Heinrich S. Gompf

Circadian Dynamics of Cytosolic and Nuclear Ca2+ in Single Suprachiasmatic Nucleus Neurons

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.

Locus Ceruleus and Anterior Cingulate Cortex Sustain Wakefulness in a Novel Environment

Locus ceruleus (LC) neuronal activity is correlated with the waking state, yet LC lesions produce only minor alterations in daily wakefulness. Here, we report that sustained elevations in neurobehavioral and EEG arousal in response to exposure to an environment with novel stimuli, including social interaction, are prevented by selective chemical lesions of the LC in rats. Similar results are seen when the anterior cingulate cortex (ACC), which receives especially dense LC innervation, is selectively denervated of LC input or is ablated by the cell-specific neurotoxin ibotenic acid. Anterograde tracing combined with tyrosine hydroxylase immunohistochemistry demonstrates ACC terminals in apposition with the distal dendrites of LC neurons. Our data implicate the ACC as both a source of input to the LC as well as one of its targets and suggests that the two structures engage in a dialog that may provide a critical neurobiological substrate for sustained attention.

Sleep disturbance induces neuroinflammation and impairment of learning and memory.

Hospitalized patients can develop cognitive function decline, the mechanisms of which remain largely to be determined. Sleep disturbance often occurs in hospitalized patients, and neuroinflammation can induce learning and memory impairment. We therefore set out to determine whether sleep disturbance can induce neuroinflammation and impairment of learning and memory in rodents. Five to 6-month-old wild-type C57BL/6J male mice were used in the studies. The mice were placed in rocking cages for 24 h, and two rolling balls were present in each cage. The mice were tested for learning and memory function using the Fear Conditioning Test one and 7 days post-sleep disturbance. Neuroinflammation in the mouse brain tissues was also determined. Of the Fear Conditioning studies at one day and 7 days after sleep disturbance, twenty-four hour sleep disturbance decreased freezing time in the context test, which assesses hippocampus-dependent learning and memory; but not the tone test, which assesses hippocampus-independent learning and memory. Sleep disturbance increased pro-inflammatory cytokine IL-6 levels and induced microglia activation in the mouse hippocampus, but not the cortex. These results suggest that sleep disturbance induces neuroinflammation in the mouse hippocampus, and impairs hippocampus-dependent learning and memory in mice. Pending further studies, these findings suggest that sleep disturbance-induced neuroinflammation and impairment of learning and memory may contribute to the development of cognitive function decline in hospitalized patients.

Blockade of astrocytic glutamate uptake in the prefrontal cortex induces anhedonia.

Major depression is associated with both dysregulated glutamatergic neurotransmission and fewer astrocytes in limbic areas including the prefrontal cortex (PFC). These deficits may be functionally related. Notably, astrocytes regulate glutamate levels by removing glutamate from the synapse via the glutamate transporter (GLT-1). Previously, we demonstrated that central blockade of GLT-1 induces anhedonia and c-Fos expression in the PFC. Given the role of the PFC in regulating mood, we hypothesized that GLT-1 blockade in the PFC alone would be sufficient to induce anhedonia in rats. We microinjected the GLT-1 inhibitor, dihydrokainic acid (DHK), into the PFC and examined the effects on mood using intracranial self-stimulation (ICSS). At lower doses, intra-PFC DHK produced modest increases in ICSS thresholds, reflecting a depressive-like effect. At higher doses, intra-PFC DHK resulted in cessation of responding. We conducted further tests to clarify whether this total cessation of responding was related to an anhedonic state (tested by sucrose intake), a nonspecific result of motor impairment (measured by the tape test), or seizure activity (measured with electroencephalogram (EEG)). The highest dose of DHK increased latency to begin drinking without altering total sucrose intake. Furthermore, neither motor impairment nor evidence of seizure activity was observed in the tape test or EEG recordings. A decrease in reward value followed by complete cessation of ICSS responding suggests an anhedonic-like effect of intra-PFC DHK; a conclusion that was substantiated by an increased latency to begin sucrose drinking. Overall, these results suggest that blockade of astrocytic glutamate uptake in the PFC is sufficient to produce anhedonia, a core symptom of depression.

Role of orexin input in the diurnal rhythm of locus coeruleus impulse activity

Activation of noradrenergic locus coeruleus (LC) neurons promotes wakefulness and behavioral arousal. In rats, LC neurons receive circadian inputs via a circuit that originates in the suprachiasmatic nucleus (SCN) and relays through the dorsomedial hypothalamus (DMH) to LC; this circuit input increases LC activity during the active period. DMH neurons expressing the peptide neurotransmitter orexin/hypocretin are ideally situated to act as a relay between SCN and LC due to their synaptic inputs from SCN and innervation of LC. Here, we examined the hypothesis that orexin is involved in transmitting circadian signals to LC using single-unit recordings of LC neurons in anesthetized rats maintained in 12:12 light-dark housing. We replicated earlier findings from this lab that LC neurons fire significantly faster on average during the active compared to rest periods. Local microinjection of an orexin antagonist, SB-334867-A attenuated the impulse activities of the fastest firing population of LC neurons during the active period. We also found that DMH orexin neurons project preferentially to LC and express a diurnal rhythm of activation that correlates with LC neuronal firing frequency. Therefore, we propose that DMH orexin neurons play a role in modulating the day-night differences of LC impulse activity.

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.

Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals

Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

Halothane-induced Hypnosis Is Not Accompanied by Inactivation of Orexinergic Output in Rodents

Background:One underexploited property of anesthetics is their ability to probe neuronal regulation of arousal. At appropriate doses, anesthetics reversibly obtund conscious perception. However, individual anesthetic agents may accomplish this by altering the function of distinct neuronal populations. Previously the authors showed that isoflurane and sevoflurane inhibit orexinergic neurons, delaying reintegration of sensory perception as denoted by emergence. Here the authors study the effects of halothane. As a halogenated alkane, halothane differs structurally, has a nonoverlapping series of molecular binding partners, and differentially modulates electrophysiologic properties of several ion channels when compared with its halogenated ether relatives. Methods:c-Fos immunohistochemistry and in vivo electrophysiology were used to assess neuronal activity. Anesthetic induction and emergence were determined behaviorally in narcoleptic orexin/ataxin-3 mice and control siblings exposed to halothane. Results:Halothane-induced hypnosis occurred despite lack of inhibition of orexinergic neurons in mice. In rats, extracellular single-unit recordings within the locus coeruleus showed significantly greater activity during halothane than during a comparable dose of isoflurane. Microinjection of the orexin-1 receptor antagonist SB-334867-A during the active period slowed firing rates of locus coeruleus neurons in halothane-anesthetized rats, but had no effect on isoflurane-anesthetized rats. Surprisingly, orexin/ataxin-3 transgenic mice, which develop narcolepsy with cataplexy because of loss of orexinergic neurons, did not show delayed emergence from halothane. Conclusion:Coordinated inhibition of hypothalamic orexinergic and locus coeruleus noradrenergic neurons is not required for anesthetic induction. Normal emergence from halothane-induced hypnosis in orexin-deficient mice suggests that additional wake-promoting systems likely remain active during general anesthesia produced by halothane.

3-Monoiodothyronamine: The rationale for its action as an endogenous adrenergic-blocking neuromodulator

The investigations reported here were designed to gain insights into the role of 3-monoiodothyronamine (T1AM) in the brain, where the amine was originally identified and characterized. Extensive deiodinase studies indicated that T1AM was derived from the T4 metabolite, reverse triiodothyronine (revT3), while functional studies provided well-confirmed evidence that T1AM has strong adrenergic-blocking effects. Because a state of adrenergic overactivity prevails when triiodothyronine (T3) concentrations become excessive, the possibility that T3s metabolic partner, revT3, might give rise to an antagonist of those T3 actions was thought to be reasonable. All T1AM studies thus far have required use of pharmacological doses. Therefore we considered that choosing a physiological site of action was a priority and focused on the locus coeruleus (LC), the major noradrenergic control center in the brain. Site-directed injections of T1AM into the LC elicited a significant, dose-dependent neuronal firing rate change in a subset of adrenergic neurons with an EC(50)=2.7 microM, a dose well within the physiological range. Further evidence for its physiological actions came from autoradiographic images obtained following intravenous carrier-free (125)I-labeled T1AM injection. These showed that the amine bound with high affinity to the LC and to other selected brain nuclei, each of which is both an LC target and a known T3 binding site. This new evidence points to a physiological role for T1AM as an endogenous adrenergic-blocking neuromodulator in the central noradrenergic system.

GABAergic synapses of the suprachiasmatic nucleus exhibit a diurnal rhythm of short‐term synaptic plasticity

We examined synaptic plasticity at intrasuprachiasmatic nucleus (SCN) γ‐aminobutyric acid (GABA)ergic synapses by measuring the paired‐pulse ratio between pairs of evoked inhibitory postsynaptic currents (IPSCs). Interstimulus intervals were chosen to represent the range of spontaneous action potential firing frequencies found in SCN neurons. A majority of synapses studied during the day exhibited paired‐pulse depression (PPD), whereas a majority of synapses studied during the night showed no PPD. Two types of PPD were found. Type 1 PPD expresses the greatest inhibition at shorter interstimulus intervals, is predominant in the early morning and is likely to be a result of vesicle depletion. Type 2 showed the greatest inhibition at interstimulus intervals between 175 and 225 ms, is found throughout the day yet rarely at night and is likely to be a result of a Ca2+‐dependent mechanism that is independent of pertussis toxin‐sensitive G‐proteins. Thus, multiple mechanisms of synaptic plasticity modulate intra‐SCN communication throughout the diurnal cycle.