Abstract Pacemaking neurons, often found in mammalian nervous systems, integrate their inputs differently than quiescent neurons. Rhythmic single-spike pacemaking that is robust to noise can be achieved with a slow process that enforces a “resting potential” at each point along a ramp-like interspike interval (ISI) coupled with a fast restorative component. To demonstrate this phenomenon, we modeled previously identified distinct subpopulations of midbrain dopamine neurons that differed in projection target and in the regularity of their pacemaking. In the model of the more regularly-firing subpopulation projecting to the dorsomedial striatum, K V 4 current was recruited by a deep after-hyperpolarizing potential (AHP) mediated by the SK channel. In the model of the less regularly-firing subpopulation projecting to the medial shell of the nucleus accumbens, the AHP was too shallow to recruit the K V 4 current. In the more regularly firing population, the trajectory in the phase space of membrane potential and slow inactivation of K V 4 was confined to move slowly through a narrow channel during the ramp-like portion of the ISI. Noisy perturbations from this channel were quickly damped by fast activation of K V 4. In contrast, the smaller AHP in the model of the subpopulation projecting to the medial shell of the nucleus accumbens failed to recruit Kv4-mediated current, therefore the narrow channel was never entered, greatly decreasing the regularity in the presence of noise. This mechanism may be broadly applicable to single-spike pacemakers and explains how slow pacemaking with small net currents can be robust to fluctuations in single channel openings. Author Summary Pacemaking cells spike at regular intervals without the need for external input. There are numerous examples of pacemaking cells in the nervous system. We show that a process with slow dynamics relative to the individual spikes can make regular pacemaking robust to the noise that is always present in biological systems.

Abstract Synaptopodin (SP) is a plasticity-related actin-modulating protein found in a subpopulation of mature cortical spines. Its presence within spines is essential for the formation of the spine apparatus (SA) organelle, a local calcium store. SP has been functionally linked to enhanced synaptic strength and long-term spine stability, i.e., indicators of potentiated synapses involved in memory trace formation. As SP is transcriptionally upregulated under contextual learning conditions in dentate granule cells (GCs), we generated a gain-of-function mouse model (Cyan Fluorescent Protein-SP transgenic mouse; CSPtg) with a ~threefold overexpression of SP to study the cellular effects of an increased availability of SP in GCs. Like the wild-type (WT) protein, transgenic SP was sorted to spines and rescued the SA organelle in GCs of SP-deficient mice. In CSPtg mice, SP was found in twice as many spines compared to WT. At the ultrastructural level, a ~threefold increase in the number of SAs was observed. Other structural properties, including spine density, average spine head size, and average SP puncta size, were not significantly altered. Patch-clamp recordings of GCs in CSPtg mice revealed an ~ 3.5-fold increase in miniature excitatory postsynaptic current frequency, indicating that the increased expression of SP/SA in spines resulted in a larger number of active synapses. Thus, higher levels of SP in GCs may increase the number of synapses available for strengthening and plasticity. This could enhance the ability of GCs to be recruited into new ensembles under contextual learning conditions.

Previously, electrophysiological differences between subpopulations of midbrain dopamine (DA) neurons were identified based on projection targets, including distinct responses to hyperpolarization and in the regularity of pacemaking. Here we explored single-compartment models of three subpopulations of DA neurons, projecting to medial shell of the nucleus accumbens (VTA-mNAcc), dorsomedial striatum (SNc-DMS) or dorsolateral striatum (SNc-DLS). We reduced the dimensionality to a phase plane consisting of membrane potential and one slow variable, either total slow potassium conductance or Kv4 channel inactivation. Nullclines are curves on which the rate of change of each variable is zero, given the value of the other variable. The voltage nullclines had three branches: upper spiking, unstable middle, and lower quiescent branch. Recruitment of Kv4 channels by the more prominent after-hyperpolarizing potential (AHP) in the DA-DMS and DA-DLS models channels stabilized pacemaking by creating a restorative moving fixed point along the quiescent branch. The slow inactivation of KV4 channels dominated and regularized the dynamics during the interspike interval; a dominant slow process may be a general mechanisn for stable regular pacemaking in a frequency range between 1-10 Hz. In contrast, the smaller AHP in VTA-mNAcc models prevented recruitment of this Kv4-mediated moving fixed point, which increased the sensitivity to synaptic inputs. On rebound from hyperpolarization the ability to produce robust ramps reverses between the DA neurons: now VTA-mNAcc projecting DA models fully recruited Kv4 channels and produced stable ramp-like pauses, whereas SNc-DLS projecting cells recruited significant regenerative inward CaV3 channels that overwhelmed Kv4 channels and produced ‘rebound’ bursts. ### Competing Interest Statement The authors have declared no competing interest.

Midbrain dopamine (DA) neurons are diverse with distinct subpopulations being essential for key functions of the brain: nigrostriatal DA neurons for voluntary movement and mesolimbic DA neurons for learning from reward prediction errors. In addition to being primarily associated with distinct senso-motor or limbic cortical-striatal circuits, DA subpopulations also directly communicate with each other via local DA release in the midbrain. While the inhibitory synaptic nature of this dopamine-to-dopamine signaling has been well established, the pre- and postsynaptic identity and logic of connectivity among DA subpopulations are still unresolved. To fill this gap, we combined retrograde tracing with projection-specific optogenetic stimulation of DA neurons and patch-clamp recordings in vitro in the adult mouse of either sex. We functionally identified a unidirectional, motor-to-limbic design of the DA synapse in the midbrain. This motor-to-limbic negative feedback connection in the midbrain was independently confirmed by monosynaptic rabies tracing of projection-defined DA subpopulations. This DA synapse might complement the limbic-to-motor striato-nigro-striatal feedforward architecture of the basal ganglia.

Dopamine neurons projecting to distinct brain regions have unique biophysical properties, which are thought to reflect functional specialization. One of these defining properties is the duration of hyperpolarization-induced firing pauses, or rebound delays, which are longer in atypical dopamine neurons targeting ventromedial striatal areas like the nucleus accumbens core. Differences in rebound delay are determined by Kv4 channel-mediated A-type currents, but the mechanisms underlying these differences and their functional implications are not well understood. We hypothesized that KChIP4a, a unique Kv4 β-subunit splice variant, determines rebound delay duration in atypical dopamine neurons. To test this, we generated a transgenic mouse line with dopamine neuron-selective removal of KChIP4a. We found that KChIP4a deletion shortened rebound delays in core-projecting atypical dopamine neurons by changing A-type current kinetics, without affecting conventional dopamine neurons. This indicates that KChIP4a acts as a selective biophysical amplifier of hyperpolarizing inhibition in core-projecting dopamine neurons, and computational modeling suggests that this effect is robust across a wide range of in vivo-like conditions for excitation/inhibition balance. Since firing pauses in core-projecting dopamine neurons are thought to drive learning from reward omission, or negative prediction errors, we tested the effect of KChIP4a deletion on different types of behavior in male and female mice. We found that removal of KChIP4a from dopamine neurons selectively accelerated learning from negative prediction errors, without affecting learning from positive prediction errors or other behavioral variables. Together, our results suggest that KChIP4a fine-tunes subthreshold excitability in projection-defined dopamine neuron populations to regulate specific types of learning.

Dopamine (DA) midbrain neurons are involved in a wide array of key brain functions including movement control and reward-based learning. They are also critical for major brain disorders such as Parkinson's disease or schizophrenia. DA neurons projecting to distinct striatal territories are diverse with regard to their molecular makeup and cellular physiology, which are likely to contribute to the observed differences in temporal DA dynamics. Among these regions, the dorsolateral striatum (DLS) displays the fastest DA dynamics, which might control the moment-to-moment vigor and variability of voluntary movements. However, the underlying mechanisms for these DLS-specific fast DA fluctuations are unresolved. Here, we show that DLS-projecting DA neurons in the substantia nigra (SN) possess a unique biophysical profile allowing immediate 10-fold accelerations in discharge frequency via rebound bursting. By using a combination of in vitro patch-clamp recordings in projection-defined DA SN subpopulations from adult male mice and developing matching projection-specific computational models, we demonstrate that a strong interaction of Cav3 and SK channels specific for DLS-projecting Aldh1a1-positive DA SN (DLS-DA) neurons controls the gain of fast rebound bursting, while Kv4 and HCN channels mediate timing of rebound excitability. In addition, GIRK channels activated by D2 and GABAB receptors prevent rebound bursting in these DLS-DA neurons. Furthermore, our in vivo patch-clamp recordings and matching in vivo computational models provide evidence that these unique rebound properties might be preserved in the intact brain, where they might endow specific computational properties well suited for the generation of fast DA dynamics present in DLS.

OBJECTIVE:Degeneration of hilar mossy cells in the dentate gyrus is an important hallmark of hippocampal sclerosis and is often observed in patients with temporal lobe epilepsy. To understand the pathogenesis of hippocampal sclerosis and develop novel neuroprotective treatments, it is critical to determine the mechanistic processes of mossy cell degeneration and factors that influence cell vulnerability or resilience. However, suitable in vitro approaches are currently lacking. We have developed and validated an organotypic slice culture-based in vitro model that facilitates mechanistic studies of activity-dependent mossy cell vulnerability and resilience. METHODS:A model was developed using entorhino-hippocampal slice cultures. Dentate gyrus granule cells were transduced with adeno-associated viruses to express channelrhodopsin2. Transduced cultures were chronically stimulated by light, and resulting cell damage was assessed by propidium iodide staining. Spontaneous synaptic activity before and after optical stimulation was recorded using whole-cell patch-clamp. RESULTS:Selective and dose-dependent hilar neuron degeneration was observed following chronic optogenetic stimulation of organotypic slice cultures expressing channelrhodopsin-2 in granule cells. Treatment with the anticonvulsant retigabine reduced stimulation-induced hilar neuron loss in a dose-dependent manner. This demonstrates the suitability of our optogenetic in vitro model for drug screening. Patch-clamp recordings verified strong synaptic activation of mossy cells during optical stimulation and a reduction in spontaneous excitatory synaptic activity after stimulation. SIGNIFICANCE:The role of mossy cells in the context of epileptic seizures has been a controversial topic of discussion. The presented in vitro model allows the study of mossy cell vulnerability on a single-cell level and provides the first evidence for changes in synaptic activity after stimulation. This model will facilitate our mechanistic understanding of temporal lobe epilepsy, providing a foundation for novel therapeutic interventions aimed at preserving mossy cell function in epilepsy patients.

Dopamine (DA) neurons in the substantia nigra (SN) control several essential functions, including the voluntary movement, learning, and motivated behavior. Healthy DA SN neurons show diverse firing patterns in vivo, ranging from slow pacemaker-like activity (1-10 Hz) to transient high-frequency bursts (<100 Hz), interspersed with pauses that can last hundreds of milliseconds. Recent in vivo patch experiments have started to reveal the subthreshold mechanisms underlying this physiological diversity, but the impact of challenges like cell loss on the in vivo activity of adult DA SN neurons, and how these may relate to behavioral disturbances, is still largely unknown. We investigated the in vivo electrophysiological properties of surviving SN DA neurons after partial unilateral 6-OHDA lesions, a single-hit, non-progressive model of neuronal cell loss. We show that mice subjected to this model have an initial motor impairment, measured by asymmetrical rotations in the open field test, which recovered over time. At 3 weeks post-lesion, when open field locomotion was strongly impaired, surviving DA SN neurons showed a compressed in vivo dynamic firing range, characterized by a 10-fold reduction of in vivo burst firing compared to controls. This in vivo phenotype was accompanied by pronounced in vitro pacemaker instability. In contrast, in the chronic post-lesion phase (>2 months), where turning symmetry in open field locomotion had recovered, surviving SN DA neurons displayed the full dynamic range of in vivo firing, including in vivo bursting, similar to controls. The normalized in vivo firing pattern was associated with a twofold acceleration of stable in vitro pacemaking, mediated by Kv4.3 potassium channel downregulation. Our findings demonstrate the existence of a homeostatic pacemaker plasticity mechanism in surviving DA SN neurons after pronounced cell loss.

Organotypic slice cultures (OTCs) are versatile tools for studying long-term structure-function relationships of neurons within a defined network (e.g. hippocampus). We developed a method for repeated experimenter-controlled activation of hippocampal granule cells (GCs) in OTCs within the incubator. After several days of contact-free photonic stimulation, we were able to ameliorate entorhinal denervation-induced structural damage in GCs. To achieve this outcome, we had to calibrate the intensity and duration of optogenetic (light) pulses using whole-cell electrophysiological recordings and multi-cell calcium imaging. Our findings showed that ChR2-expressing cells generated action potentials (APs) or calcium transients in response to illumination but were otherwise functionally indistinguishable from non-transduced GCs within the same neural circuit. However, the threshold for AP firing in single GCs varied based on the stimulus light intensity and the expression levels of ChR2. This information allowed us to calibrate light intensity for chronic stimulations. Denervated GCs exhibited significant spine loss four days post-denervation, but this detrimental effect was mitigated when AP firing was induced at a physiological GC bursting rate. Phototoxic damage caused by chronic light exposure was significantly reduced if illuminated with longer wavelength and by adding antioxidants to the culture medium. Our study presents a versatile approach for concurrent non-invasive manipulation and observation of neural circuit activity and remodeling in vitro.

Slow pacemaking is a key physiological process in specific excitable cells, yet its underlying mechanisms remain debated. Here we identify a conserved, voltage-dependent pacemaker current that is essential for slow, regular firing in both midbrain dopaminergic neurons and sinoatrial node cells. Conductance-based models incorporating this current reproduce stable pacemaking, requiring a fast-activating, small-amplitude current. This is further confirmed by dynamic-clamp experiments in dopaminergic neurons. Replacing the pacemaker current in a model with a voltage-independent conductance such as the non-selective sodium leak channels fails to sustain slow rhythmicity, highlighting the necessity for an adequate voltage dependence. Our results suggest a novel and shared biophysical mechanism for slow pacemaking in neuronal and cardiac systems. ### Competing Interest Statement The authors have declared no competing interest.

Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum–projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system.

Dissecting the diversity of midbrain dopamine (DA) neurons by optotagging is a promising addition to better identify their functional properties and contribution to motivated behavior. Retrograde molecular targeting of DA neurons with specific axonal projection allows further refinement of this approach. Here, we focus on adult mouse DA neurons in the substantia nigra pars compacta (SNc) projecting to dorsal striatum (DS) by demonstrating the selectivity of a floxed AAV9-based retrograde channelrhodopsin-eYFP (ChR-eYFP) labeling approach in DAT-cre mice. Furthermore, we show the utility of a sparse labeling version for anatomical single-cell reconstruction and demonstrate that ChR-eYFR expressing DA neurons retain intrinsic functional properties indistinguishable from conventionally retrogradely red-beads-labeled neurons. We systematically explore the properties of optogenetically evoked action potentials (o APs ) and their interaction with intrinsic pacemaking in this defined subpopulation of DA neurons. We found that the shape of the o AP and its first derivative, as a proxy for extracellularly recorded APs, is highly distinct from spontaneous APs (sAPs) of the same neurons and systematically varies across the pacemaker duty cycle. The timing of the o AP also affects the backbone oscillator of the intrinsic pacemaker by introducing transient “compensatory pauses”. Characterizing this systematic interplay between oAPs and sAPs in defined DA neurons will also facilitate a refinement of DA neuron optotagging in vivo.

The low-threshold L-type calcium channel Ca v 1.3 accelerates the pacemaker rate in the heart, but its functional role for the extended dynamic range of neuronal firing is still unresolved. Here, we show that Ca v 1.3 calcium channels act as unexpectedly simple, full-range linear amplifiers of firing rates for lateral dopamine substantia nigra (DA SN) neurons in mice. This means that they boost in vitro or in vivo firing frequencies between 2 and 50 hertz by about 30%. Furthermore, we demonstrate that clinically relevant, low nanomolar concentrations of the L-type channel inhibitor isradipine selectively reduce the in vivo firing activity of these nigrostriatal DA SN neurons at therapeutic plasma concentrations. Thus, our study identifies the pacemaker function of neuronal Ca v 1.3 channels and provides direct evidence that repurposing dihydropyridines such as isradipine is feasible to selectively modulate the in vivo activity of highly vulnerable DA SN subpopulations in Parkinson’s disease.