Ion channels and information processing in GnRH neuron dendrites.

Recent findings indicate that a majority of action potentials originate from dendrites of GnRH neurons. This localization of the dendrite as the principle site of action potential initiation has sparked considerable interest in the nature of ionic channels throughout GnRH neurons. This paper will review the ionic conductances described within GnRH neurons and their implications for physiological output, such as sensitivity to steroids and diurnal state. To date, a majority of information regarding ionic conductances in GnRH neurons pertains to somata and the first 50-100 µm of dendrite length. Thus, unraveling the tapestry created by the nature and distribution of dendritic conductances in GnRH neurons lies at the forefront of understanding the control of reproductive hormone secretion.

Simulated GABA synaptic input and L-type calcium channels form functional microdomains in hypothalamic gonadotropin-releasing hormone neurons.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons integrate the multiple internal and external cues that regulate sexual reproduction. In contrast to other neurons that exhibit extensive dendritic arbors, GnRH neurons usually have a single dendrite with relatively little branching. This largely precludes the integration strategy in which a single dendritic branch serves as a unit of integration. In the present study, we identify a gradient in L-type calcium channels in dendrites of mouse GnRH neurons and its interaction with GABAergic and glutamatergic inputs. Higher levels of L-type calcium channels are in somata/proximal dendrites (i.e., 0-26 µm) and distal dendrites (∼130 µm dendrite length), but intervening midlengths of dendrite (∼27-130 µm) have reduced L-type calcium channels. Using uncaging of GABA, there is a decreasing GABAergic influence along the dendrite and the impact of GABA(A) receptors is dependent on activation of L-type calcium channels. This results in amplification of proximal GABAergic signals and attenuation of distal dendritic signals. Most interestingly, the intervening dendritic regions create a filter through which only relatively high-amplitude, low-frequency GABAergic signaling to dendrites elicits action potentials. The findings of the present study suggest that GnRH dendrites adopt an integration strategy whereby segments of single nonbranching GnRH dendrites create functional microdomains and thus serve as units of integration.

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons.

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.

Spatially selective, testosterone-independent remodeling of dendrites in gonadotropin-releasing hormone (GnRH) neurons prepubertally in male rats.

Adult GnRH neurons exhibit a stereotypic morphology with a small soma, single axon, and single dendrite arising from the soma with little branching. The adult morphology of GnRH neurons in mice reflects an anatomical consolidation of dendrites over postnatal development. We examined this issue in rat GnRH neurons with biocytin filling in live hypothalamic slices from infant males, as adult littermates and in gonad-intact males, castrated males, and in males with one of three levels of testosterone (T) treatment. Somatic area and total dendritic length were significantly greater in infant males than in adults. Moreover, total numbers of dendrite branches were greater in infant males as compared with adults. The number of higher order branches and the lengths of higher order branches were also greater in infant males than in adults. Most interestingly, in adults a single dendrite arose from the somata, consistently at 180° from the axon. In contrast, prepubertal animals had an average of 2.2 ± 0.2 primary dendrites arising from somata (range, one to seven primary dendrites). Angles relative to the axon at which dendrites in prepubertal males emanated from GnRH somata were highly variable. Castration at 25 d of age and castration at 25 d of age with one of three levels of T treatment did not influence morphological parameters when GnRH neurons were examined between 40 d and 48 d of age. Thus, a spatially selective remodeling of primary dendrites and consolidation of distal GnRH dendritic arbors occurs during postnatal development and is largely independent of T.

Dual somatic recordings from gonadotropin-releasing hormone (GnRH) neurons identified by green fluorescent protein (GFP) in hypothalamic slices.

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3). Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons. The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dendritic action potential initiation in hypothalamic gonadotropin-releasing hormone neurons.

It is dogma that action potentials are initiated at the soma/axon hillock of neurons. However, dendrites often exhibit conductances necessary for spike generation and represent functionally independent processing compartments within neurons. GnRH neurons provide an interesting neuronal phenotype with simple, relatively unbranched, unipolar or bipolar dendrites of extensive lengths (>1000 microm) covered in spines. These neurons control fertility and must integrate a variety of internal homeostatic and external environmental cues. We used imaging, electrophysiological, and modeling studies to understand how they integrate and process information along dendrites. Simultaneous recordings from distal dendrites and somata of individual GnRH neurons indicate distal dendrites are the primary site of spike initiation in these cells. Compartmental modeling indicates that sites of spike initiation depend upon location of excitatory input and dendrite geometry. Together, these studies demonstrate a novel pattern of spike generation in mammalian neurons and indicate that afferent inputs within distal dendritic microdomains directly initiate action potentials.

Emerging methodologies for the study of hypothalamic gonadotropin-releasing-hormone (GnRH) neurons.

Gonadotropin-releasing-hormone (GnRH) neurons form part of a central neural oscillator that controls sexual reproduction through intermittent release of the GnRH peptide. Activity of GnRH neurons, and by extension release of GnRH, has been proposed to reflect intrinsic properties and synaptic input of GnRH neurons. To study GnRH neurons, we used traditional electrophysiology and computational methods. These emerging methodologies enhance the elucidation of processing in GnRH neurons. We used dynamic current-clamping to understand how living GnRH somata process input from glutamate and GABA, two key neurotransmitters in the neuroendocrine hypothalamus. In order to study the impact of synaptic integration in dendrites and neuronal morphology, we have developed full-morphology models of GnRH neurons. Using dynamic clamping, we have demonstrated that small-amplitude glutamatergic currents can drive repetitive firing in GnRH neurons. Furthermore, application of simulated GABAergic synapses with a depolarized reversal potential have revealed two functional subpopulations of GnRH neurons: one population in which GABA chronically depolarizes membrane potential (without inducing action potentials) and a second population in which GABAergic excitation results in slow spiking. Finally, when AMPA-type and GABA-type simulated inputs are applied together, action potentials occur when the AMPA-type conductance occurs during the descending phase of GABAergic excitation and at the nadir of GABAergic inhibition. Compartmental computer models have shown that excitatory synapses at >300 microns from somtata are unable to drive spiking with purely passive dendrites. In models with active dendrites, distal synapses are more efficient at driving spiking than somatic inputs. We then used our models to extend the results from dynamic current clamping at GnRH somata to distribute synaptic inputs along the dendrite. We show that propagation delays for dendritic synapses alter synaptic integration in GnRH neurons by widening the temporal window of interaction for the generation of action potentials. Finally, we have shown that changes in dendrite morphology can modulate the output of GnRH neurons by altering the efficacy of action potential generation in response to after-depolarization potentials (ADPs). Taken together, the methodologies of dynamic current clamping and multi-compartmental modeling can make major contributions to the study of synaptic integration and structure-function relationships in hypothalamic GnRH neurons. Use of these methodological approaches will continue to provide keen insights leading to conceptual advances in our understanding of reproductive hormone secretion in normal and pathological physiology and open the door to understanding whether the mechanisms of pulsatile GnRH release are conserved across species.