of both drugs was remarkably different, with ketamine effects peaking earlier than PCP effects. Although this study is compatible with the glutamate hypothesis for fast-acting antidepressant action, it does not support a local disinhibition mechanism as the source for the increased pyramidal neuron activity in mPFC. The short-lasting increase in thalamo-cortical activity is likely associated with the rapid psychotomimetic action of both agents but could also be part of a cascade of events ultimately leading to the persistent antidepressant effects of ketamine. Changes in spectral contents of high-frequency bands by the drugs show potential as translational biomarkers for target engagement of NMDA-R modulators.

Synergistic Nigral Output Pathways Shape Movement.

Locomotion relies on the activity of basal ganglia networks, where, as the output, the substantia nigra pars reticulata (SNr) integrates incoming signals and relays them to downstream areas. The cellular and circuit substrates of such a complex function remain unclear. We hypothesized that the SNr controls different aspects of locomotion through coordinated cell-type-specific sub-circuits. Using anatomical mapping, single-cell qPCR, and electrophysiological techniques, we identified two SNr sub-populations: the centromedial-thalamo projectors (CMps) and the SN compacta projectors (SNcps), which are genetically targeted based on vesicular transporter for gamma-aminobutyric acid (VGAT) or parvalbumin (PV) expression, respectively. Optogenetic manipulation of these two sub-types across a series of motor tests provided evidence that they govern different aspects of motor behavior. While CMp activity supports the continuity of motor patterns, SNcp modulates the immediate motor drive behind them. Collectively, our data suggest that at least two different sub-circuits arise from the SNr, engage different behavioral motor components, and collaborate to produce correct locomotion.

Distinct Cortical-Thalamic-Striatal Circuits through the Parafascicular Nucleus.

The thalamic parafascicular nucleus (PF), an excitatory input to the basal ganglia, is targeted with deep-brain stimulation to alleviate a range of neuropsychiatric symptoms. Furthermore, PF lesions disrupt the execution of correct motor actions in uncertain environments. Nevertheless, the circuitry of the PF and its contribution to action selection are poorly understood. We find that, in mice, PF has the highest density of striatum-projecting neurons among all sub-cortical structures. This projection arises from transcriptionally and physiologically distinct classes of PF neurons that are also reciprocally connected with functionally distinct cortical regions, differentially innervate striatal neurons, and are not synaptically connected in PF. Thus, mouse PF contains heterogeneous neurons that are organized into parallel and independent associative, limbic, and somatosensory circuits. Furthermore, these subcircuits share motifs of cortical-PF-cortical and cortical-PF-striatum organization that allow each PF subregion, via its precise connectivity with cortex, to coordinate diverse inputs to striatum.

Postnatal development of cholinergic input to the thalamic reticular nucleus of the mouse.

The thalamic reticular nucleus (TRN), a shell-like structure comprised of GABAergic neurons, gates signal transmission between thalamus and cortex. While TRN is innervated by axon collaterals of thalamocortical and corticothalamic neurons, other ascending projections modulate activity during different behavioral states such as attention, arousal, and sleep-wake cycles. One of the largest arise from cholinergic neurons of the basal forebrain and brainstem. Despite its integral role, little is known about how or when cholinergic innervation and synapse formation occurs. We utilized genetically modified mice, which selectively express fluorescent protein and/or channelrhodopsin-2 in cholinergic neurons, to visualize and stimulate cholinergic afferents in the developing TRN. Cholinergic innervation of TRN follows a ventral-to-dorsal progression, with nonvisual sensory sectors receiving input during week 1, and the visual sector during week 2. By week 3, the density of cholinergic fibers increases throughout TRN and forms a reticular profile. Functional patterns of connectivity between cholinergic fibers and TRN neurons progress in a similar manner, with weak excitatory nicotinic responses appearing in nonvisual sectors near the end of week 1. By week 2, excitatory responses become more prevalent and arise in the visual sector. Between weeks 3-4, inhibitory muscarinic responses emerge, and responses become biphasic, exhibiting a fast excitatory, and a long-lasting inhibitory component. Overall, the development of cholinergic projections in TRN follows a similar plan as the rest of sensory thalamus, with innervation of nonvisual structures preceding visual ones, and well after the establishment of circuits conveying sensory information from the periphery to the cortex.

Daily, circadian and seasonal changes of rhodopsin-like encephalic photoreceptor and its involvement in mediating photoperiodic responses of Gambel's white-crowned Sparrow, Zonotrichia leucophrys gambelii.

Extra-retinal, non-pineal, encephalic photoreceptors (EP) play important roles in mediating development of the reproductive system by the annual change in day length (photoperiodic gonadal response - PGR) in birds. However, the distribution of rhodopsin-like EPs and their functional daily, circadian and seasonal changes are still unclear in the avian brain. This study identifies two novel groups of rhodopsin-immunoreactive cells in the nucleus paraventricularis magnocellularis (PVN) of the hypothalamus and in the medial basal hypothalamus (MBH) in a seasonally breeding species, Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii). In the PVN, rhodopsin-ir cell number showed both daily and circadian changes with more labeled cells apparent in the night phase in photosensitive birds, while only circadian changes were observed involving fewer labeled cells in the night phase in photorefractory birds. Single long day photo-stimulation significantly decreased the rhodopsin-ir cell number only in photosensitive birds, coincident with a rise in plasma levels of luteinizing hormone (LH). In the MBH, rhodopsin-ir cell number did not show daily, circadian or single long day induced changes in either photoperiodic states. But, overall these rhodopsin expressing neurons significantly increased from photosensitive to photorefractory states. In the median eminence (ME), more intense rhodopsin-ir was detected in photorefractory birds compared to photosensitive birds. For expression of GnRH and vasoactive intestinal polypeptide (VIP), seasonal differences were found with opposite relationships, consistent with previous studies. Our results suggest different roles of the two groups of rhodopsin-like EPs in the regulation of PGR in white-crowned sparrows.

Stimulus-induced transitions between spike-wave discharges and spindles with the modulation of thalamic reticular nucleus.

It is believed that thalamic reticular nucleus (TRN) controls spindles and spike-wave discharges (SWD) in seizure or sleeping processes. The dynamical mechanisms of spatiotemporal evolutions between these two types of activity, however, are not well understood. In light of this, we first use a single-compartment thalamocortical neural field model to investigate the effects of TRN on occurrence of SWD and its transition. Results show that the increasing inhibition from TRN to specific relay nuclei (SRN) can lead to the transition of system from SWD to slow-wave oscillation. Specially, it is shown that stimulations applied in the cortical neuronal populations can also initiate the SWD and slow-wave oscillation from the resting states under the typical inhibitory intensity from TRN to SRN. Then, we expand into a 3-compartment coupled thalamocortical model network in linear and circular structures, respectively, to explore the spatiotemporal evolutions of wave states in different compartments. The main results are: (i) for the open-ended model network, SWD induced by stimulus in the first compartment can be transformed into sleep-like slow UP-DOWN and spindle states as it propagates into the downstream compartments; (ii) for the close-ended model network, weak stimulations performed in the first compartment can result in the consistent experimentally observed spindle oscillations in all three compartments; in contrast, stronger periodic single-pulse stimulations applied in the first compartment can induce periodic transitions between SWD and spindle oscillations. Detailed investigations reveal that multi-attractor coexistence mechanism composed of SWD, spindles and background state underlies these state evolutions. What's more, in order to demonstrate the state evolution stability with respect to the topological structures of neural network, we further expand the 3-compartment coupled network into 10-compartment coupled one, with linear and circular structures, and nearest-neighbor (NN) coupled network as well as its realization of small-world (SW) topology via random rewiring, respectively. Interestingly, for the cases of linear and circular connetivities, qualitatively similar results were obtained in addition to the more irregularity of firings. However, SWD can be eventually transformed into the consistent low-amplitude oscillations for both NN and SW networks. In particular, SWD evolves into the slow spindling oscillations and background tonic oscillations within the NN and SW network, respectively. Our modeling and simulation studies highlight the effect of network topology in the evolutions of SWD and spindling oscillations, which provides new insights into the mechanisms of cortical seizures development.

Activation of the thalamic parafascicular nucleus by electrical stimulation of the peripheral vestibular nerve in rats.

The parafascicular nucleus (PFN) of the thalamus is a primary structure in the feedback circuit of the basal ganglia-thalamo-cortical system, as well as in the neural circuit of the vestibulo-thalamo-striatal pathway. We investigated the characteristics of the functional connectivity between the peripheral vestibular system and the PFN in rats. A single electrical stimulation was applied to the horizontal semicircular canal nerve in the peripheral vestibular end-organs. This resulted in polysynaptic local field potentials (LFPs) in the PFN, which were composed of long-lasting multiple waves. The LFPs were prominently seen contralateral to the stimulation site. The PFN LFPs were suppressed by transient chemical de-afferentation of peripheral vestibular activity using a 5% lidocaine injection into the middle ear. The spontaneous firing rate of the single units increased after electrical stimulation to the horizontal canal nerve in a frequency-dependent manner. The induction of cFos protein was more prominent in the contralateral PFN than in the ipsilateral PFN following horizontal semicircular canal nerve stimulation. The functional vestibulo-parafascicular connection is a neural substrate for the transmission of vestibular sensory information to the basal ganglia.

Thalamic reticular nucleus in Caiman crocodilus: forebrain connections.

Forebrain connections of the thalamic reticular nucleus associated with the lateral forebrain bundle were analyzed in Caiman crocodilus. Both the compact portion, the dorsal peduncular nucleus, and the diffuse part, the perireticular region, associated with the lateral forebrain bundle, were studied. A small tracer injection into the dorsal peduncular nucleus demonstrated reciprocal connections with a restricted portion of the dorsal thalamus. Tracer placements into this nucleus retrogradely labeled cells in a caudal portion of the ventrolateral area of the telencephalon. These results are compared with similar studies in other amniotes.

Early postnatal switch in GABAA receptor α-subunits in the reticular thalamic nucleus.

The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology, and immunohistochemistry in mice, we characterized postsynaptic developmental changes in these inhibitory projection neurons. First, at postnatal days 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8 and then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABA(A) receptor (GABA(A)R)-mediated IPSCs differed between neonatal and mature nRt neurons, and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABA(A)Rs, augmented only mature IPSCs, whereas clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708, but only in immature neurons. In α3(H126R) mice, in which α3-subunits were mutated to become benzodiazepine insensitive, IPSCs were enhanced compared with those in wild-type animals in early development. Third, tonic GABA(A)R activation in nRt is age dependent and more prominent in immature neurons, which correlates with early expression of α5-containing GABA(A)Rs. Thus neonatal nRt neurons show relatively high expression of α5-subunits, which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABA(A)R subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development.

Ascending parabrachio-thalamo-striatal pathways: potential circuits for integration of gustatory and oral motor functions.

The medial parabrachial nucleus (MPB) and external part of the medial parabrachial nucleus (MPBE) relay gustatory, oral mechanosensory and other visceral information in the rat brain and reportedly project not only to the parvicellular part of the posteromedial ventral thalamic nucleus (VPMpc) but also to the ventrocaudal part of the intralaminar thalamic nuclei. Generally, the intralaminar thalamic nuclei project topographically to the caudate putamen (CPu); however, it is unclear where the ventrocaudal part of the intralaminar thalamic nuclei projects within the CPu. Thus, we visualized neural pathways from the MPB and MPBE to the CPu via the ventrocaudal part of the intralaminar thalamic nuclei using an anterograde tracer, biotinylated dextran amine, and a retrograde tracer, cholera toxin B subunit. We found that the MPB and MPBE sent a relatively stronger input to the ventrocaudal part of the intralaminar thalamic nuclei such as the oval paracentral thalamic nucleus (OPC), central medial thalamic nucleus (CM) and parafascicular thalamic nucleus (PF) and retroreuniens area (RRe) as compared to the VPMpc. In turn, these thalamic nuclei projected to the ventral part of the CPu with the topographical arrangement as follows: the OPC to the ventrocentral part of the CPu; ventrolateral part of the PF to the ventrolateral part of the CPu; and the caudal part of the CM, ventromedial part of the PF and RRe to the ventromedial part of the CPu. Further, we found that the VPMpc rather projected to the interstitial nucleus of the posterior limb of the anterior commissure than the CPu. The ventral part of the CPu is reported to be involved in jaw movement as well as food and water intake functions. Therefore, these parabrachio-thalamo-striatal pathways that we demonstrated here suggest that gustatory and oral mechanosensory information affects feeding behavior within the ventral part of the CPu.

The nonspecific thalamus: A place in a wedding bed for making memories last?

We summarize anatomical, electrophysiological and behavioral evidence that the rostral intralaminar (ILN) and the reuniens and rhomboid (ReRh) nuclei that belong to the nonspecific thalamus, might be part of a hippocampo-cortico-thalamic network underlying consolidation of enduring declarative(-like) memories at systems level. The first part of this review describes the anatomical and functional organization of these thalamic nuclei. The second part presents the theoretical models supporting the active systems-level consolidation, a process that relies upon sleep specific field-potential oscillations occurring during both slow-wave sleep (SWS) and rapid eye movement (REM) sleep. The last part presents data in the rat showing that the lesion of the rostral ILN or of the ReRh specifically hinders the formation of remote spatial memories without affecting task acquisition or retrieval of a recent memory. These results showing a critical role of the ILN and ReRh nuclei in the transformation of a recent memory into a remote one are discussed in the context of their control of cortical arousal (ARAS) and of thalamo-cortico-thalamic synchronization.

Asymmetry and modulation of spike timing in electrically coupled neurons.

Electrical coupling mediates interactions between neurons of the thalamic reticular nucleus (TRN), which play a critical role in regulating thalamocortical and corticothalamic communication by inhibiting thalamic relay cells. Accumulating evidence has shown that asymmetry of electrical synapses is a fundamental and dynamic property, but the effect of asymmetry on coupled networks is unexplored. Recording from patched pairs in rat brain slices, we investigate asymmetry in the subthreshold regime and show that electrical synapses can exert powerful effects on the spike times of coupled neighbors. Electrical synaptic signaling modulates spike timing by 10-20 ms, in an effect that also exhibits asymmetry. Furthermore, we show through modeling that coupling asymmetry expands the set of outputs for pairs of coupled neurons through enhanced regions of synchrony and reversals of spike order. These results highlight the power and specificity of signaling exerted by electrical synapses, which contribute to information flow across the brain.

Two functionally distinct networks of gap junction-coupled inhibitory neurons in the thalamic reticular nucleus.

Gap junctions (GJs) electrically couple GABAergic neurons of the forebrain. The spatial organization of neuron clusters coupled by GJs is an important determinant of network function, yet it is poorly described for nearly all mammalian brain regions. Here we used a novel dye-coupling technique to show that GABAergic neurons in the thalamic reticular nucleus (TRN) of mice and rats form two types of GJ-coupled clusters with distinctive patterns and axonal projections. Most clusters are elongated narrowly along functional modules within the plane of the TRN, with axons that selectively inhibit local groups of relay neurons. However, some coupled clusters have neurons arrayed across the thickness of the TRN and target their axons to both first- and higher-order relay nuclei. Dye coupling was reduced, but not abolished, among cells of connexin36 knock-out mice. Our results suggest that GJs form two distinct types of inhibitory networks that correlate activity either within or across functional modules of the thalamus.

Burst firing of neurons in the thalamic reticular nucleus during locomotion.

This study examined the burst firing of neurons in the motor sector of the thalamic reticular nucleus (RE) of the cat. These neurons are inhibitory cells that project to the motor thalamus. The firing activity of RE neurons was studied during four behaviors: sleep, standing, walking on a flat surface, and accurate stepping on crosspieces of a horizontal ladder. Extracellularly recorded firing activity was analyzed in 58 neurons that were identified according to their receptive fields on the contralateral forelimb. All neurons generated bursts of spikes during sleep, half generated bursts of spikes during standing, and one-third generated bursts of spikes during walking. The majority of bursts were sequences of spikes with an exponential buildup of the firing rate followed by exponential decay with time constants in the range of 10-30 ms. We termed them "full-scale" bursts. All neurons also generated "atypical" bursts, in which the buildup of the firing rate deviated from the characteristic order. Burst firing was most likely to occur in neurons with receptive fields on the distal forelimb and least likely in neurons related to the proximal limb. Full-scale bursts were more frequent than atypical bursts during unconstrained walking on the flat surface. Bursts of both types occurred with similar probability during accurate stepping on the horizontal ladder, a task that requires forebrain control of locomotion. We suggest that transformations of the temporal pattern of bursts in the inhibitory RE neurons facilitate the tuning of thalamo-cortical signals to the complexity of ongoing locomotor tasks.

Astrocytes potentiate GABAergic transmission in the thalamic reticular nucleus via endozepine signaling.

Emerging evidence indicates that diazepam-binding inhibitor (DBI) mediates an endogenous benzodiazepine-mimicking (endozepine) effect on synaptic inhibition in the thalamic reticular nucleus (nRT). Here we demonstrate that DBI peptide colocalizes with both astrocytic and neuronal markers in mouse nRT, and investigate the role of astrocytic function in endozepine modulation in this nucleus by testing the effects of the gliotoxin fluorocitrate (FC) on synaptic inhibition and endozepine signaling in the nRT using patch-clamp recordings. FC treatment reduced the effective inhibitory charge of GABAA receptor (GABAAR)-mediated spontaneous inhibitory postsynaptic currents in WT mice, indicating that astrocytes enhance GABAAR responses in the nRT. This effect was abolished by both a point mutation that inhibits classical benzodiazepine binding to GABAARs containing the α3 subunit (predominant in the nRT) and a chromosomal deletion that removes the Dbi gene. Thus, astrocytes are required for positive allosteric modulation via the α3 subunit benzodiazepine-binding site by DBI peptide family endozepines. Outside-out sniffer patches pulled from neurons in the adjacent ventrobasal nucleus, which does not contain endozepines, show a potentiated response to laser photostimulation of caged GABA when placed in the nRT. FC treatment blocked the nRT-dependent potentiation of this response, as did the benzodiazepine site antagonist flumazenil. When sniffer patches were placed in the ventrobasal nucleus, however, subsequent treatment with FC led to potentiation of the uncaged GABA response, suggesting nucleus-specific roles for thalamic astrocytes in regulating inhibition. Taken together, these results suggest that astrocytes are required for endozepine actions in the nRT, and as such can be positive modulators of synaptic inhibition.

Developmental GnRH signaling is not required for sexual differentiation of kisspeptin neurons but is needed for maximal Kiss1 gene expression in adult females.

Kisspeptin, encoded by Kiss1, stimulates reproduction. In rodents, one Kiss1 population resides in the hypothalamic anterior ventral periventricular nucleus and neighboring rostral periventricular nucleus (AVPV/PeN). AVPV/PeN Kiss1 neurons are sexually dimorphic (greater in females), yet the mechanisms regulating their development and sexual differentiation remain poorly understood. Neonatal estradiol (E2) normally defeminizes AVPV/PeN kisspeptin neurons, but emerging evidence suggests that developmental E2 may also influence feminization of kisspeptin, although exactly when in development this process occurs is unknown. In addition, the obligatory role of GnRH signaling in governing sexual differentiation of Kiss1 or other sexually dimorphic traits remains untested. Here, we assessed whether AVPV/PeN Kiss1 expression is permanently impaired in adult hpg (no GnRH or E2) or C57BL6 mice under different E2 removal or replacement paradigms. We determined that 1) despite lacking GnRH signaling in development, marked sexual differentiation of Kiss1 still occurs in hpg mice; 2) adult hpg females, who lack lifetime GnRH and E2 exposure, have reduced AVPV/PeN Kiss1 expression compared to wild-type females, even after chronic adulthood E2 treatment; 3) E2 exposure to hpg females during the pubertal period does not rescue their submaximal adult Kiss1 levels; and 4) in C57BL6 females, removal of ovarian E2 before the pubertal or juvenile periods does not impair feminization and maximal adult AVPV/PeN Kiss1 expression nor the ability to generate LH surges, indicating that puberty is not a critical period for Kiss1 development. Thus, sexual differentiation still occurs without GnRH, but GnRH or downstream E2 signaling is needed sometime before juvenile development for complete feminization and maximal Kiss1 expression in adult females.