Developmental and evolutionary constraints on olfactory circuit selection.

SignificanceIn this work, we explore the hypothesis that biological neural networks optimize their architecture, through evolution, for learning. We study early olfactory circuits of mammals and insects, which have relatively similar structure but a huge diversity in size. We approximate these circuits as three-layer networks and estimate, analytically, the scaling of the optimal hidden-layer size with input-layer size. We find that both longevity and information in the genome constrain the hidden-layer size, so a range of allometric scalings is possible. However, the experimentally observed allometric scalings in mammals and insects are consistent with biologically plausible values. This analysis should pave the way for a deeper understanding of both biological and artificial networks.

Serotonergic afferents from the dorsal raphe decrease the excitability of pyramidal neurons in the anterior piriform cortex.

The olfactory system receives extensive serotonergic inputs from the dorsal raphe, a nucleus involved in control of behavior, regulation of mood, and modulation of sensory processing. Although many studies have investigated how serotonin modulates the olfactory bulb, few have focused on the anterior piriform cortex (aPC), a region important for olfactory learning and encoding of odor identity and intensity. Specifically, the mechanism and functional significance of serotonergic modulation of the aPC remain largely unknown. Here we used pharmacologic, optogenetic, and fiber photometry techniques to examine the serotonergic modulation of neural activity in the aPC in vitro and in vivo. We found that serotonin (5-HT) reduces the excitability of pyramidal neurons directly via 5-HT2C receptors, phospholipase C, and calcium-activated potassium (BK) channels. Furthermore, endogenous serotonin attenuates odor-evoked calcium responses in aPC pyramidal neurons. These findings identify the mechanism underlying serotonergic modulation of the aPC and shed light on its potential role.

Target-specific control of piriform cortical output via distinct inhibitory circuits.

Information represented by principal neurons in anterior piriform cortex (APC) is regulated by local, recurrent excitation and inhibition, but the circuit mechanisms remain elusive. Two types of layer 2 (L2) principal neurons, semilunar (SL), and superficial pyramidal (SP) cells, are parallel output channels, and the control of their activity gates the output of APC. Here, we examined the hypothesis that recurrent inhibition differentially regulates SL and SP cells. Patterned optogenetic stimulation revealed that the strength of recurrent inhibition is target- and layer-specific: L1 > L3 for SL cells, but L3 > L1 for SP cells. This target- and layer-specific inhibition was largely attributable to the parvalbumin (PV), but not somatostatin, interneurons. Intriguingly, olfactory experience selectively modulated the PV to SP microcircuit while maintaining the overall target and laminar specificity of inhibition. Together, these results indicate the importance of target-specific inhibitory wiring for odor processing, implicating these mechanisms in gating the output of piriform cortex.

Rapid Learning of Odor-Value Association in the Olfactory Striatum.

Rodents can successfully learn multiple novel stimulus-response associations after only a few repetitions when the contingencies predict reward. The circuits modified during such reinforcement learning to support decision-making are not known, but the olfactory tubercle (OT) and posterior piriform cortex (pPC) are candidates for decoding reward category from olfactory sensory input and relaying this information to cognitive and motor areas. Through single-cell recordings in behaving male and female C57BL/6 mice, we show here that an explicit representation for reward category emerges in the OT within minutes of learning a novel odor-reward association, whereas the pPC lacks an explicit representation even after weeks of overtraining. The explicit reward category representation in OT is visible in the first sniff (50-100 ms) of an odor on each trial, and precedes the motor action. Together, these results suggest that the coding of stimulus information required for reward prediction does not occur within olfactory cortex, but rather in circuits involving the olfactory striatum.SIGNIFICANCE STATEMENT Rodents are olfactory specialists and can use odors to learn contingencies quickly and well. We have found that mice can readily learn to place multiple odors into rewarded and unrewarded categories. Once they have learned the rule, they can do such categorization in a matter of minutes (<10 trials). We found that neural activity in olfactory cortex largely reflects sensory coding, with very little explicit information about categories. By contrast, neural activity in a brain region in the ventral striatum is rapidly modified in a matter of minutes to reflect reward category. Our experiments set up a paradigm for studying rapid sensorimotor reinforcement in a circuit that is right at the interface of sensory input and reward areas.

Temporal Differences in Interneuron Invasion of Neocortex and Piriform Cortex during Mouse Cortical Development.

Establishing a balance between excitation and inhibition is critical for brain functions. However, how inhibitory interneurons (INs) generated in the ventral telencephalon integrate with the excitatory neurons generated in the dorsal telencephalon remains elusive. Previous studies showed that INs migrating tangentially to enter the neocortex (NCx), remain in the migratory stream for days before invading the cortical plate during late corticogenesis. Here we show that in developing mouse cortices, INs in the piriform cortex (PCx; the major olfactory cortex) distribute differently from those in the NCx. We provide evidence that during development INs invade and mature earlier in PCx than in NCx, likely owing to the lack of CXCR4 expression in INs from PCx compared to those in NCx. We analyzed IN distribution patterns in Lhx2 cKO mice, where projection neurons in the lateral NCx are re-fated to generate an ectopic PCx (ePCx). The PCx-specific IN distribution patterns found in ePCx suggest that properties of PCx projection neurons regulate IN distribution. Collectively, our results show that the timing of IN invasion in the developing PCx fundamentally differs from what is known in the NCx. Further, our results suggest that projection neurons instruct the PCx-specific pattern of IN distribution.

Differential inhibition of pyramidal cells and inhibitory interneurons along the rostrocaudal axis of anterior piriform cortex.

The spatial representation of stimuli in sensory neocortices provides a scaffold for elucidating circuit mechanisms underlying sensory processing. However, the anterior piriform cortex (APC) lacks topology for odor identity as well as afferent and intracortical excitation. Consequently, olfactory processing is considered homogenous along the APC rostral-caudal (RC) axis. We recorded excitatory and inhibitory neurons in APC while optogenetically activating GABAergic interneurons along the RC axis. In contrast to excitation, we find opposing, spatially asymmetric inhibition onto pyramidal cells (PCs) and interneurons. PCs are strongly inhibited by caudal stimulation sites, whereas interneurons are strongly inhibited by rostral sites. At least two mechanisms underlie spatial asymmetries. Enhanced caudal inhibition of PCs is due to increased synaptic strength, whereas rostrally biased inhibition of interneurons is mediated by increased somatostatin-interneuron density. Altogether, we show differences in rostral and caudal inhibitory circuits in APC that may underlie spatial variation in odor processing along the RC axis.

The Laminar Organization of Piriform Cortex Follows a Selective Developmental and Migratory Program Established by Cell Lineage.

Piriform cortex (PC) is a 3-layer paleocortex receiving primary afferent input from the olfactory bulb. The past decade has seen significant progress in understanding the synaptic, cellular and functional organization of PC, but PC embryogenesis continues to be enigmatic. Here, using birthdating strategies and clonal analyses, we probed the early development and laminar specificity of neurogenesis/gliogenesis as it relates to the organization of the PC. Our data demonstrate a temporal sequence of laminar-specific neurogenesis following the canonical "inside-out" pattern, with the notable exception of PC Layer II which exhibited an inverse "outside-in" temporal neurogenic pattern. Of interest, we found no evidence of a neurogenic gradient along the anterior to posterior axis, although the timing of neuronal migration and laminar development was delayed rostrally by approximately 24 h. To begin probing if lineage affected cell fate in the PC, we labeled PC neuroblasts using a multicolor technique and analyzed their laminar organization. Our results suggested that PC progenitors were phenotypically committed to reach specific layers early in the development. Collectively, these studies shed new light on the determinants of the laminar specificity of neuronal/glial organization in PC and the likely role of subpopulations of committed progenitors in regulating PC embryogenesis.

Spontaneous activity in the piriform cortex extends the dynamic range of cortical odor coding.

Neurons in the neocortex exhibit spontaneous spiking activity in the absence of external stimuli, but the origin and functions of this activity remain uncertain. Here, we show that spontaneous spiking is also prominent in a sensory paleocortex, the primary olfactory (piriform) cortex of mice. In the absence of applied odors, piriform neurons exhibit spontaneous firing at mean rates that vary systematically among neuronal classes. This activity requires the participation of NMDA receptors and is entirely driven by bottom-up spontaneous input from the olfactory bulb. Odor stimulation produces two types of spatially dispersed, odor-distinctive patterns of responses in piriform cortex layer 2 principal cells: Approximately 15% of cells are excited by odor, and another approximately 15% have their spontaneous activity suppressed. Our results show that, by allowing odor-evoked suppression as well as excitation, the responsiveness of piriform neurons is at least twofold less sparse than currently believed. Hence, by enabling bidirectional changes in spiking around an elevated baseline, spontaneous activity in the piriform cortex extends the dynamic range of odor representation and enriches the coding space for the representation of complex olfactory stimuli.

Cellular Plasticity in the Adult Murine Piriform Cortex: Continuous Maturation of Dormant Precursors Into Excitatory Neurons.

Neurogenesis in the healthy adult murine brain is based on proliferation and integration of stem/progenitor cells and is thought to be restricted to 2 neurogenic niches: the subventricular zone and the dentate gyrus. Intriguingly, cells expressing the immature neuronal marker doublecortin (DCX) and the polysialylated-neural cell adhesion molecule reside in layer II of the piriform cortex. Apparently, these cells progressively disappear along the course of ageing, while their fate and function remain unclear. Using DCX-CreERT2/Flox-EGFP transgenic mice, we demonstrate that these immature neurons located in the murine piriform cortex do not vanish in the course of aging, but progressively resume their maturation into glutamatergic (TBR1+, CaMKII+) neurons. We provide evidence for a putative functional integration of these newly differentiated neurons as indicated by the increase in perisomatic puncta expressing synaptic markers, the development of complex apical dendrites decorated with numerous spines and the appearance of an axonal initial segment. Since immature neurons found in layer II of the piriform cortex are generated prenatally and devoid of proliferative capacity in the postnatal cortex, the gradual maturation and integration of these cells outside of the canonical neurogenic niches implies that they represent a valuable, but nonrenewable reservoir for cortical plasticity.

Balanced feedforward inhibition and dominant recurrent inhibition in olfactory cortex.

Throughout the brain, the recruitment of feedforward and recurrent inhibition shapes neural responses. However, disentangling the relative contributions of these often-overlapping cortical circuits is challenging. The piriform cortex provides an ideal system to address this issue because the interneurons responsible for feedforward and recurrent inhibition are anatomically segregated in layer (L) 1 and L2/3 respectively. Here we use a combination of optical and electrical activation of interneurons to profile the inhibitory input received by three classes of principal excitatory neuron in the anterior piriform cortex. In all classes, we find that L1 interneurons provide weaker inhibition than L2/3 interneurons. Nonetheless, feedforward inhibitory strength covaries with the amount of afferent excitation received by each class of principal neuron. In contrast, intracortical stimulation of L2/3 evokes strong inhibition that dominates recurrent excitation in all classes. Finally, we find that the relative contributions of feedforward and recurrent pathways differ between principal neuron classes. Specifically, L2 neurons receive more reliable afferent drive and less overall inhibition than L3 neurons. Alternatively, L3 neurons receive substantially more intracortical inhibition. These three features--balanced afferent drive, dominant recurrent inhibition, and differential recruitment by afferent vs. intracortical circuits, dependent on cell class--suggest mechanisms for olfactory processing that may extend to other sensory cortices.

Sodium and potassium conductances in principal neurons of the mouse piriform cortex: a quantitative description.

KEY POINTS: The primary olfactory (or piriform) cortex is a promising model system for understanding how the cerebral cortex processes sensory information, although an investigation of the piriform cortex is hindered by a lack of detailed information about the intrinsic electrical properties of its component neurons. In the present study, we quantify the properties of voltage-dependent sodium currents and voltage- and calcium-dependent potassium currents in two important classes of excitatory neurons in the main input layer of the piriform cortex. We identify several classes of these currents and show that their properties are similar to those found in better-studied cortical regions. Our detailed quantitative descriptions of these currents will be valuable to computational neuroscientists who aim to build models that explain how the piriform cortex encodes odours. ABSTRACT: The primary olfactory cortex (or piriform cortex, PC) is an anatomically simple palaeocortex that is increasingly used as a model system for investigating cortical sensory processing. However, little information is available on the intrinsic electrical conductances in neurons of the PC, hampering efforts to build realistic computational models of this cortex. In the present study, we used nucleated macropatches and whole-cell recordings to rigorously quantify the biophysical properties of voltage-gated sodium (NaV ), voltage-gated potassium (KV ) and calcium-activated potassium (KCa ) conductances in two major classes of glutamatergic neurons in layer 2 of the PC, semilunar (SL) cells and superficial pyramidal (SP) cells. We found that SL and SP cells both express a fast-inactivating NaV current, two types of KV current (A-type and delayed rectifier-type) and three types of KCa current (fast-, medium- and slow-afterhyperpolarization currents). The kinetic and voltage-dependent properties of the NaV and KV conductances were, with some exceptions, identical in SL and SP cells and similar to those found in neocortical pyramidal neurons. The KCa conductances were also similar across the different types of neurons. Our results are summarized in a series of empirical equations that should prove useful to computational neuroscientists seeking to model the PC. More broadly, our findings indicate that, at the level of single-cell electrical properties, this palaeocortex is not so different from the neocortex, vindicating efforts to use the PC as a model of cortical sensory processing in general.

Learning-induced modulation of the effect of neuroglial transmission on synaptic plasticity.

Training rats in a complex olfactory discrimination task results in acquisition of "rule learning" (learning how to learn), a term describing the capability to perform the task superbly. Such rule learning results in strengthening of both excitatory and inhibitory synaptic connections between neurons in the piriform cortex. Moreover, intrinsic excitability is also enhanced throughout the pyramidal neuron population. Surprisingly, the cortical network retains its stability under these long-term modifications. In particular, the susceptibility for long-term potentiation (LTP) induction, while decreased for a short time window, returns to almost its pretraining value, although significant strengthening of AMPA receptor-mediated glutamatergic transmission remains. Such network balance is essential for maintaining the single-cell modifications that underlie long-term memory while preventing hyperexcitability that would result in runaway synaptic activity. However, the mechanisms underlying the long-term maintenance of such balance have yet to be described. In this study, we explored the role of astrocyte-mediated gliotransmission in long-term maintenance of learning-induced modifications in susceptibility for LTP induction and control of the strength of synaptic inhibition. We show that blocking connexin 43 hemichannels, which form gap junctions between astrocytes, decreases significantly the ability to induce LTP by stimulating the excitatory connections between piriform cortex pyramidal neurons after learning only. In parallel, spontaneous miniature inhibitory postsynaptic current amplitude is reduced in neurons from trained rats only, to the level of prelearning. Thus gliotransmission has a key role in maintaining learning-induced cortical stability by a wide-ranged control on synaptic transmission and plasticity. NEW & NOTEWORTHY We explore the role of astrocyte-mediated gliotransmission in maintenance of olfactory discrimination learning-induced modifications. We show that blocking gap junctions between astrocytes decreases significantly the ability to induce long-term potentiation in the piriform cortex after learning only. In parallel, synaptic inhibition is reduced in neurons from trained rats only, to the level of prelearning. Thus gliotransmission has a key role in maintaining learning-induced cortical stability by a wide-ranged control on synaptic transmission and plasticity.

Optogenetic Mapping of Intracortical Circuits Originating from Semilunar Cells in the Piriform Cortex.

Despite its comparatively simple trilaminar architecture, the primary olfactory (piriform) cortex of mammals is capable of performing sophisticated sensory processing, an ability that is thought to depend critically on its extensive associational (intracortical) excitatory circuits. Here, we used a novel transgenic mouse model and optogenetics to measure the connectivity of associational circuits that originate in semilunar (SL) cells in layer 2a of the anterior piriform cortex (aPC). We generated a mouse line (48L) in which channelrhodopsin-2 (ChR) could be selectively expressed in a subset of SL cells. Light-evoked excitatory postsynaptic currents (EPSCs) could be evoked in superficial pyramidal cells (17.4% of n = 86 neurons) and deep pyramidal cells (33.3%, n = 9) in the aPC, but never in ChR- SL cells (0%, n = 34). Thus, SL cells monosynaptically excite pyramidal cells, but not other SL cells. Light-evoked EPSCs were also selectively elicited in 3 classes of GABAergic interneurons in layer 3 of the aPC. Our results show that SL cells are specialized for providing feedforward excitation of specific classes of neurons in the aPC, confirming that SL cells comprise a functionally distinctive input layer.

Response Patterns of GABAergic Neurons in the Anterior Piriform Cortex of Awake Mice.

Local inhibition by γ-amino butyric acid (GABA)-containing neurons is of vital importance for the operation of sensory cortices. However, the physiological response patterns of cortical GABAergic neurons are poorly understood, especially in the awake condition. Here, we utilized the recently developed optical tagging technique to specifically record GABAergic neurons in the anterior piriform cortex (aPC) in awake mice. The identified aPC GABAergic neurons were stimulated with robotic delivery of 32 distinct odorants, which covered a broad range of functional groups. We found that aPC GABAergic neurons could be divided into 4 types based on their response patterns. Type I, type II, and type III neurons displayed broad excitatory responses to test odorants with different dynamics. Type I neurons were constantly activated during odorant stimulation, whereas type II neurons were only transiently activated at the onset of odorant delivery. In addition, type III neurons displayed transient excitatory responses both at the onset and termination of odorant presentation. Interestingly, type IV neurons were broadly inhibited by most of the odorants. Taken together, aPC GABAergic neurons adopt different strategies to affect the cortical circuitry. Our results will allow for better understanding of the role of cortical GABAergic interneurons in sensory information processing.

Enhanced neurogenesis and possible synaptic reorganization in the piriform cortex of adult rat following kainic acid-induced status epilepticus.

Epileptic seizure has been reported to enhance adult neurogenesis and induce aberrant synaptic reorganization in the human dentate gyrus in the hippocampal formation. However, adult neurogenesis in the extrahippocampal regions has not been well studied. To investigate seizure-enhanced neurogenesis in the extrahippocampal regions, we performed histological and immunohistochemical as well as western blot analyses on the cerebrum of Sprague-Dawley rats (n = 51, male, 7 weeks old, body weight 250-300 g) treated with intraperitoneal injection of kainic acid (KA, 10 mg/kg) to induce status epilepticus (SE) (n = 36) or normal saline solution (n = 15) followed by 5'-bromo-2-deoxyuridine (BrdU) injection to label newborn cells. Even though severe neuronal damage was found in the piriform cortex of rats having SE, immunohistochemistry for double cortin (DCX) revealed an increase in the number of immature neurons in the piriform cortex. Double immunofluorescence staining demonstrated that DCX-positive cells in the piriform cortex were positive for both BrdU and neuronal nuclear antigen. Immunohistochemistry and western blotting revealed increased expressions of synaptophysin and postsynaptic density protein 95 in the piriform cortex of rat having SE. These results suggested the enhanced neurogenesis and possible synaptic reorganization in the piriform cortex of the KA-treated rat.

ß-Adrenoceptor activation enhances L-type calcium channel currents in anterior piriform cortex pyramidal cells of neonatal mice: implication for odor learning.

Early odor preference learning occurs in one-week-old rodents when a novel odor is paired with a tactile stimulation mimicking maternal care. ß-Adrenoceptors and L-type calcium channels (LTCCs) in the anterior piriform cortex (aPC) are critically involved in this learning. However, whether ß-adrenoceptors interact directly with LTCCs in aPC pyramidal cells is unknown. Here we show that pyramidal cells expressed significant LTCC currents that declined with age. ß-Adrenoceptor activation via isoproterenol age-dependently enhanced LTCC currents. Nifedipine-sensitive, isoproterenol enhancement of calcium currents was only observed in post-natal day 7-10 mice. APC ß-adrenoceptor activation induced early odor preference learning was blocked by nifedipine coinfusion.

Quantitative analysis of axon collaterals of single pyramidal cells of the anterior piriform cortex of the guinea pig.

BACKGROUND: The role of the piriform cortex (PC) in olfactory information processing remains largely unknown. The anterior part of the piriform cortex (APC) has been the focus of cortical-level studies of olfactory coding, and associative processes have attracted considerable attention as an important part in odor discrimination and olfactory information processing. Associational connections of pyramidal cells in the guinea pig APC were studied by direct visualization of axons stained and quantitatively analyzed by intracellular biocytin injection in vivo. RESULTS: The observations illustrated that axon collaterals of the individual cells were widely and spatially distributed within the PC, and sometimes also showed a long associational projection to the olfactory bulb (OB). The data showed that long associational axons were both rostrally and caudally directed throughout the PC, and the intrinsic associational fibers of pyramidal cells in the APC are omnidirectional connections in the PC. Within the PC, associational axons typically followed rather linear trajectories and irregular bouton distributions. Quantitative data of the axon collaterals of two pyramidal cells in the APC showed that the average length of axonal collaterals was 101 mm, out of which 79 mm (78% of total length) were distributed in the PC. The average number of boutons was 8926 and 7101, respectively, with 79% of the total number of boutons being distributed in the PC. The percentage of the total area of the APC and the posterior piriform cortex occupied by the average distribution region of the axon collaterals of two superficial pyramidal (SP) cells was about 18 and 5%, respectively. CONCLUSION: Our results demonstrate that omnidirectional connection of pyramidal cells in the APC provides a substrate for recurrent processes. These findings indicate that the axon collaterals of SP cells in the PC could make synaptic contacts with all granule cells in the OB. This study provides the morphological evidence for understanding the mechanisms of information processing and associative memory in the APC.

Nonsensory target-dependent organization of piriform cortex.

The piriform cortex (PCX) is the largest component of the olfactory cortex and is hypothesized to be the locus of odor object formation. The distributed odorant representation found in PCX contrasts sharply with the topographical representation seen in other primary sensory cortices, making it difficult to test this view. Recent work in PCX has focused on functional characteristics of these distributed afferent and association fiber systems. However, information regarding the efferent projections of PCX and how those may be involved in odor representation and object recognition has been largely ignored. To investigate this aspect of PCX, we have used the efferent pathway from mouse PCX to the orbitofrontal cortex (OFC). Using double fluorescent retrograde tracing, we identified the output neurons (OPNs) of the PCX that project to two subdivisions of the OFC, the agranular insula and the lateral orbitofrontal cortex (AI-OPNs and LO-OPNs, respectively). We found that both AI-OPNs and LO-OPNs showed a distinct spatial topography within the PCX and fewer than 10% projected to both the AI and the LO as judged by double-labeling. These data revealed that the efferent component of the PCX may be topographically organized. Further, these data suggest a model for functional organization of the PCX in which the OPNs are grouped into parallel output circuits that provide olfactory information to different higher centers. The distributed afferent input from the olfactory bulb and the local PCX association circuits would then ensure a complete olfactory representation, pattern recognition capability, and neuroplasticity in each efferent circuit.

Arc-Expressing Neuronal Ensembles Supporting Pattern Separation Require Adrenergic Activity in Anterior Piriform Cortex: An Exploration of Neural Constraints on Learning.

Arc ensembles in adult rat olfactory bulb (OB) and anterior piriform cortex (PC) were assessed after discrimination training on highly similar odor pairs. Nonselective α- and ß-adrenergic antagonists or saline were infused in the OB or anterior PC during training. OB adrenergic blockade slowed, but did not prevent, odor discrimination learning. After criterion performance, Arc ensembles in anterior piriform showed enhanced stability for the rewarded odor and pattern separation for the discriminated odors as described previously. Anterior piriform adrenergic blockade prevented acquisition of similar odor discrimination and of OB ensemble changes, even with extended overtraining. Mitral and granule cell Arc ensembles in OB showed enhanced stability for rewarded odor only in the saline group. Pattern separation was not seen in the OB. Similar odor discrimination co-occurs with increased stability in rewarded odor representations and pattern separation to reduce encoding overlap. The difficulty of similar discriminations may relate to the necessity to both strengthen rewarded representations and weaken overlap across similar representations. SIGNIFICANCE STATEMENT: We show for the first time that adrenoceptors in anterior piriform cortex (aPC) must be engaged for adult rats to learn to discriminate highly similar odors. Loss of adrenergic activation in olfactory bulb (OB) slows, but does not prevent, discrimination learning. Both increased stability of the rewarded odor representation and increased pattern separation of the rewarded and unrewarded odors in aPC accompany successful discrimination. In the OB, rewarded odors increase in ensemble stability, but there is no evidence of pattern separation. We suggest that the slow acquisition of similar odor discriminations is related to the differing plasticity requirements for increased stability and pattern separation.

Expression of macrophage migration inhibitory factor in the mouse neocortex and posterior piriform cortices during postnatal development.

Macrophage migration inhibitory factor (MIF) functions as a pleiotropic protein, participating in a vast array of cellular and biological processes. Abnormal expression of MIF has been implicated in many neurological diseases, including Parkinson's disease, epilepsy, Alzheimer's Disease, stroke, and neuropathic pain. However, the expression patterns of mif transcript and MIF protein from the early postnatal period through adulthood in the mouse brain are still poorly understood. We therefore investigated the temporal and spatial expression of MIF in the mouse neocortex during postnatal development in detail and partially in posterior piriform cortices (pPC). As determined by quantitative real-time PCR (qPCR), mif transcript gradually increased during development, with the highest level noted at postnatal day 30 (P30) followed by a sharp decline at P75. In contrast, Western blotting results showed that MIF increased constantly from P7 to P75. The highest level of MIF was at P75, while the lowest level of MIF was at P7. Immunofluorescence histochemistry revealed that MIF-immunoreactive (ir) cells were within the entire depth of the developed neocortex, and MIF was heterogeneously distributed among cortical cells, especially at P7, P14, P30, and P75; MIF was abundant in the pyramidal layer within pPC. Double immunostaining showed that all the mature neurons were MIF-ir and all the intensely stained MIF-ir cells were parvalbumin positive (Pv +) at adult. Moreover, it was demonstrated that MIF protein localized in the perikaryon, processes, presynaptic structures, and the nucleus in neurons. Taken together, the developmentally regulated expression and the subcellular localization of MIF should form a platform for an analysis of MIF neurodevelopmental biology and MIF-related nerve diseases.