Biochemical and Hematologic Reference Intervals for Anesthetized, Female, Juvenile Yorkshire Swine.

Swine are widely used in biomedical research, translational research, xenotransplantation, and agriculture. For these uses, physiologic reference intervals are extremely important for assessing the health status of the swine and diagnosing disease. However, few biochemical and hematologic reference intervals that comply with guidelines from the Clinical and Laboratory Standards Institute and the American Society for Veterinary Clinical Pathology are available for swine. These guidelines state that reference intervals should be determined by using 120 subjects or more. The aim of this study was to generate hematologic and biochemical reference intervals for female, juvenile Yorkshire swine (Sus scrofa domesticus) and to compare these values with those for humans and baboons (Papio hamadryas). Blood samples were collected from the femoral artery or vein of female, juvenile Yorkshire swine, and standard hematologic and biochemical parameters were analyzed in multiple studies. Hematologic and biochemical reference intervals were calculated for arterial blood samples from Yorkshire swine (n = 121 to 124); human and baboon reference intervals were obtained from the literature. Arterial reference intervals for Yorkshire swine differed significantly from those for humans and baboons in all commonly measured parameters except platelet count, which did not differ significantly from the human value, and glucose, which was not significantly different from the baboon value. These data provide valuable information for investigators using female, juvenile Yorkshire swine for biomedical re- search, as disease models, and in xenotransplantation studies as well as useful physiologic information for veterinarians and livestock producers. Our findings highlight the need for caution when comparing data and study outcomes between species.

Effects of a patient-derived de novo coding alteration of CACNA1I in mice connect a schizophrenia risk gene with sleep spindle deficits.

CACNA1I, a schizophrenia risk gene, encodes a subtype of voltage-gated T-type calcium channel CaV3.3. We previously reported that a patient-derived missense de novo mutation (R1346H) of CACNA1I impaired CaV3.3 channel function. Here, we generated CaV3.3-RH knock-in animals, along with mice lacking CaV3.3, to investigate the biological impact of R1346H (RH) variation. We found that RH mutation altered cellular excitability in the thalamic reticular nucleus (TRN), where CaV3.3 is abundantly expressed. Moreover, RH mutation produced marked deficits in sleep spindle occurrence and morphology throughout non-rapid eye movement (NREM) sleep, while CaV3.3 haploinsufficiency gave rise to largely normal spindles. Therefore, mice harboring the RH mutation provide a patient derived genetic model not only to dissect the spindle biology but also to evaluate the effects of pharmacological reagents in normalizing sleep spindle deficits. Importantly, our analyses highlighted the significance of characterizing individual spindles and strengthen the inferences we can make across species over sleep spindles. In conclusion, this study established a translational link between a genetic allele and spindle deficits during NREM observed in schizophrenia patients, representing a key step toward testing the hypothesis that normalizing spindles may be beneficial for schizophrenia patients.

Functional Topography and Development of Inhibitory Reticulothalamic Barreloid Projections.

The thalamic reticular nucleus (TRN) is the main source of inhibition to the somatosensory thalamus (ventrobasal nucleus, VB) in mice. However, the functional topography and development of these projections with respect to the VB barreloids has been largely unexplored. In this respect, to assist in the study of these projections, we have utilized a vesicular gamma-aminobutryic acid (GABA) transporter (VGAT)-Venus transgenic mouse line to develop a brain slice preparation that enables the rapid identification of inhibitory neurons and projections. We demonstrate the utility of our in vitro brain slice preparation for physiologically mapping inhibitory reticulothalamic (RT) topography, using laser-scanning photostimulation via glutamate uncaging. Furthermore, we utilized this slice preparation to compare the development of excitatory and inhibitory projections to VB. We found that excitatory projections to the barreloids, created by ascending projections from the brain stem, develop by postnatal day 2-3 (P2-P3). By contrast, inhibitory projections to the barreloids lag ~5 days behind excitatory projections to the barreloids, developing by P7-P8. We probed this lag in inhibitory projection development through early postnatal whisker lesions. We found that in whisker-lesioned animals, the development of inhibitory projections to the barreloids closed by P4, in register with that of the excitatory projections to the barreloids. Our findings demonstrate both developmental and topographic organizational features of the RT projection to the VB barreloids, whose mechanisms can now be further examined utilizing the VGAT-Venus mouse slice preparation that we have characterized.

Wisteria Floribunda Agglutinin-Labeled Perineuronal Nets in the Mouse Inferior Colliculus, Thalamic Reticular Nucleus and Auditory Cortex.

Perineuronal nets (PNNs) are specialized extracellular matrix molecules that are associated with the closing of the critical period, among other functions. In the adult brain, PNNs surround specific types of neurons, however the expression of PNNs in the auditory system of the mouse, particularly at the level of the midbrain and forebrain, has not been fully described. In addition, the association of PNNs with excitatory and inhibitory cell types in these structures remains unknown. Therefore, we sought to investigate the expression of PNNs in the inferior colliculus (IC), thalamic reticular nucleus (TRN) and primary auditory cortex (A1) of the mouse brain by labeling with wisteria floribunda agglutinin (WFA). To aid in the identification of inhibitory neurons in these structures, we employed the vesicular GABA transporter (VGAT)-Venus transgenic mouse strain, which robustly expresses an enhanced yellow-fluorescent protein (Venus) natively in nearly all gamma-amino butyric acid (GABA)-ergic inhibitory neurons, thus enabling a rapid and unambiguous assessment of inhibitory neurons throughout the nervous system. Our results demonstrate that PNNs are expressed throughout the auditory midbrain and forebrain, but vary in their local distribution. PNNs are most dense in the TRN and least dense in A1. Furthermore, PNNs are preferentially associated with inhibitory neurons in A1 and the TRN, but not in the IC of the mouse. These data suggest regionally specific roles for PNNs in auditory information processing.

Central projections of auditory receptor neurons of crickets.

We describe the central projections of physiologically characterized auditory receptor neurons of crickets as revealed by confocal microscopy. Receptors tuned to ultrasonic frequencies (similar to those produced by echolocating, insectivorous bats), to a mid-range of frequencies, and a subset of those tuned to low, cricket-like frequencies have similar projections, terminating medially within the auditory neuropile. Quantitative analysis shows that despite the general similarity of these projections they are tonotopic, with receptors tuned to lower frequencies terminating more medially. Another subset of cricket-song-tuned receptors projects more laterally and posteriorly than the other types. Double-fills of receptors and identified interneurons show that the three medially projecting receptor types are anatomically well positioned to provide monosynaptic input to interneurons that relay auditory information to the brain and to interneurons that modify this ascending information. The more laterally and posteriorly branching receptor type may not interact directly with this ascending pathway, but is well positioned to provide direct input to an interneuron that carries auditory information to more posterior ganglia. These results suggest that information about cricket song is segregated into functionally different pathways as early as the level of receptor neurons. Ultrasound-tuned and mid-frequency tuned receptors have approximately twice as many varicosities, which are sites of transmitter release, per receptor as either anatomical type of cricket-song-tuned receptor. This may compensate in part for the numerical under-representation of these receptor types.

Commissural functional topography of the inferior colliculus assessed in vitro.

The inferior colliculus (IC) receives ascending and descending information from several convergent neural sources. As such, exploring the neural pathways that converge in the IC is crucial to uncovering their multi-varied roles in the integration of auditory and other sensory information. Among these convergent pathways, the IC commissural connections represent an important route for the integration of bilateral information in the auditory system. Here, we describe the preparation and validation of a novel in vitro slice preparation for examining the functional topography and synaptic properties of the commissural and intrinsic projections in the IC of the mouse. This preparation, in combination with modern genetic approaches in the mouse, enables the specific examination of these pathways, which potentially can reveal cell-type specific processing channels in the auditory midbrain.

Nicotinic alteration of functional thalamocortical topography.

The thalamocortical pathways form highly topographic connections from the primary sensory thalamic nuclei to the primary cortical areas. The synaptic properties of these thalamocortical connections are modifiable by activation from various neuromodulators, such as acetylcholine. Cholinergic activation can alter functional properties in both the developing and the mature nervous system. Moreover, environmental factors, such as nicotine, can activate these receptors, although the circuit-level alterations resulting from such nicotinic activation of sensory neural circuits remain largely unexplored. Therefore, we examined alterations to the functional topography of thalamocortical circuits in the developing sensory pathways of the mouse. Photostimulation by uncaging of glutamate was used to map these functional thalamocortical alterations in response to nicotinic receptor activation. As a result, we found that activation of forebrain nicotinic acetylcholine receptors results in an expansion and enhancement of functional thalamocortical topographies as assessed in brain slice preparations using laser-scanning photostimulation by uncaging of glutamate. These physiological changes were correlated with the neuroanatomical expression of nicotinic acetylcholine receptor subtypes (α7 and ß2). These circuit-level alterations may provide a neural substrate underlying the plastic development and reshaping of thalamocortical circuitry in response to nicotinic receptor activation.

Frequency transformation in the auditory lemniscal thalamocortical system.

The auditory lemniscal thalamocortical (TC) pathway conveys information from the ventral division of the medial geniculate body to the primary auditory cortex (A1). Although their general topographic organization has been well characterized, functional transformations at the lemniscal TC synapse still remain incompletely codified, largely due to the need for integration of functional anatomical results with the variability observed with various animal models and experimental techniques. In this review, we discuss these issues with classical approaches, such as in vivo extracellular recordings and tracer injections to physiologically identified areas in A1, and then compare these studies with modern approaches, such as in vivo two-photon calcium imaging, in vivo whole-cell recordings, optogenetic methods, and in vitro methods using slice preparations. A surprising finding from a comparison of classical and modern approaches is the similar degree of convergence from thalamic neurons to single A1 neurons and clusters of A1 neurons, although, thalamic convergence to single A1 neurons is more restricted from areas within putative thalamic frequency lamina. These comparisons suggest that frequency convergence from thalamic input to A1 is functionally limited. Finally, we consider synaptic organization of TC projections and future directions for research.

Functional convergence of thalamic and intrinsic projections to cortical layers 4 and 6.

Ascending sensory information is conveyed from the thalamus to layers 4 and 6 of sensory cortical areas. Interestingly, receptive field properties of cortical layer 6 neurons are different from those in layer 4. Do such differences reflect distinct inheritance patterns from the thalamus or are they derived instead from local cortical circuits? To distinguish between these possibilities, we utilized in vitro slice preparations containing the thalamocortical pathways in the auditory and somatosensory systems. Responses from neurons in layers 4 and 6 that resided in the same column were recorded using whole-cell patch clamp. Laser-scanning photostimulation via uncaging of glutamate in the thalamus and cortex was used to map the functional topography of thalamocortical and intracortical inputs to each layer. In addition, we assessed the functional divergence of thalamocortical inputs by optical imaging of flavoprotein autofluorescence. We found that the thalamocortical inputs to layers 4 and 6 originated from the same thalamic domain, but the intracortical projections to the same neurons differed dramatically. Our results suggest that the intracortical projections, rather than the thalamic inputs, to each layer contribute more to the differences in their receptive field properties.

Global hyper-synchronous spontaneous activity in the developing optic tectum.

Studies of patterned spontaneous activity can elucidate how the organization of neural circuits emerges. Using in vivo two-photon Ca(2+) imaging, we studied spatio-temporal patterns of spontaneous activity in the optic tectum of Xenopus tadpoles. We found rhythmic patterns of global synchronous spontaneous activity between neurons, which depends on visual experience and developmental stage. By contrast, synchronous spontaneous activity between non-neuronal cells is mediated more locally. To understand the source of the neuronal spontaneous activity, input to the tectum was systematically removed. Whereas removing input from the visual or mechanosensory system alone had little effect on patterned spontaneous activity, removing input from both systems drastically altered it. These results suggest that either input is sufficient to maintain the intrinsically generated spontaneous activity and that patterned spontaneous activity results from input from multisensory systems. Thus, the amphibian midbrain differs from the mammalian visual system, whose spontaneous activity is controlled by retinal waves.

Laser-scanning photostimulation of optogenetically targeted forebrain circuits.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

Spatial organization of repetition rate processing in cat anterior auditory field.

Auditory cortex updates incoming information on a segment by segment basis for human speech and animal communication. Measuring repetition rate transfer functions (RRTFs) captures temporal responses to repetitive sounds. In this study, we used repetitive click trains to describe the spatial distribution of RRTF responses in cat anterior auditory field (AAF) and to discern potential variations in local temporal processing capacity. A majority of RRTF filters are band-pass. Temporal parameters estimated from RRTFs and corrected for characteristic frequency or latency dependencies are non-homogeneously distributed across AAF. Unlike the shallow global gradient observed in spectral receptive field parameters, transitions from loci with high to low temporal parameters are steep. Quantitative spatial analysis suggests non-uniform, circumscribed local organization for temporal pattern processing superimposed on global organization for spectral processing in cat AAF.

Encoding of temporal information by timing, rate, and place in cat auditory cortex.

A central goal in auditory neuroscience is to understand the neural coding of species-specific communication and human speech sounds. Low-rate repetitive sounds are elemental features of communication sounds, and core auditory cortical regions have been implicated in processing these information-bearing elements. Repetitive sounds could be encoded by at least three neural response properties: 1) the event-locked spike-timing precision, 2) the mean firing rate, and 3) the interspike interval (ISI). To determine how well these response aspects capture information about the repetition rate stimulus, we measured local group responses of cortical neurons in cat anterior auditory field (AAF) to click trains and calculated their mutual information based on these different codes. ISIs of the multiunit responses carried substantially higher information about low repetition rates than either spike-timing precision or firing rate. Combining firing rate and ISI codes was synergistic and captured modestly more repetition information. Spatial distribution analyses showed distinct local clustering properties for each encoding scheme for repetition information indicative of a place code. Diversity in local processing emphasis and distribution of different repetition rate codes across AAF may give rise to concurrent feed-forward processing streams that contribute differently to higher-order sound analysis.

Spatial interaction between spectral integration and frequency gradient in primary auditory cortex.

Primary sensory cortical areas are characterized by orderly and largely independent representations of several receptive field properties. This is expressed in multiple, spatially overlaying parameter distributions, such as orientation preference, spatial frequency, and ocular dominance maps in the primary visual cortex. In the auditory cortex, two main and presumably independent representational parameters are the center frequency and the frequency extent of spectral tuning curves. Here we demonstrate interactions between cortical tonotopic gradient and spectral bandwidth modules in cat primary auditory cortex (AI). First, the spatial representation of spectral integration is not equally expressed across the whole frequency range in AI. Narrow-bandwidth modules are found only in the mid-frequency region (5-20 kHz). Thus spectral integration properties delineate three frequency regions (<5, 5-20, and >20 kHz) in cat AI. Second, the extent of spectral integration covaries with the local tonotopic gradient in the low- and mid-frequency ranges. Regions with a shallow frequency gradient tend to have narrower spectral integration than those with a steep gradient. These relationships between spectral selectivity and frequency gradient constrain forebrain models of thalamo- and corticocortical convergence and connectivity and may reflect the processing of behaviorally relevant stimulus constellations.

Development of spectral and temporal response selectivity in the auditory cortex.

The mechanisms by which hearing selectivity is elaborated and refined in early development are very incompletely determined. In this study, we documented contributions of progressively maturing inhibitory influences on the refinement of spectral and temporal response properties in the primary auditory cortex. Inhibitory receptive fields (IRFs) of infant rat auditory cortical neurons were spectrally far broader and had extended over far longer duration than did those of adults. The selective refinement of IRFs was delayed relative to that of excitatory receptive fields by an approximately 2-week period that corresponded to the critical period for plasticity. Local application of a GABA(A) receptor antagonist revealed that intracortical inhibition contributes to this progressive receptive field maturation for response selectivity in frequency. Conversely, it had no effect on the duration of IRFs or successive-signal cortical response recovery times. The importance of exposure to patterned acoustic inputs was suggested when both spectral and temporal IRF maturation were disrupted in rat pups reared in continuous, moderate-intensity noise. They were subsequently renormalized when animals were returned to standard housing conditions as adults.

Concurrent tonotopic processing streams in auditory cortex.

The basis for multiple representations of equivalent frequency ranges in auditory cortex was studied with physiological and anatomical methods. Our goal was to trace the convergence of thalamic, commissural, and corticocortical information upon two tonotopic fields in the cat, the primary auditory cortex (AI) and the anterior auditory field (AAF). Both fields are among the first cortical levels of processing. After neurophysiological mapping of characteristic frequency, we injected different retrograde tracers at separate, frequency-matched loci in AI and AAF. We found differences in their projections that support the notion of largely segregated parallel processing streams in the auditory thalamus and cerebral cortex. In each field, ipsilateral cortical input amounts to approximately 70% of the number of cells projecting to an isofrequency domain, while commissural and thalamic sources are each approximately 15%. Labeled thalamic and cortical neurons were concentrated in tonotopically predicted regions and in smaller loci far from their spectrally predicted positions. The few double-labeled thalamic neurons (<2%) are consistent with the hypothesis that information to AI and AAF travels along independent processing streams despite widespread regional overlap of thalamic input sources. Double labeling is also sparse in both the corticocortical and commissural systems ( approximately 1%), confirming their independence. The segregation of frequency-specific channels within thalamic and cortical systems is consistent with a model of parallel processing in auditory cortex. The global convergence of cells outside the targeted frequency domain in AI and AAF could contribute to context-dependent processing and to intracortical plasticity and reorganization.

Modular functional organization of cat anterior auditory field.

Two tonotopic areas, the primary auditory cortex (AI) and the anterior auditory field (AAF), are the primary cortical fields in the cat auditory system. They receive largely independent, concurrent thalamocortical projections from the different thalamic divisions despite their hierarchical equivalency. The parallel streams of thalamic inputs to AAF and AI suggest that AAF neurons may differ from AI neurons in physiological properties. Although a modular functional organization in cat AI has been well documented, little is known about the internal organization of AAF beyond tonotopy. We studied how basic receptive field parameters (RFPs) are spatially organized in AAF with single- and multiunit recording techniques. A distorted tonotopicity with an underrepresentation in midfrequencies (1 and 5 kHz) and an overrepresentation in the high-frequency range was found. Spectral bandwidth (Q-values) and response threshold were significantly correlated with characteristic frequency (CF). To understand whether AAF has a modular organization of RFPs, CF dependencies were eliminated by a nonparametric, local regression model, and the residuals (difference between the model and observed values) were evaluated. In a given isofrequency domain, clusters of low or high residual RFP values were interleaved for threshold, spectral bandwidth, and latency, suggesting a modular organization. However, RFP modules in AAF were not expressed as robustly as in AI. A comparison of RFPs between AAF and AI shows that AAF neurons were more broadly tuned and had shorter latencies than AI neurons. These physiological field differences are consistent with anatomical evidence of largely independent, concurrent thalamocortical projections in AI and AAF, which strongly suggest field-specific processing.