Effect of size on expression of bistability in mouse spinal motoneurons.

Bistability in spinal motoneurons supports tonic spike activity in the absence of excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons innervating slow antigravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers display bistability is still controversial. To address this, we examined the relationship between soma size and bistability in lumbar (L4-L5) ventrolateral α-motoneurons of choline acetyltransferase (ChAT)-green fluorescent protein (GFP) and Hb9-GFP mice during the first 4 wk of life. We found that as neuron size increases, the prevalence of bistability rises. Smaller α-motoneurons lack bistability, whereas larger fast α-motoneurons [matrix metalloproteinase-9 (MMP-9)+/Hb9+] with a soma area ≥ 400 µm2 exhibit significantly higher bistability. Ionic currents associated with bistability, including the persistent Nav1.6 current, the thermosensitive Trpm5 Ca2+-activated Na+ current, and the slowly inactivating Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable properties but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size correlates with bistability in mice.NEW & NOTEWORTHY Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow motoneurons but present in most large, fast motoneurons. This difference results from differential expression of ionic currents that enable bistability, which are highly expressed in large motoneurons but small or absent in small motoneurons. These results support a possible role for fast motoneurons in maintenance of tonic posture in addition to their known roles in fast movements.

A size principle for bistability in mouse spinal motoneurons.

Bistability in spinal motoneurons supports tonic spike activity in the absence of excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons innervating slow antigravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers display bistability related to postural tone is still controversial. To address this, we examined the relationship between soma size and bistability in lumbar ventrolateral α-motoneurons of ChAT-GFP and Hb9-GFP mice across different developmental stages: neonatal (P2-P7), young (P7-P14) and mature (P21-P25). We found that as neuron size increases, the prevalence of bistability rises. Smaller α-motoneurons lack bistability, while larger fast α-motoneurons (MMP-9 + /Hb9 + ) with a soma area ≥ 400µm 2 exhibit significantly higher bistability. Ionic currents associated with bistability, including the persistent Nav1.6 current, thermosensitive Trpm5 Ca 2+ -activated Na + current and the slowly inactivating Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable properties, but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size dictates this process. New and Noteworthy: Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow motoneurons but present in most large, fast motoneurons. This difference results from differential expression of ionic currents that enable bistability, which are highly expressed in large motoneurons but small or absent in small motoneurons. These results support a possible role for fast motoneurons in maintenance of tonic posture in addition to their known roles in fast movements.

Effects of Chronic Spinal Cord Injury on Relationships among Ion Channel and Receptor mRNAs in Mouse Lumbar Spinal Cord.

Spinal cord injury (SCI) causes widespread changes in gene expression of the spinal cord, even in the undamaged spinal cord below the level of the lesion. Less is known about changes in the correlated expression of genes after SCI. We investigated gene co-expression networks among voltage-gated ion channel and neurotransmitter receptor mRNA levels using quantitative RT-PCR in longitudinal slices of the mouse lumbar spinal cord in control and chronic SCI animals. These longitudinal slices were made from the ventral surface of the cord, thus forming slices relatively enriched in motor neurons or interneurons. We performed absolute quantitation of mRNA copy number for 50 ion channel or receptor transcripts from each sample, and used multiple correlation analyses to detect patterns in correlated mRNA levels across all pairs of genes. The majority of channels and receptors changed in expression as a result of chronic SCI, but did so differently across slice levels. Furthermore, motor neuron-enriched slices experienced an overall loss of correlated channel and receptor expression, while interneuron slices showed a dramatic increase in the number of positively correlated transcripts. These correlation profiles suggest that spinal cord injury induces distinct changes across cell types in the organization of gene co-expression networks for ion channels and transmitter receptors.

Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomotion.

Spinal motoneurons are endowed with nonlinear spiking behaviors manifested by a spike acceleration whose functional significance remains uncertain. Here, we show in rodent lumbar motoneurons that these nonlinear spiking properties do not rely only on activation of dendritic nifedipine-sensitive L-type Ca2+ channels, as assumed for decades, but also on the slow inactivation of a nifedipine-sensitive K+ current mediated by Kv1.2 channels that are highly expressed in axon initial segments. Specifically, the pharmacological and computational inhibition of Kv1.2 channels occluded the spike acceleration of rhythmically active motoneurons and the correlated slow buildup of rhythmic motor output recorded at the onset of locomotor-like activity. This study demonstrates that slow inactivation of Kv1.2 channels provides a potent gain control mechanism in mammalian spinal motoneurons and has a behavioral role in enhancing locomotor drive during the transition from immobility to steady-state locomotion.

Organization of left-right coordination of neuronal activity in the mammalian spinal cord: Insights from computational modelling.

KEY POINTS: Coordination of neuronal activity between left and right sides of the mammalian spinal cord is provided by several sets of commissural interneurons (CINs) whose axons cross the midline. Genetically identified inhibitory V0D and excitatory V0V CINs and ipsilaterally projecting excitatory V2a interneurons were shown to secure left-right alternation at different locomotor speeds. We have developed computational models of neuronal circuits in the spinal cord that include left and right rhythm-generating centres interacting bilaterally via three parallel pathways mediated by V0D , V2a-V0V and V3 neuron populations. The models reproduce the experimentally observed speed-dependent left-right coordination in normal mice and the changes in coordination seen in mutants lacking specific neuron classes. The models propose an explanation for several experimental results and provide insights into the organization of the spinal locomotor network and parallel CIN pathways involved in gait control at different locomotor speeds. ABSTRACT: Different locomotor gaits in mammals, such as walking or galloping, are produced by coordinated activity in neuronal circuits in the spinal cord. Coordination of neuronal activity between left and right sides of the cord is provided by commissural interneurons (CINs), whose axons cross the midline. In this study, we construct and analyse two computational models of spinal locomotor circuits consisting of left and right rhythm generators interacting bilaterally via several neuronal pathways mediated by different CINs. The CIN populations incorporated in the models include the genetically identified inhibitory (V0D ) and excitatory (V0V ) subtypes of V0 CINs and excitatory V3 CINs. The model also includes the ipsilaterally projecting excitatory V2a interneurons mediating excitatory drive to the V0V CINs. The proposed network architectures and CIN connectivity allow the models to closely reproduce and suggest mechanistic explanations for several experimental observations. These phenomena include: different speed-dependent contributions of V0D and V0V CINs and V2a interneurons to left-right alternation of neural activity, switching gaits between the left-right alternating walking-like activity and the left-right synchronous hopping-like pattern in mutants lacking specific neuron classes, and speed-dependent asymmetric changes of flexor and extensor phase durations. The models provide insights into the architecture of spinal network and the organization of parallel inhibitory and excitatory CIN pathways and suggest explanations for how these pathways maintain alternating and synchronous gaits at different locomotor speeds. The models propose testable predictions about the neural organization and operation of mammalian locomotor circuits.

Sensitization of neonatal rat lumbar motoneuron by the inflammatory pain mediator bradykinin.

Bradykinin (Bk) is a potent inflammatory mediator that causes hyperalgesia. The action of Bk on the sensory system is well documented but its effects on motoneurons, the final pathway of the motor system, are unknown. By a combination of patch-clamp recordings and two-photon calcium imaging, we found that Bk strongly sensitizes spinal motoneurons. Sensitization was characterized by an increased ability to generate self-sustained spiking in response to excitatory inputs. Our pharmacological study described a dual ionic mechanism to sensitize motoneurons, including inhibition of a barium-sensitive resting K(+) conductance and activation of a nonselective cationic conductance primarily mediated by Na(+). Examination of the upstream signaling pathways provided evidence for postsynaptic activation of B2 receptors, G protein activation of phospholipase C, InsP3 synthesis, and calmodulin activation. This study questions the influence of motoneurons in the assessment of hyperalgesia since the withdrawal motor reflex is commonly used as a surrogate pain model.

Adult spinal V2a interneurons show increased excitability and serotonin-dependent bistability.

In mice, most studies of the organization of the spinal central pattern generator (CPG) for locomotion, and its component neuron classes, have been performed on neonatal [postnatal day (P)2-P4] animals. While the neonatal spinal cord can generate a basic locomotor pattern, it is often argued that the CPG network is in an immature form whose detailed properties mature with postnatal development. Here, we compare intrinsic properties and serotonergic modulation of the V2a class of excitatory spinal interneurons in behaviorally mature (older than P43) mice to those in neonatal mice. Using perforated patch recordings from genetically tagged V2a interneurons, we revealed an age-dependent increase in excitability. The input resistance increased, the rheobase values decreased, and the relation between injected current and firing frequency (F/I plot) showed higher excitability in the adult neurons, with almost all neurons firing tonically during a current step. The adult action potential (AP) properties became narrower and taller, and the AP threshold hyperpolarized. While in neonates the AP afterhyperpolarization was monophasic, most adult V2a interneurons showed a biphasic afterhyperpolarization. Serotonin increased excitability and depolarized most neonatal and adult V2a interneurons. However, in ∼30% of adult V2a interneurons, serotonin additionally elicited spontaneous intrinsic membrane potential bistability, resulting in alternations between hyperpolarized and depolarized states with a dramatically decreased membrane input resistance and facilitation of evoked plateau potentials. This was never seen in younger animals. Our findings indicate a significant postnatal development of the properties of locomotor-related V2a interneurons, which could alter their interpretation of synaptic inputs in the locomotor CPG.

TTX-resistant NMDA receptor-mediated membrane potential oscillations in neonatal mouse Hb9 interneurons.

Conditional neuronal membrane potential oscillations have been identified as a potential mechanism to help support or generate rhythmogenesis in neural circuits. A genetically identified population of ventromedial interneurons, called Hb9, in the mouse spinal cord has been shown to generate TTX-resistant membrane potential oscillations in the presence of NMDA, serotonin and dopamine, but these oscillatory properties are not well characterized. Hb9 interneurons are rhythmically active during fictive locomotor-like behavior. In this study, we report that exogenous N-Methyl-D-Aspartic acid (NMDA) application is sufficient to produce membrane potential oscillations in Hb9 interneurons. In contrast, exogenous serotonin and dopamine application, alone or in combination, are not sufficient. The properties of NMDA-induced oscillations vary among the Hb9 interneuron population; their frequency and amplitude increase with increasing NMDA concentration. NMDA does not modulate the T-type calcium current (I(Ca(T))), which is thought to be important in generating locomotor-like activity, in Hb9 neurons. These results suggest that NMDA receptor activation is sufficient for the generation of TTX-resistant NMDA-induced membrane potential oscillations in Hb9 interneurons.

A comparison of serotonin neuromodulation of mouse spinal V2a interneurons using perforated patch and whole cell recording techniques.

Whole cell recordings (WCRs) are frequently used to study neuronal properties, but may be problematic when studying neuromodulatory responses, due to dialysis of the cell's cytoplasm. Perforated patch recordings (PPR) avoid cellular dialysis and might reveal additional modulatory effects that are lost during WCR. We have previously used WCR to characterize the responses of the V2a class of Chx10-expressing neurons to serotonin (5-HT) in the neonatal mouse spinal cord (Zhong et al., 2010). Here we directly compare multiple aspects of the responses to 5-HT using WCR and PPR in Chx10-eCFP neurons in spinal cord slices from 2 to 4 day old mice. Cellular properties recorded in PPR and WCR were similar, but high-quality PP recordings could be maintained for significantly longer. Both WCR and PPR cells could respond to 5-HT, and although neurons recorded by PPR showed a significantly greater response to 5-HT in some parameters, the absolute differences between PPR and WCR were small. We conclude that WCR is an acceptable recording method for short-term recordings of neuromodulatory effects, but the less invasive PPR is preferable for detailed analyses and is necessary for stable recordings lasting an hour or more.

Spinal cord injury induces serotonin supersensitivity without increasing intrinsic excitability of mouse V2a interneurons.

Denervation-induced plastic changes impair locomotor recovery after spinal cord injury (SCI). Spinal motoneurons become hyperexcitable after SCI, but the plastic responses of locomotor network interneurons (INs) after SCI have not been studied. Using an adult mouse SCI model, we analyzed the effects of complete spinal cord lesions on the intrinsic electrophysiological properties, excitability, and neuromodulatory responses to serotonin (5-HT) in mouse lumbar V2a spinal INs, which help regulate left-right alternation during locomotion. Four weeks after SCI, V2a INs showed almost no changes in baseline excitability or action potential properties; the only parameter that changed was a reduced input resistance. However, V2a INs became 100- to 1000-fold more sensitive to 5-HT. Immunocytochemical analysis showed that SCI caused a coordinated loss of serotonergic fibers and the 5-HT transporter (SERT). Blocking the SERT with citalopram in intact mice did not increase 5-HT sensitivity to the level seen after SCI. SCI also evoked an increase in 5-HT(2C) receptor cluster number and intensity, suggesting that several plastic changes cooperate in increasing 5-HT sensitivity. Our results suggest that different components of the spinal neuronal network responsible for coordinating locomotion are differentially affected by SCI, and highlight the importance of understanding these changes when considering therapies targeted at functional recovery.

Neuronal activity in the isolated mouse spinal cord during spontaneous deletions in fictive locomotion: insights into locomotor central pattern generator organization.

We explored the organization of the spinal central pattern generator (CPG) for locomotion by analysing the activity of spinal interneurons and motoneurons during spontaneous deletions occurring during fictive locomotion in the isolated neonatal mouse spinal cord, following earlier work on locomotor deletions in the cat. In the isolated mouse spinal cord, most spontaneous deletions were non-resetting, with rhythmic activity resuming after an integer number of cycles. Flexor and extensor deletions showed marked asymmetry: flexor deletions were accompanied by sustained ipsilateral extensor activity, whereas rhythmic flexor bursting was not perturbed during extensor deletions. Rhythmic activity on one side of the cord was not perturbed during non-resetting spontaneous deletions on the other side, and these deletions could occur with no input from the other side of the cord. These results suggest that the locomotor CPG has a two-level organization with rhythm-generating (RG) and pattern-forming (PF) networks, in which only the flexor RG network is intrinsically rhythmic. To further explore the neuronal organization of the CPG, we monitored activity of motoneurons and selected identified interneurons during spontaneous non-resetting deletions. Motoneurons lost rhythmic synaptic drive during ipsilateral deletions. Flexor-related commissural interneurons continued to fire rhythmically during non-resetting ipsilateral flexor deletions. Deletion analysis revealed two classes of rhythmic V2a interneurons. Type I V2a interneurons retained rhythmic synaptic drive and firing during ipsilateral motor deletions, while type IIV2a interneurons lost rhythmic synaptic input and fell silent during deletions. This suggests that the type I neurons are components of the RG, whereas the type II neurons are components of the PF network.We propose a computational model of the spinal locomotor CPG that reproduces our experimental results. The results may provide novel insights into the organization of spinal locomotor networks.

Neuromodulator-evoked synaptic metaplasticity within a central pattern generator network.

Synapses show short-term activity-dependent dynamics that alter the strength of neuronal interactions. This synaptic plasticity can be tuned by neuromodulation as a form of metaplasticity. We examined neuromodulator-induced metaplasticity at a graded chemical synapse in a model central pattern generator (CPG), the pyloric network of the spiny lobster stomatogastric ganglion. Dopamine, serotonin, and octopamine each produce a unique motor pattern from the pyloric network, partially through their modulation of synaptic strength in the network. We characterized synaptic depression and its amine modulation at the graded synapse from the pyloric dilator neuron to the lateral pyloric neuron (PD→LP synapse), driving the PD neuron with both long square pulses and trains of realistic waveforms over a range of presynaptic voltages. We found that the three amines can differentially affect the amplitude of graded synaptic transmission independently of the synaptic dynamics. Low concentrations of dopamine had weak and variable effects on the strength of the graded inhibitory postsynaptic potentials (gIPSPs) but reliably accelerated the onset of synaptic depression and recovery from depression independently of gIPSP amplitude. Octopamine enhanced gIPSP amplitude but decreased the amount of synaptic depression; it slowed the onset of depression and accelerated its recovery during square pulse stimulation. Serotonin reduced gIPSP amplitude but increased the amount of synaptic depression and accelerated the onset of depression. These results suggest that amine-induced metaplasticity at graded chemical synapses can alter the parameters of synaptic dynamics in multiple and independent ways.

Postnatal emergence of serotonin-induced plateau potentials in commissural interneurons of the mouse spinal cord.

Most studies of the mouse hindlimb locomotor network have used neonatal (P0-5) mice. In this study, we examine the postnatal development of intrinsic properties and serotonergic modulation of intersegmental commissural interneurons (CINs) from the neonatal period (P0-3) to the time the animals bear weight (P8-10) and begin to show adult walking (P14-16). CINs show an increase in excitability with age, associated with a decrease in action potential halfwidth and appearance of a fast component to the afterhyperpolarization at P14-16. Serotonin (5-HT) depolarizes and increases the excitability of most CINs at all ages. The major developmental difference is that serotonin can induce plateau potential capability in P14-16 CINs, but not at younger ages. These plateau potentials are abolished by nifedipine, suggesting that they are mediated by an L-type calcium current, I(Ca(L)). Voltage-clamp analysis demonstrates that 5-HT increases a nifedipine-sensitive voltage-activated calcium current, I(Ca(V)), in P14-16 CINs but does not increase I(Ca(V)) in P8-10 CINs. These results, together with earlier work on 5-HT effects on neonatal CINs, suggest that 5-HT increases the excitability of CINs at all ages studied, but by opposite effects on calcium currents, decreasing N- and P/Q-type calcium currents and, indirectly, calcium-activated potassium current, at P0-3 but increasing I(Ca(L)) at P14-16.

Serotonin modulates multiple calcium current subtypes in commissural interneurons of the neonatal mouse.

Calcium currents are critical to the intrinsic properties of neurons and the networks that contain them. These currents make attractive targets for neuromodulation. Here, we examine the serotonergic modulation of specific calcium current subtypes in neonatal (P0-5) intersegmental commissural interneurons (CINs), members of the hindlimb locomotor central pattern generator in the mouse spinal cord. Previous work in our lab showed that serotonin (5-HT) excited CINs in part by reducing a calcium current and thus indirectly reducing the calcium-activated potassium current (Diaz-Rios et al. 2007). We have determined which calcium currents are targets of serotonin modulation. Utilizing whole cell voltage clamp and toxins to specific calcium current subtypes, we found that N- and P/Q-type currents comprise over 60% of the overall calcium current. Blockade of each of these subtypes alone with either ω-conotoxin GVIA or ω-agatoxin TK was unable to occlude 5-HT's reduction of the calcium current. However, coapplication of both blockers together fully occluded 5-HT's reduction of the calcium current. Thus, 5-HT decreases both N- and P/Q-type calcium current to excite neonatal CINs.

Long-duration perforated patch recordings from spinal interneurons of adult mice.

It has been very difficult to record from interneurons in acute slices of the lumbar spinal cord from mice >3 wk of age. The low success rate and short recording times limit in vitro experimentation on mouse spinal networks to neonatal and early postnatal periods when locomotor networks are still developmentally immature. To overcome this limitation and enable investigation of mature locomotor network neurons, we have established a reliable procedure to record from spinal cord neurons in slices from adult, behaviorally mature mice of any age. Two key changes to the established neonate procedure were implemented. First, we remove the cord by a dorsal laminectomy from a deeply anesthetized animal. This enables respiration and other vital functions to continue up to the moment the maximally oxygenated lumbar spinal cord is removed, improving the health of the slices. Second, since adult spinal cord interneurons appear more sensitive to the intracellular dialysis that occurs during whole cell recordings, we introduced perforated patch recordings to the procedure. Stable recordings up to 12 h in duration were obtained with our new method. This will allow investigation of changes in mature neuronal properties in disease states or after spinal cord injury and allow prolonged recordings of responses to drug application that were previously impossible.

Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores.

Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network. We analyzed the mechanisms by which DA evokes bursting in the AB neuron. Previous work showed that DA-evoked bursting is critically dependent on external calcium (Harris-Warrick RM, Flamm RE. J Neurosci 7: 2113-2128, 1987). Using two-photon microscopy and calcium imaging, we show that DA evokes the release of calcium from intracellular stores well before the emergence of voltage oscillations. When this release from intracellular stores is blocked by antagonists of ryanodine or inositol trisphosphate (IP(3)) receptor channels, DA fails to evoke AB bursting. We further demonstrate that DA enhances the calcium-activated inward current, I(CAN), despite the fact that it significantly reduces voltage-activated calcium currents. This suggests that DA-induced release of calcium from intracellular stores activates I(CAN), which provides a depolarizing ramp current that underlies endogenous bursting in the AB neuron.

Neuromodulation and flexibility in Central Pattern Generator networks.

Central Pattern Generator (CPG) networks, which organize rhythmic movements, have long served as models for neural network organization. Modulatory inputs are essential components of CPG function: neuromodulators set the parameters of CPG neurons and synapses to render the networks functional. Each modulator acts on the network by many effects which may oppose one another; this may serve to stabilize the modulated state. Neuromodulators also determine the active neuronal composition in the CPG, which varies with state changes such as locomotor speed. The pattern of gene expression which determines the electrophysiological personality of each CPG neuron is also under modulatory control. It is not possible to model the function of neural networks without including the actions of neuromodulators.

Localization and function of Ih channels in a small neural network.

Subthreshold ionic currents, which activate below the firing threshold and shape the cell's firing properties, play important roles in shaping neural network activity. We examined the distribution and synaptic roles of the hyperpolarization-activated inward current (I(h)) in the pyloric network of the lobster stomatogastric ganglion (STG). I(h) channels are expressed throughout the STG in a patchy distribution and are highly expressed in the fine neuropil, an area that is rich in synaptic contacts. We performed double labeling for I(h) protein and for the presynaptic marker synaptotagmin. The large majority of labeling in the fine neuropil was adjacent but nonoverlapping, suggesting that I(h) is localized in close proximity to synapses but not in the presynaptic terminals. We compared the pattern of I(h) localization with Shal transient potassium channels, whose expression is coregulated with I(h) in many STG neurons. Unlike I(h), we found significant levels of Shal protein in the soma membrane and the primary neurite. Both proteins were found in the synaptic fine neuropil, but with little evidence of colocalization in individual neurites. We performed electrophysiological experiments to study a potential role for I(h) in regulating synaptic transmission. At a synapse between two identified pyloric neurons, the amplitude of inhibitory postsynaptic potentials (IPSPs) decreased with increasing postsynaptic activation of I(h). Pharmacological block of I(h) restored IPSP amplitudes to levels seen when I(h) was not activated. These experiments suggest that modulation of postsynaptic I(h) might play an important role in the control of synaptic strength in this rhythmogenic neural network.

Frequency-dependent recruitment of V2a interneurons during fictive locomotion in the mouse spinal cord.

The principles governing the recruitment of interneurons during acceleration in vertebrate locomotion are unknown. In the mouse, the V2a spinal interneurons are dispensable for left-right coordination at low locomotor frequencies, but their function is essential for maintaining left-right coordination at high frequencies. Here we explore the mechanisms driving this frequency-dependent role using four methods to determine how V2a interneurons are recruited at different locomotor frequencies. We show that half of the V2a interneurons receive rhythmic locomotor synaptic drive, which increases with cycle frequency, recruiting more of the neurons to fire at higher frequencies. The other V2a interneurons do not receive locomotion-related synaptic drive and are not recruited into the locomotor network at any frequency. The increased role of V2a interneurons at higher locomotor frequencies arises from increased synaptic drive to recruit subthreshold oscillating V2a neurons, and not from recruitment of a second set of silent V2a interneurons.

VIP, CRF, and PACAP act at distinct receptors to elicit different cAMP/PKA dynamics in the neocortex.

The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase-activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription-polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects.