Peter J. Hemond

Distinct classes of pyramidal cells exhibit mutually exclusive firing patterns in hippocampal area CA3b

It is thought that CA3 pyramidal neurons communicate mainly through bursts of spikes rather than so‐called trains of regular firing action potentials. Reports of both burst firing and nonburst firing CA3 cells suggest that they may fire with more than one output pattern. With the use of whole‐cell recording methods we studied the firing properties of rat hippocampal pyramidal neurons in vitro within the CA3b subregion and found three distinct types of firing patterns. Approximately 37% of cells were regular firing where spikes generated by minimal current injection (rheobase) were elicited with a short latency and with stronger current intensities trains of spikes exhibited spike frequency adaptation (SFA). Another 46% of neurons exhibited a delayed onset at rheobase with a weakly‐adapting firing pattern upon stronger stimulation. The remaining 17% of cells showed a burst‐firing pattern, though only elicited in response to strong current injection and spontaneous bursts were never observed. Control experiments indicated that the distinct firing patterns were not due to our particular slicing methods or recording techniques. Finally, computer modeling was used to identify how relative differences in K+ conductances, specifically KC, KM, and KD, between cells contribute to the different characteristics of the three types of firing patterns observed experimentally.

Caloric restriction prevents aging-associated changes in spike-mediated Ca2+ accumulation and the slow afterhyperpolarization in hippocampal CA1 pyramidal neurons.

In hippocampal pyramidal neurons from aged animals voltage-gated Ca2+ entry and the slow, post-burst afterhyperpolarization are enhanced. As a result, there is a decrease in neuronal excitability and, in turn, an alteration in synaptic plasticity. Restricting the caloric intake of a rodent is a well-known paradigm for increasing lifespan and ameliorating a number of neurodegenerative features of aging, including deficits in synaptic plasticity and cognition. Here we show in rat CA1 pyramidal neurons from aged animals (18-20 months old) that a restricted diet prevents the enhancement of dendritic spike-mediated Ca2+ accumulation. In contrast, no significant changes in the rates of Ca2+ recovery were observed suggesting that Ca2+ clearance mechanisms are not affected by aging or caloric restriction. Lastly, we found that caloric restriction also prevented the aging-associated increase in the slow, post-burst afterhyperpolarization. Our results suggest that caloric restriction-sensitive changes in Ca2+ accumulation and membrane excitability may in part account for the protective effects of dietary restriction on synaptic plasticity and learning deficits in aged animals.

Synaptic integration in hypothalamic gonadotropin releasing hormone (GnRH) neurons

The impact of the A-type GABA (GABA-A) receptor in gonadotropin releasing hormone (GnRH) neurons is controversial. In adult GnRH neurons, the GABA-A receptor conductance has been reported to either hyperpolarize or depolarize GnRH neurons. Regardless of whether GABA is inhibitory or excitatory in GnRH neurons, GABAergic input would be integrated with post-synaptic potentials generated by other synaptic inputs. We used dynamic current clamping and compartmental computer modeling to examine the integration of AMPA-type glutamatergic input and GABA-mediated input in both the hyperpolarizing (inhibitory) and depolarizing (excitatory) modes in GnRH neurons from transgenic mice (Mus Musculus) generated on a C57BL6 background. In both living and model neurons, action potentials were most likely a few ms after a maximum in AMPA conductance coincided with a minimum in inhibitory GABA. Excitatory GABA interacted differently with AMPA, with spikes most likely, in both dynamic clamping of living neurons and in model neurons, when a maximum in AMPA coincided with the decay from peak of a maximum in GABA. Distributing synapses along the dendrite maximized the temporal relationship between AMPA and GABA conductances and therefore, the potential for spiking. Thus, these two dominant neurotransmitters could interact in multiple frames to generate action potentials in GnRH neurons.

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.

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 GABAA 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.

The membrane response of hippocampal CA3b pyramidal neurons near rest: Heterogeneity of passive properties and the contribution of hyperpolarization-activated currents

Pyramidal neurons in the CA3 region of the hippocampal formation integrate synaptic information arriving in the dendrites within discrete laminar regions. At potentials near or below the resting potential integration of synaptic signals is most affected by the passive properties of the cell and hyperpolarization-activated currents (I(h)). Here we focused specifically on a subset of neurons within the CA3b subregion of the rat hippocampus in order to better understand their membrane response within subthreshold voltage ranges. Using a combined experimental and computational approach we found that the passive properties of these neurons varied up to fivefold between cells. Likewise, there was a large variance in the expression of I(h) channels. However, the contribution of I(h) was minimal at resting potentials endowing the membrane with an apparent linear response to somatic current injection within +/-10 mV. Unlike in CA1 pyramidal neurons, however, I(h) activation was not potentiated in an activity-dependent manner. Computer modeling, based on a combination of voltage- and current-clamp data, suggested that an increasing density of these channels with distance from the soma, compared with a uniform distribution, would have no significant effect on the general properties of the cell because of their relatively lower expression. Nonetheless, temporal summation of excitatory inputs was affected by the presence of I(h) in the dendrites in a frequency- and distance-dependent fashion.

IGF2 knockout mice are resistant to kainic acid-induced seizures and neurodegeneration.

Insulin-like growth factor 2 (Igf2), a member of the insulin gene family, is important for brain development and has known neurotrophic properties. Though Igf2, its receptors, and binding proteins, are expressed in the adult CNS, their role in the adult brain is less well-understood. Here we studied how Igf2 deficiency affects brains of adult Igf2 knockout (Igf2(-/-)) mice following neurotoxic insult produced by the glutamate analog kainic acid (KA). Igf2(-/-) mice exhibited attenuated epileptiform activity in response to KA and were less susceptible to hippocampal neurodegeneration compared with Igf2(+/+) mice. Other brain areas protected by the lack of Igf2 included the amygdala complex, septal nuclei, and thalamic region. Apoptosis, as determined by TUNEL and Hoechst 33342 staining, was accordingly less for Igf2(-/-) mice. Hippocampal slices from Igf2(-/-) mice also were protected against the effects epileptogenic effects of KA compared to Igf2(+/+) mice suggesting that neuroprotection afforded by a lack of Igf2 may be developmental in origin and experiments demonstrating enhanced synaptic inhibition in slices taken from Igf2(-/-) mice support this hypothesis. Taken together, these results suggest that Igf2 may be important for mechanisms and circuits that contribute to neurodegeneration and epilepsy.

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.

Changes in dendritic architecture: not your "usual suspect" in control of the onset of puberty in male rats.

Until the recent past, the search for the underlying drive for the pubertal increase in gonadotropin-releasing hormone (GnRH) hormone from the GnRH-containing neurons in the hypothalamus was largely focused on extrinsic factors. The most recent evidence however indicates changes in the structure of GnRH neurons themselves may contribute to this fundamental event in development. Based on our studies in males, dendritic architecture is not static from birth until adulthood. Instead, dendrites undergo a dramatic remodeling during the postnatal period which is independent of testosterone and occurs before the pubertal increase in GnRH release. First, the number of dendrites emanating from somata is reduced between infancy and adulthood. Moreover, a dendrite of adult GnRH neurons invariability arises at angle of 180°from the axon as opposed to the extraordinary variability in location during infancy. In fact, in some neurons from infants, no dendrite even resides in the adult location. Thus, there is a spatially selective remodeling of primary dendrites. Secondly, dendrites of GnRH neurons from infants were highly branched prior to assuming the compact morphology of adults. Finally, other morphological aspects of GnRH neurons such as total dendritic length, the numbers of dendrite branches and the lengths of higher order branches were significantly greater in infants than adults, indicating a consolidation of dendritic arbors. Activity in multi-compartment models of GnRH neurons, suggest the impact of structure on neuronal activity is exerted with both active and passive dendrites. Thus, passive properties make a defining contribution to function. Accordingly, changes in morphology alone are likely to have functional consequences for the pattern of activity in GnRH neurons. Our findings suggest structural remodeling of dendrites during the postnatal period likely facilitates repetitive action potentials and thus, GnRH release at the time of puberty.

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.